Publications

2025 Scientific Article in Redox Biology Redox Biol. 79:103452 (2025)

Radiosensitizing capacity of fenofibrate in glioblastoma cells depends on lipid metabolism.

Despite advances in multimodal therapy approaches such as resection, chemotherapy and radiotherapy, the overall survival of patients with grade 4 glioblastoma (GBM) remains extremely poor (average survival time <2 years). Altered lipid metabolism, which increases fatty acid synthesis and thereby contributes to radioresistance in GBM, is a hallmark of cancer. Therefore, we explored the radiosensitizing effect of the clinically approved, lipid-lowering drug fenofibrate (FF) in different GBM cell lines (U87, LN18). Interestingly, FF (50 μM) significantly radiosensitizes U87 cells by inducing DNA double-strand breaks through oxidative stress and impairing mitochondrial membrane integrity, but radioprotects LN18 cells by reducing the production of reactive oxygen species (ROS) and stabilizing the mitochondrial membrane potential. A comparative protein and lipid analysis revealed striking differences in the two GBM cell lines: LN18 cells exhibited a significantly higher membrane expression density of the fatty acid (FA) cluster protein transporter CD36 than U87 cells, a higher expression of glycerol-3-phosphate acyltransferase 4 (GPAT4) which supports the production of large lipid droplets (LDs), and a lower expression of diacylglycerol O-acyltransferase 1 (DGAT1) which regulates the formation of small LDs. Consequently, large LDs are predominantly found in LN18 cells, whereas small LDs are found in U87 cells. After a combined treatment of FF and irradiation, the number of large LDs significantly increased in radioresistant LN18 cells, whereas the number of small LDs decreased in radiosensitive U87 cells. The radioprotective effect of FF in LN18 cells could be associated with the presence of large LDs, which act as a sink for the lipophilic drug FF. To prevent uptake of FF by large LDs and to ameliorate its function as a radiosensitizer, FF was encapsulated in biomimetic cell membrane extracellular lipid vesicles (CmEVs) which alter the intracellular trafficking of the drug. In contrast to the free drug, CmEV-encapsulated FF was predominantly enriched in the lysosomal compartment, causing necrosis by impairing lysosomal membrane integrity. Since the stability of plasma and lysosomal membranes is maintained by the presence of the stress-inducible heat shock protein 70 (Hsp70) which has a strong affinity to tumor-specific glycosphingolipids, necrosis occurs predominantly in LN18 cells having a lower membrane Hsp70 expression density than U87 cells. In summary, our findings indicate that the lipid metabolism of tumor cells can affect the radiosensitizing capacity of FF when encountered either as a free drug or as a drug loaded in biomimetic lipid vesicles.

2025 Scientific Article in Journal of Biomedical Optics J. Biomed. Opt. 30:S13708 (2025)

Systematic comparison of fluorescence imaging in the near-infrared and shortwave-infrared spectral range using clinical tumor samples containing cetuximab-IRDye800CW.

SIGNIFICANCE: Shortwave-infrared (SWIR) imaging is reported to yield better contrast in fluorescence-guided surgery than near-infrared (NIR) imaging, due to a reduction in scattering. This benefit of SWIR was shown in animal studies, however not yet in clinical studies with patient samples. AIM: We investigate the potential benefit of SWIR to NIR imaging in clinical samples containing cetuximab-IRDye800CW in fluorescence-guided surgery. APPROACH: The potential of the epidermal growth factor-targeted NIR dye cetuximab-IRDye800CW in the shortwave range was examined by recording the absorption and emission spectrum. An ex vivo comparison of NIR and SWIR images using clinical tumor samples of patients with penile squamous cell carcinoma (PSCC) and head and neck squamous cell carcinoma (HNSCC) containing cetuximab-IRDye800CW was performed. The comparison was based on the tumor-to-background ratio and an adapted contrast-to-noise ratio (aCNR) using the standard of care pathology tissue assessment as the golden standard. RESULTS: Based on the emission spectrum, cetuximab-IRDye800CW can be detected in the SWIR range. In clinical PSCC samples, overall SWIR imaging was found to perform similarly to NIR imaging (NIR imaging is better than SWIR in the 2/7 criteria examined, and SWIR is better than NIR in the 3/7 criteria). However, when inspecting HNSCC data, NIR is better than SWIR in nearly all (5/7) examined criteria. This difference seems to originate from background autofluorescence overwhelming the off-peak SWIR fluorescence signal in HNSCC tissue. CONCLUSION: SWIR imaging using the targeted tracer cetuximab-IRDye800CW currently does not provide additional benefit over NIR imaging in ex vivo clinical samples. Background fluorescence in the SWIR region, resulting in a higher background signal, limits SWIR imaging in HNSCC samples. However, SWIR shows potential in increasing the contrast of tumor borders in PSCC samples, as shown by a higher aCNR over a line.

2025 Scientific Article in Journal of Biomedical Optics J. Biomed. Opt. 30:S13703 (2025)

Impact of signal-to-noise ratio and contrast definition on the sensitivity assessment and benchmarking of fluorescence molecular imaging systems.

SIGNIFICANCE: Standardization of fluorescence molecular imaging (FMI) is critical for ensuring quality control in guiding surgical procedures. To accurately evaluate system performance, two metrics, the signal-to-noise ratio (SNR) and contrast, are widely employed. However, there is currently no consensus on how these metrics can be computed. AIM: We aim to examine the impact of SNR and contrast definitions on the performance assessment of FMI systems. APPROACH: We quantified the SNR and contrast of six near-infrared FMI systems by imaging a multi-parametric phantom. Based on approaches commonly used in the literature, we quantified seven SNRs and four contrast values considering different background regions and/or formulas. Then, we calculated benchmarking (BM) scores and respective rank values for each system. RESULTS: We show that the performance assessment of an FMI system changes depending on the background locations and the applied quantification method. For a single system, the different metrics can vary up to ∼ 35    dB (SNR), ∼ 8.65    a . u . (contrast), and ∼ 0.67    a . u . (BM score). CONCLUSIONS: The definition of precise guidelines for FMI performance assessment is imperative to ensure successful clinical translation of the technology. Such guidelines can also enable quality control for the already clinically approved indocyanine green-based fluorescence image-guided surgery.

2024 Review in Inverse Problems Inverse Probl. 40:125030 (2024)

Wirtinger gradient descent methods for low-dose Poisson phase retrieval.

The problem of phase retrieval has many applications in the field of optical imaging. Motivated by imaging experiments with biological specimens, we primarily consider the setting of low-dose illumination where Poisson noise plays the dominant role. In this paper, we discuss gradient descent algorithms based on different loss functions adapted to data affected by Poisson noise, in particular in the low-dose regime. Starting from the maximum log-likelihood function for the Poisson distribution, we investigate different regularizations and approximations of the problem to design an algorithm that meets the requirements that are faced in applications. In the course of this, we focus on low-count measurements. Based on an improved version of a variance-stabilizing transform for the Poisson distribution, we derive a decision rule for the regularization parameter in an averaged amplitude-based loss function. For all discussed loss functions, we study the convergence of the respective gradient descent algorithms to stationary points and find constant step sizes that guarantee descent of the loss in each iteration. Numerical experiments in the low-dose regime are performed to corroborate the theoretical observations.

2024 Scientific Article in Communications Biology Comm. Biol. 7:1627 (2024)

ICU patient-on-a-chip emulating orchestration of mast cells and cerebral organoids in neuroinflammation.

Propofol and midazolam are the current standard of care for prolonged sedation in Intensive Care Units (ICUs). However, the effects and mechanism of these sedatives in brain tissue are unclear. Herein, the development of an ICU patient-on-a-chip platform to elucidate those effects is reported. The humanized neural tissue compartment combines mast cells differentiated from human induced pluripotent stem cells (hiPSCs) with cerebral organoids in a three-dimensional (3D) matrix, which is covered with a membrane populated with human cerebral microvascular endothelial cells (hCMEC/D3) that separates the tissue chamber from the vascular lumen, where sedatives were infused for four days to evaluate neurotoxicity and cell-mediated immune responses. Subsequent to propofol administration, gene expressions of CD40 and TNF-α in mast cells, AIF1 in microglia and GFAP/S100B/OLIG2/MBP in macroglia were elevated, as well as NOS2, CD80, CD40, CD68, IL6 and TNF-α mediated proinflammation is noted in cerebral organoids, which resulted in higher expressions of GJB1, GABA-A and NMDAR1 in the tissue construct of the platform. Besides, midazolam administration stimulated expression of CD40 and CD203c+ reactivated mast cell proliferation and compromised BBB permeability and decreased TEER values with higher barrier disruption, whereas increased populations of CD11b+ microglia, higher expressions of GFAP/DLG4/GJB1 and GABA-A-/NMDAR1- identities, as well as glutamate related neurotoxicity and IL1B, IFNG, IFNA1, IL6 genes mediated proinflammation, resulting in increased apoptotic zones are observed in cerebral organoids. These results suggest that different sedatives cause variations in cell type activation that modulate different pathways related to neuroinflammation and neurotoxicity in the ICU patient-on-chip platform.

2024 Scientific Article in Cell Host & Microbe Cell Host Microbe 32, 2112-2130.e10 (2024)

Persistence of spike protein at the skull-meninges-brain axis may contribute to the neurological sequelae of COVID-19.

SARS-CoV-2 infection is associated with long-lasting neurological symptoms, although the underlying mechanisms remain unclear. Using optical clearing and imaging, we observed the accumulation of SARS-CoV-2 spike protein in the skull-meninges-brain axis of human COVID-19 patients, persisting long after viral clearance. Further, biomarkers of neurodegeneration were elevated in the cerebrospinal fluid from long COVID patients, and proteomic analysis of human skull, meninges, and brain samples revealed dysregulated inflammatory pathways and neurodegeneration-associated changes. Similar distribution patterns of the spike protein were observed in SARS-CoV-2-infected mice. Injection of spike protein alone was sufficient to induce neuroinflammation, proteome changes in the skull-meninges-brain axis, anxiety-like behavior, and exacerbated outcomes in mouse models of stroke and traumatic brain injury. Vaccination reduced but did not eliminate spike protein accumulation after infection in mice. Our findings suggest persistent spike protein at the brain borders may contribute to lasting neurological sequelae of COVID-19.

2024 Scientific Article in Nature Methods Nat. Methods 21, 2260–2270 (2024)

Probe set selection for targeted spatial transcriptomics.

Targeted spatial transcriptomic methods capture the topology of cell types and states in tissues at single-cell and subcellular resolution by measuring the expression of a predefined set of genes. The selection of an optimal set of probed genes is crucial for capturing the spatial signals present in a tissue. This requires selecting the most informative, yet minimal, set of genes to profile (gene set selection) for which it is possible to build probes (probe design). However, current selections often rely on marker genes, precluding them from detecting continuous spatial signals or new states. We present Spapros, an end-to-end probe set selection pipeline that optimizes both gene set specificity for cell type identification and within-cell type expression variation to resolve spatially distinct populations while considering prior knowledge as well as probe design and expression constraints. We evaluated Spapros and show that it outperforms other selection approaches in both cell type recovery and recovering expression variation beyond cell types. Furthermore, we used Spapros to design a single-cell resolution in situ hybridization on tissues (SCRINSHOT) experiment of adult lung tissue to demonstrate how probes selected with Spapros identify cell types of interest and detect spatial variation even within cell types.

2024 Scientific Article in SIAM Journal on Imaging Sciences SIAM J. Imaging Sci. 17, 1978-2014 (2024)

Background removal for ptychography via wigner distribution deconvolution.

Ptychography is a computational imaging technique that aims to reconstruct the object of interest from a set of diffraction patterns. Each of these is obtained by a localized illumination of the object, which is shifted after each illumination to cover its whole domain. Because in the resulting measurements the phase information is lost, ptychography gives rise to solving a phase retrieval problem. In this work, we consider ptychographic measurements contaminated by a background signal. Such a background is caused by imperfections in the experimental setup and appears as a signal that is added to the diffraction patterns. The background is assumed to be independent of the shift of the object, i.e., it is the same for all diffraction patterns. Two algorithms are provided, for arbitrary objects and for so-called phase objects that do not absorb the light but only scatter it. For the second type, a uniqueness of reconstruction is established for almost every object. Our approach is based on the Wigner distribution deconvolution, which lifts the object to a higher-dimensional matrix space where the recovery can be reformulated as a linear problem. The background only affects a few equations of the linear system that are therefore discarded. The lost information is then restored using redundancy in the higher-dimensional space.

2024 Scientific Article in Cancers Cancers 16:3589 (2024)

Cannabidiol (CBD) protects lung endothelial cells from irradiation-induced oxidative stress and inflammation in vitro and in vivo.

Objective: Radiotherapy, which is commonly used for the local control of thoracic cancers, also induces chronic inflammatory responses in the microvasculature of surrounding normal tissues such as the lung and heart that contribute to fatal radiation-induced lung diseases (RILDs) such as pneumonitis and fibrosis. In this study, we investigated the potential of cannabidiol (CBD) to attenuate the irradiation damage to the vasculature. Methods: We investigated the ability of CBD to protect a murine endothelial cell (EC) line (H5V) and primary lung ECs isolated from C57BL/6 mice from irradiation-induced damage in vitro and lung ECs (luECs) in vivo, by measuring the induction of oxidative stress, DNA damage, apoptosis (in vitro), and induction of inflammatory and pro-angiogenic markers (in vivo). Results: We demonstrated that a non-lethal dose of CBD reduces the irradiation-induced oxidative stress and early apoptosis of lung ECs by upregulating the expression of the cytoprotective mediator heme-oxygenase-1 (HO-1). The radiation-induced increased expression of inflammatory (ICAM-2, MCAM) and pro-angiogenic (VE-cadherin, Endoglin) markers was significantly reduced by a continuous daily treatment of C57BL/6 mice with CBD (i.p. 20 mg/kg body weight), 2 weeks before and 2 weeks after a partial irradiation of the lung (less than 20% of the lung volume) with 16 Gy. Conclusions: CBD has the potential to improve the clinical outcome of radiotherapy by reducing toxic side effects on the microvasculature of the lung.

2024 Scientific Article in Clinical Cancer Research Clin. Cancer Res., DOI: 10.1158/1078-0432.CCR-24-1522 (2024)

Targeting c-MET for endoscopic detection of dysplastic lesions within Barrett's esophagus using EMI-137 fluorescence imaging.

PURPOSE: Esophageal cancer (EC) carries a poor prognosis with 5-year overall survival of less than 20%. Barrett's esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). The aim of this study was to investigate the ability of EMI-137, a mesenchymal-epithelial transition factor (c-MET)-targeting optical imaging tracer, to detect dysplasia in BE. EXPERIMENTAL DESIGN: c-MET expression in human esophageal tissue was investigated using Gene Expression Omnibus (GEO) datasets, tissue microarrays and BE biopsies. EMI-137 was tested in a dual xenograft mouse model bearing OE33 (c-MET high expression) and FLO-1 (c-MET low expression) tumors. Fluorescence molecular endoscopy (FME) was performed in a mouse model of Barrett's-like metaplasia and dysplasia (L2-IL1β). Tumors and organs-of-interest were evaluated through ex vivo fluorescence imaging. RESULTS: MET mRNA expression analyses and c-MET immunostaining confirmed upregulation of c-MET in BE and EAC compared to normal epithelium. There was strong accumulation of EMI-137 in OE33 xenografts 3 h post injection decreasing by more than 50% on co-injection of a 10-fold molar excess of unlabeled EMI-137. The target-to background ratio (TBR) at 3 h p.i. for OE33 and FLO-1 tumors was 10.08 and 1.42, respectively. FME of L2-IL1β mice showed uptake of EMI-137 in dysplastic lesions within BE with a TBR of 1.9 in vivo, and greater than 2 in ex vivo fluorescence imaging. CONCLUSIONS: EMI-137 accumulates in dysplastic lesions within BE and in c-MET positive EAC. EMI-137 imaging has potential as a screening and surveillance tool for patients with BE and as a means to detecting dysplasia and EAC.

2024 Scientific Article in Photoacoustics Photoacoustics 40:100660 (2024)

Multiscale optoacoustic assessment of skin microvascular reactivity in carotid artery disease.

Microvascular endothelial dysfunction may provide insights into systemic diseases, such as carotid artery disease. Raster-scan optoacoustic mesoscopy (RSOM) can produce images of skin microvasculature during endothelial dysfunction challenges via numerous microvascular features. Herein, RSOM was employed to image the microvasculature of 26 subjects (13 patients with single carotid artery disease, 13 healthy participants) to assess the dynamics of 18 microvascular features at three scales of detail, i.e., the micro- (<100 μm), meso- (≈100–1000 μm) and macroscale (<1000 μm), during post-occlusive reactive hyperemia challenges. The proposed analysis identified a subgroup of 9 features as the most relevant to carotid artery disease because they achieved the most efficient classification (AUC of 0.93) between the two groups in the first minute of hyperemia (sensitivity/specificity: 0.92/0.85). This approach provides a non-invasive solution to microvasculature quantification in carotid artery disease, a main form of cardiovascular disease, and further highlights the possible link between systemic disease and microvascular dysfunction.

2024 Scientific Article in Journal of Biophotonics J. Biophotonics:e202400330 (2024)

Hybrid Raman and partial wave spectroscopy microscope for the characterization of molecular and structural alterations in tissue.

We present a hybrid Raman spectroscopy (RS) and partial wave spectroscopy (PWS) microscope for the characterization of molecular and structural tissue alterations. The PWS performance was assessed with surface roughness standards, while the Raman performance with a silicon crystal standard. We also validated the system on stomach and intestinal mouse tissues, two closely-related tissue types, and demonstrate that the addition of PWS information improves RS data classification for these tissue types from R2 = 0.892 to R2 = 0.964 (norm of residuals 0.863 and 0.497, respectively). Then, in a proof-of-concept experiment, we show that the hybrid system can detect changes in intestinal tissues harvested from a tumorigenic Villin-Cre, Apcfl/wt mouse. We discuss how the hybrid modality offers new abilities to identify the relative roles of PWS morphological features and Raman molecular fingerprints, possibly allowing for their combination to enhance the study of carcinogenesis and early cancer diagnostics in the future.

2024 Scientific Article in Nature Neuroscience Nat. Neurosci., DOI: 10.1038/s41593-024-01755-8 (2024)

Tonic and burst-like locus coeruleus stimulation distinctly shift network activity across the cortical hierarchy.

Noradrenaline (NA) release from the locus coeruleus (LC) changes activity and connectivity in neuronal networks across the brain, modulating multiple behavioral states. NA release is mediated by both tonic and burst-like LC activity. However, it is unknown whether the functional changes in target areas depend on these firing patterns. Using optogenetics, photometry, electrophysiology and functional magnetic resonance imaging in mice, we show that tonic and burst-like LC firing patterns elicit brain responses that hinge on their distinct NA release dynamics. During moderate tonic LC activation, NA release engages regions associated with associative processing, while burst-like stimulation biases the brain toward sensory processing. These activation patterns locally couple with increased astrocytic and inhibitory activity and change the brain's topological configuration in line with the hierarchical organization of the cerebral cortex. Together, these findings reveal how the LC-NA system achieves a nuanced regulation of global circuit operations.

2024 Review in Molecular Imaging and Biology Mol. Imaging Biol., DOI: 10.1007/s11307-024-01954-6 (2024)

Neurotoxin-derived optical probes for elucidating molecular and developmental biology of neurons and synaptic connections : Toxin-derived optical probes for neuroimaging.

Botulinum neurotoxins (BoNTs) and tetanus toxin (TeTX) are the deadliest biological substances that cause botulism and tetanus, respectively. Their astonishing potency and capacity to enter neurons and interfere with neurotransmitter release at presynaptic terminals have attracted much interest in experimental neurobiology and clinical research. Fused with reporter proteins or labelled with fluorophores, BoNTs and TeTX and their non-toxic fragments also offer remarkable opportunities to visualize cellular processes and functions in neurons and synaptic connections. This study presents the state-of-the-art optical probes derived from BoNTs and TeTX and discusses their applications in molecular and synaptic biology and neurodevelopmental research. It reviews the principles of the design and production of probes, revisits their applications with advantages and limitations and considers prospects for future improvements. The versatile characteristics of discussed probes and reporters make them an integral part of the expanding toolkit for molecular neuroimaging, promoting the discovery process in neurobiology and translational neurosciences.

2024 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 30, 4618-4634 (2024)

Remote neuroinflammation in newly diagnosed glioblastoma correlates with unfavorable clinical outcome.

PURPOSE: Current therapy strategies still provide only limited success in the treatment of glioblastoma, the most frequent primary brain tumor in adults. In addition to the characterization of the tumor microenvironment, global changes in the brain of patients with glioblastoma have been described. However, the impact and molecular signature of neuroinflammation distant of the primary tumor site have not yet been thoroughly elucidated. EXPERIMENTAL DESIGN: We performed translocator protein (TSPO)-PET in patients with newly diagnosed glioblastoma (n = 41), astrocytoma WHO grade 2 (n = 7), and healthy controls (n = 20) and compared TSPO-PET signals of the non-lesion (i.e., contralateral) hemisphere. Back-translation into syngeneic SB28 glioblastoma mice was used to characterize Pet alterations on a cellular level. Ultimately, multiplex gene expression analyses served to profile immune cells in remote brain. RESULTS: Our study revealed elevated TSPO-PET signals in contralateral hemispheres of patients with newly diagnosed glioblastoma compared to healthy controls. Contralateral TSPO was associated with persisting epileptic seizures and shorter overall survival independent of the tumor phenotype. Back-translation into syngeneic glioblastoma mice pinpointed myeloid cells as the predominant source of contralateral TSPO-PET signal increases and identified a complex immune signature characterized by myeloid cell activation and immunosuppression in distant brain regions. CONCLUSIONS: Neuroinflammation within the contralateral hemisphere can be detected with TSPO-PET imaging and associates with poor outcome in patients with newly diagnosed glioblastoma. The molecular signature of remote neuroinflammation promotes the evaluation of immunomodulatory strategies in patients with detrimental whole brain inflammation as reflected by high TSPO expression.

2024 Review in The Journal of Open Source Software JOSS, DOI: 10.21105/joss.06593 (2024)

Re-envisioning numerical information field theory (NIFTy.re): A library for gaussian processes and variational inference.

maging is the process of transforming noisy, incomplete data into a space that humans can interpret. NIFTy is a Bayesian framework for imaging and has already successfully been applied to many fields in astrophysics. Previous design decisions held the performance and the development of methods in NIFTy back. We present a rewrite of NIFTy, coined NIFTy.re, which reworks the modeling principle, extends the inference strategies, and outsources much of the heavy lifting to JAX. The rewrite dramatically accelerates models written in NIFTy, lays the foundation for new types of inference machineries, improves maintainability, and enables interoperability between NIFTy and the JAX machine learning ecosystem.

2024 Scientific Article in NPJ Cardiovascular Health NPJ Cardiovasc. Health 1:14 (2024)

Intravascular ICG-enhanced NIRF-IVUS imaging to assess progressive atherosclerotic lesions in excised human coronary arteries.

Indocyanine green (ICG)-enhanced intravascular near-infrared fluorescence (NIRF) imaging enhances the information obtained with intravascular ultrasound (IVUS) by visualizing pathobiological characteristics of atherosclerotic plaques. To advance our understanding of this hybrid method, we aimed to assess the potential of NIRF-IVUS to identify different stages of atheroma progression by characterizing ICG uptake in human pathological specimens. After excision, 15 human coronary specimens from 13 adult patients were ICG-perfused and imaged with NIRF-IVUS. All specimens were then histopathologically and immunohistochemically assessed. NIRF-IVUS imaging revealed colocalization of ICG-deposition to plaque areas of lipid accumulation, endothelial disruption, neovascularization and inflammation. Moreover, ICG concentrations were significantly higher in advanced coronary artery disease stages (p < 0.05) and correlated significantly to plaque macrophage burden (r = 0.67). Current intravascular methods fail to detect plaque biology. Thus, we demonstrate how human coronary atheroma stage can be assessed based on pathobiological characteristics uniquely captured by ICG-enhanced intravascular NIRF.

2024 Review in Nature Methods Nat. Methods, DOI: 10.1038/s41592-024-02396-2 (2024)

Controlling the sound of light: photoswitching optoacoustic imaging.

Optoacoustic (photoacoustic) imaging advances allow high-resolution optical imaging much deeper than optical microscopy. However, while label-free optoacoustics have already entered clinical application, biological imaging is in need of ubiquitous optoacoustic labels for use in ways that are similar to how fluorescent proteins propelled optical microscopy. We review photoswitching advances that shine a new light or, in analogy, 'bring a new sound' to biological optoacoustic imaging. Based on engineered labels and novel devices, switching uses light or other energy forms and enables signal modulation and synchronous detection for maximizing contrast and detection sensitivity over other optoacoustic labels. Herein, we explain contrast enhancement in the spectral versus temporal domains and review labels and key concepts of switching and their properties to modulate optoacoustic signals. We further outline systems and applications and discuss how switching can enable optoacoustic imaging of cellular or molecular contrast at depths and resolutions beyond those of other optical methods.

2024 Scientific Article in Skin Research and Technology Skin Res. Technol. 30:e70039 (2024)

Quantitative interpretation of infrared images of lower limbs in individuals with and without type 2 diabetes mellitus.

BACKGROUND: The quantitative interpretation of the radiometric information extracted from infrared (IR) images in individuals with and without type 2 diabetes mellitus (DM2) is an open problem yet to be solved. This is of particular value given that DM2 is a worldwide health problem and onset for evolution toward diabetic foot disease (DFD). Since DM2 causes changes at the vascular and neurological levels, the metabolic heat distribution on the outer skin is modified as a consequence of such alterations. Of particular interest in this contribution are those alterations displayed over the skin's heat patterns at the lower limbs. At the core of such alterations is the deterioration of the vascular and neurological networks responsible for procuring systemic thermoregulation. It is within this context that IR imaging is introduced as a likely aiding tool to assist with the clinical diagnosis of DM2 at stages early enough to prevent the evolution of the DFD. METHODS: IR images of lower limbs are acquired from a cohort of individuals clinically diagnosed with and without DM2. Additional inclusion criteria for patients are to be free from any visible wound or tissue-related trauma (e.g., injuries, edema, and so forth), and also free from non-metabolic comorbidities. All images and data are equally processed and analyzed using indices that evaluate the spatial and temporal evolution of temperature distribution in lower limbs. We studied the temporal response of individuals' legs after inducing an external stimulus. For this purpose, we combine the information of the asymmetry and thermal response index (ATR) and the thermal response index (TRI), computed using images at different times, improving the results previously obtained individually with ATR and TRI. RESULTS: A novel representation of the information extracted from IR images of the lower limbs in individuals with and without DM2 is presented. This representation was built using the ATR and TRI indices for the anterior and posterior views (PVs), individually and combining the information from both views. In all cases, the information of each index and each view presents linearity properties that allow said information to be interpreted quantitatively in a well-defined and limited space. This representation, built in a polar coordinate space, allows obtaining sensitivity values of 86%, 97%, and 97%, and specificity values of 83%, 72%, and 78% for the anterior view (AV), the PV, and the combined views, respectively. Additionally, it was observed that the angular variable that defines this new representation space allows to significantly (p < 0.01) differentiate the groups, while correlating with clinical variables of interest, such as glucose and glycated hemoglobin. CONCLUSION: The linearity properties that exist between the ATR and TRI indices allow a quantitative interpretation of the information extracted from IR images of the lower extremities of individuals with and without DM2, and allow the construction of a representation space that eliminates possible ambiguities in the interpretation, while simplifying it, making it accessible for clinical use.

2024 Cardiovascular Diabetology Cardiovasc. Diabetol. 23:341 (2024)

Correction: GIP receptor agonism improves dyslipidemia and atherosclerosis independently of body weight loss in preclinical mouse model for cardio-metabolic disease.

Following publication of the original article [1], the author noticed the errors in Fig. 2 and in Results section. The bar graph is mistakenly duplicated in “percentage of plaque area of the aortic valves” of Fig. 2E. The corrected figure is given below: Acyl-GIP ameliorates dyslipidemia and atherosclerotic plaque formation in LDLR-/- male mice. Plasma (A) triglycerides, (B) cholesterol and (C) lipoprotein fractions as well as (D and E) the percentage of plaque area in aortic arches and valves and along the descending aorta of male LDLR-/- mice treated daily with either vehicle or acyl-GIP via subcutaneous injections for 28 days. n = 7. Blood lipids were determined from sac plasma at the end of the study. Data represent means ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001, determined by unpaired two-sided t-test In Result section under the heading “GIPR-agonist acyl-GIP ameliorates dyslipidemia and atherosclerotic plaque formation in male LDLR-/- mice independently of weight loss”, the last sentence should read “Most importantly, acyl-GIP treatment was accompanied by reduced atherosclerotic plaque formation within the aortic valve and a trend to decrease fat streaks along the descending aorta (Fig. 2E)“ instead of “Most importantly, acyl-GIP treatment was accompanied by reduced atherosclerotic plaque formation within the aortic valve (Fig. 2G–H) and decreased fat streaks along the descending aorta (Fig. 2I)”.

2024 Scientific Article in Scientific Reports Sci. Rep. 14:21864 (2024)

Dipeptide metabolite, glutamyl-glutamate mediates microbe-host interaction to boost spermatogenesis.

The decrease in sperm count and infertility is a global issue that remains unresolved. By screening environmental bacterial isolates, we have found that a novel lactic acid bacterium, Lactiplantibacillus plantarum SNI3, increased testis size, testosterone levels, sperm count, sexual activity and fertility in mice that have consumed the bacteria for four weeks. The abundance of L. plantarum in the colon microbiome was positively associated with sperm count. Fecal microbiota transplantation (FMT) from L. plantarum SNI3-dosed mice improved testicular functions in microbiome-attenuated recipient animals. To identify mediators that confer pro-reproductive effects on the host, untargeted in situ mass spectrometry metabolomics was performed on testis samples of L. plantarum SNI3-treated and control mice. Enrichment pathway analysis revealed several perturbed metabolic pathways in the testis of treated mice. Within the testis, a dipeptide, glutamyl-glutamate (GluGlu) was the most upregulated metabolite following L. plantarum SNI3 administration. To validate the pro-reproductive feature of GluGlu, systemic and local injections of the dipeptide have been performed. γ-GluGlu increased sperm count but had no effect on testosterone. These findings highlight the role of γ-GluGlu in mediating spermatogenetic effects of L. plantarum on the male mouse host and -following relevant human clinical trials- may provide future tools for treating certain forms of male infertility.

2024 Scientific Article in Metabolism: clinical and experimental Metabolism 161:156034 (2024)

Inter-organ cross-talk in human cancer cachexia revealed by spatial metabolomics.

BACKGROUND: Cancer cachexia (CCx) presents a multifaceted challenge characterized by negative protein and energy balance and systemic inflammatory response activation. While previous CCx studies predominantly focused on mouse models or human body fluids, there's an unmet need to elucidate the molecular inter-organ cross-talk underlying the pathophysiology of human CCx. METHODS: Spatial metabolomics were conducted on liver, skeletal muscle, subcutaneous and visceral adipose tissue, and serum from cachectic and control cancer patients. Organ-wise comparisons were performed using component, pathway enrichment and correlation network analyses. Inter-organ correlations in CCx altered pathways were assessed using Circos. Machine learning on tissues and serum established classifiers as potential diagnostic biomarkers for CCx. RESULTS: Distinct metabolic pathway alteration was detected in CCx, with adipose tissues and liver displaying the most significant (P ≤ 0.05) metabolic disturbances. CCx patients exhibited increased metabolic activity in visceral and subcutaneous adipose tissues and liver, contrasting with decreased activity in muscle and serum compared to control patients. Carbohydrate, lipid, amino acid, and vitamin metabolism emerged as highly interacting pathways across different organ systems in CCx. Muscle tissue showed decreased (P ≤ 0.001) energy charge in CCx patients, while liver and adipose tissues displayed increased energy charge (P ≤ 0.001). We stratified CCx patients by severity and metabolic changes, finding that visceral adipose tissue is most affected, especially in cases of severe cachexia. Morphometric analysis showed smaller (P ≤ 0.05) adipocyte size in visceral adipose tissue, indicating catabolic processes. We developed tissue-based classifiers for cancer cachexia specific to individual organs, facilitating the transfer of patient serum as minimally invasive diagnostic markers of CCx in the constitution of the organs. CONCLUSIONS: These findings support the concept of CCx as a multi-organ syndrome with diverse metabolic alterations, providing insights into the pathophysiology and organ cross-talk of human CCx. This study pioneers spatial metabolomics for CCx, demonstrating the feasibility of distinguishing cachexia status at the organ level using serum.

2024 Scientific Article in npj Precision Oncology npj Precis. Oncol. 8:116 (2024)

Metabolic pathway-based subtypes associate glycan biosynthesis and treatment response in head and neck cancer.

Head and Neck Squamous Cell Carcinoma (HNSCC) is a heterogeneous malignancy that remains a significant challenge in clinical management due to frequent treatment failures and pronounced therapy resistance. While metabolic dysregulation appears to be a critical factor in this scenario, comprehensive analyses of the metabolic HNSCC landscape and its impact on clinical outcomes are lacking. This study utilized transcriptomic data from four independent clinical cohorts to investigate metabolic heterogeneity in HNSCC and define metabolic pathway-based subtypes (MPS). In HPV-negative HNSCCs, MPS1 and MPS2 were identified, while MPS3 was enriched in HPV-positive cases. MPS classification was associated with clinical outcome post adjuvant radio(chemo)therapy, with MPS1 consistently exhibiting the highest risk of therapeutic failure. MPS1 was uniquely characterized by upregulation of glycan (particularly chondroitin/dermatan sulfate) metabolism genes. Immunohistochemistry and pilot mass spectrometry imaging analyses confirmed this at metabolite level. The histological context and single-cell RNA sequencing data identified the malignant cells as key contributors. Globally, MPS1 was distinguished by a unique transcriptomic landscape associated with increased disease aggressiveness, featuring motifs related to epithelial-mesenchymal transition, immune signaling, cancer stemness, tumor microenvironment assembly, and oncogenic signaling. This translated into a distinct histological appearance marked by extensive extracellular matrix remodeling, abundant spindle-shaped cancer-associated fibroblasts, and intimately intertwined populations of malignant and stromal cells. Proof-of-concept data from orthotopic xenotransplants replicated the MPS phenotypes on the histological and transcriptome levels. In summary, this study introduces a metabolic pathway-based classification of HNSCC, pinpointing glycan metabolism-enriched MPS1 as the most challenging subgroup that necessitates alternative therapeutic strategies.

2024 Scientific Article in Nature Communications Nat. Commun. 15:7521 (2024)

Bragg grating etalon-based optical fiber for ultrasound and optoacoustic detection.

Fiber-based interferometers receive significant interest as they lead to miniaturization of optoacoustic and ultrasound detectors without the quadratic loss of sensitivity common to piezoelectric elements. Nevertheless, in contrast to piezoelectric crystals, current fiber-based ultrasound detectors operate with narrow ultrasound bandwidth which limits the application range and spatial resolution achieved in imaging implementations. We port the concept of silicon waveguide etalon detection to optical fibers using a sub-acoustic reflection terminator to a Bragg grating embedded etalon resonator (EER), uniquely implementing direct and forward-looking access to incoming ultrasound waves. Precise fabrication of the terminator is achieved by continuously recording the EER spectrum during polishing and fitting the spectra to a theoretically calculated spectrum for the selected thickness. Characterization of the EER inventive design reveals a small aperture (10.1 µm) and an ultra-wide bandwidth (160 MHz) that outperforms other fiber resonators and enables an active detection area and overall form factor that is smaller by more than an order of magnitude over designs based on piezoelectric transducers. We discuss how the EER paves the way for the most adept fiber-based miniaturized sound detection today, circumventing the limitations of currently available designs.

2024 Scientific Article in Molecular Immunology Mol. Immunol. 174, DOI: 10.1016/j.molimm.2024.07.008 (2024)

The immunomodulatory effects of cannabidiol on Hsp70-activated NK cells and tumor target cells.

BACKGROUND: Cannabidiol (CBD), the major non-psychoactive component of cannabis, exhibits anti-inflammatory properties, but less is known about the immunomodulatory potential of CBD on activated natural killer (NK) cells and/or their targets. Many tumor cells present heat shock protein 70 (Hsp70) on their cell surface in a tumor-specific manner and although a membrane Hsp70 (mHsp70) positive phenotype serves as a target for Hsp70-activated NK cells, a high mHsp70 expression is associated with tumor aggressiveness. This study investigated the immuno-modulatory potential of CBD on NK cells stimulated with TKD Hsp70 peptide and IL-2 (TKD+IL-2) and also on HCT116 p53wt and HCT116 p53-/- colorectal cancer cells exhibiting high and low basal levels of mHsp70 expression. RESULTS: Apart from an increase in the density of NTB-A and a reduced expression of LAMP-1, the expression of all other activatory NK cell receptors including NKp30, NKG2D and CD69 which are significantly up-regulated after stimulation with TKD+IL-2 remained unaffected after a co-treatment with CBD. However, the release of major pro-inflammatory cytokines by NK cells such as interferon-γ (IFN-γ) and the effector molecule granzyme B (GrzB) was significantly reduced upon CBD treatment. With respect to the tumor target cells, CBD significantly reduced the elevated expression of mHsp70 but had no effect on the low basal mHsp70 expression. Expression of other NK cell ligands such as MICA and MICB remained unaffected, and the NK cell ligands ULBP and B7-H6 were not expressed on these target cells. Consistent with the reduced mHsp70 expression, treatment of both effector and target cells with CBD reduced the killing of high mHsp70 expressing tumor cells by TKD+IL-2+CBD pre-treated NK cells but had no effect on the killing of low mHsp70 expressing tumor cells. Concomitantly, CBD treatment reduced the TKD+IL-2 induced increased release of IFN-γ, IL-4, TNF-α and GrzB, but CBD had no effect on the release of IFN-α when NK cells were co-incubated with tumor target cells. CONCLUSION: Cannabidiol (CBD) may potentially diminish the anti-tumor effectiveness of TKD+IL-2 activated natural killer (NK) cells.

2024 Scientific Article in Physical Review B Phys. Rev. B Condens. Matter 110:064102 (2024)

Thermal vibrations in the inversion of dynamical electron scattering.

Relativistic forward scattering of electrons at finite temperature involves the incoherent superposition of diffraction patterns formed by different snapshots of thermal atomic displacements. In experiments, thermal vibrations lead to thermal diffuse scattering (TDS), partly dominating diffraction patterns of thick specimens. This study sheds light on the effects of TDS on solutions to the inverse scattering problem using combined realand diffraction-space information acquired in a scanning transmission electron microscope (STEM) to retrieve the object's phase. Using frozen phonon multislice within the Einstein approximation, realistic ground truth data of 20-nm-thick SrTiO3 is generated and subjected to contemporary inverse multislice schemes to retrieve the projected Coulomb potential slicewise. We first classify phase retrieval algorithms as to their assumptions on periodicity along the incident beam direction, as well as pixelwise and parametrized reconstruction methods. It is found that pixelwise object reconstructions are capable of retrieving structural details qualitatively while being prone to contain TDS-related artifacts which can result in unphysical potentials. For pixelwise reconstructions of multiple independent specimen slices, we observe that the origin of TDS, i.e., thermal atomic displacements, starts to emerge naturally. However, the quantitative assessment tends to too small mean squared thermal displacements, also when reconstructing multiple object modes. Using an atomistically parametrized inversion strategy which exploits the explicit separation of thermal vibrations and potentials, temperature and chemistry of the specimen can be retrieved quantitatively with high accuracy.

2024 Review in Heliyon Heliyon 10:e36659 (2024)

Decoding the genomic enigma: Approaches to studying extrachromosomal circular DNA.

Extrachromosomal circular DNA (eccDNA), a pervasive yet enigmatic component of the eukaryotic genome, exists autonomously from its chromosomal counterparts. Ubiquitous in eukaryotes, eccDNA plays a critical role in the orchestration of cellular processes and the etiology of diseases, particularly cancers. However, the full scope of its influence on health and disease remains elusive, presenting a rich vein of research yet to be mined. Unraveling the complexities of eccDNA necessitates a distillation of methodologies — from biogenesis to functional analysis — a landscape we overview in this study with precision and clarity. Here, we systematically outline cutting-edge methodologies from high-throughput sequencing and bioinformatics to experimental validations, showcasing the intricate world of eccDNAs. We combed through a treasure trove of auxiliary research resources and analytical tools. Moreover, we chart a course for future inquiry, illuminating the horizon with potential groundbreaking strategies for designing eccDNA research projects and pioneering new methodological frontiers.

2024 Scientific Article in Nature Physics Nat. Phys., DOI: 10.1038/s41567-024-02591-0 (2024)

Flow physics guides morphology of ciliated organs.

Organs that pump luminal fluids by the coordinated beat of motile cilia are integral to animal physiology. Such organs include the human airways, brain ventricles and reproductive tracts. Although cilia organization and duct morphology vary drastically in the animal kingdom, ducts are typically classified as carpet or flame designs. The reason behind the appearance of these two different designs and how they relate to fluid pumping remain unclear. Here, we demonstrate that two structural parameters—lumen diameter and cilia-to-lumen ratio—organize the observed duct diversity into a continuous spectrum that connects carpets to flames across all animal phyla. Using a unified fluid model, we show that carpets and flames represent trade-offs between flow rate and pressure generation. We propose that the convergence of ciliated organ designs follows functional constraints rather than phylogenetic distance and offer guiding design principles for synthetic ciliary pumps.

2024 Editorial in Journal of Clinical Laboratory Analysis J. Clin. Lab. Anal. 38:e25082 (2024)

Importance of In Vitro Embryo Model Procedure Standardization.

In vivo studies offer a detailed understanding of organism functioning, surpassing the insights provided by in vitro studies. These experiments are crucial for comprehending disease emergence, progression, and associated mechanisms in humans, as well as for developing treatments. When choosing experimental models, factors such as genomic similarity, physiological relevance, ethical appropriateness, and economic feasibility must be considered. Standardized protocols enhance the reliability, and reproducibility of scientific methods, promoting the assessment of research in the scientific literature. Researchers conducting embryo studies should establish and document standardized protocols for increased data comparability. Standardization is vital for scientific validity, reproducibility, and comparability in both in vivo and in vitro studies, ensuring the accuracy and reliability of experimental results and advancing scientific knowledge.

2024 Scientific Article in Micron Micron 185:103688 (2024)

Influence of loss function and electron dose on ptychography of 2D materials using the Wirtinger flow.

Iterative phase retrieval is based on minimising a loss function as a measure of the consistency of an initial guess and underlying experimental data. Under ideal experimental conditions, real data contains Poissonian noise due to counting statistics. In this work, we use the Wirtinger Flow concept in combination with four common loss functions, being the L1 loss, the mean-squared error (MSE), the amplitude loss and the Poisson loss. Since only the latter reflects the counting statistics as an asymmetric Poisson distribution correctly, our simulation study focuses on two main cases. Firstly, high-dose momentum-resolved scanning transmission electron microscopy (STEM) of an MoS2 monolayer is considered for phase retrieval. In this case, it is found that the four losses perform differently with respect to chemical sensitivity and frequency transfer, which we interprete in terms of the substantially different signal level in the bright and dark field part of diffraction patterns. Remedies are discussed using further simulations, addressing the use of virtual ring detectors for the dark field, or restricting loss calculation to the bright field. Secondly, a dose series is presented down to 100 electrons per diffraction pattern. It is found that all losses yield qualitatively reasonable structural data in the phase, whereas only MSE and Poisson loss range at the correct amplitude level. Chemical contrast is, in general, reliably obtained using the Poisson concept, which also provides the most continuous spatial frequency transfer as to the reconstructed object transmission function.

2024 Scientific Article in Advanced Materials Technologies Adv. Mater. Technol., DOI: 10.1002/admt.202400645 (2024)

Inkjet-printed 3D electrode arrays for recording signals from cortical organoids.

Monitoring electrical activity across multiple planes in 3D cell cultures and organoids is imperative to comprehensively understand their functional connections and behavior. However, traditional planar microelectrode arrays (MEAs) are intended for surface recordings and are inadequate in addressing this aspect. The limitations, such as longer production times and limited adaptability imposed by standard clean-room techniques, constrain the design possibilities for 3D electrode arrays and potentially hinder effective cell-electrode coupling. To tackle this challenge, a novel approach is presented that leverages rapid prototyping processes and additive manufacturing in combination with wet etching and electrodeposition to enhance electrode fabrication and performance. The laser-patterned MEAs on glass, polyimide (PI) foil, or polyethylene terephthalate (PET) foil substrates incorporate high-aspect ratio (up to 44:1) ink-jet printed 3D electrode structures with heights up to 1 mm at a pitch of 200 µm, enabling precise recording within cell tissues. The specific shapes of the electrode tips and customizable 3D structures provide great flexibility in electrode placement. The versatility of the 3D MEAs is demonstrated by recording the electrophysiological activity of cortical organoids in situ, paving the way for investigating neural activity under regular or various pathologically altered conditions in vitro in a high throughput manner.

2024 Scientific Article in Bioinformatics Bioinformatics 40, i548-i557 (2024)

GraphCompass: Spatial metrics for differential analyses of cell organization across conditions.

SUMMARY: Spatial omics technologies are increasingly leveraged to characterize how disease disrupts tissue organization and cellular niches. While multiple methods to analyze spatial variation within a sample have been published, statistical and computational approaches to compare cell spatial organization across samples or conditions are mostly lacking. We present GraphCompass, a comprehensive set of omics-adapted graph analysis methods to quantitatively evaluate and compare the spatial arrangement of cells in samples representing diverse biological conditions. GraphCompass builds upon the Squidpy spatial omics toolbox and encompasses various statistical approaches to perform cross-condition analyses at the level of individual cell types, niches, and samples. Additionally, GraphCompass provides custom visualization functions that enable effective communication of results. We demonstrate how GraphCompass can be used to address key biological questions, such as how cellular organization and tissue architecture differ across various disease states and which spatial patterns correlate with a given pathological condition. GraphCompass can be applied to various popular omics techniques, including, but not limited to, spatial proteomics (e.g. MIBI-TOF), spot-based transcriptomics (e.g. 10× Genomics Visium), and single-cell resolved transcriptomics (e.g. Stereo-seq). In this work, we showcase the capabilities of GraphCompass through its application to three different studies that may also serve as benchmark datasets for further method development. With its easy-to-use implementation, extensive documentation, and comprehensive tutorials, GraphCompass is accessible to biologists with varying levels of computational expertise. By facilitating comparative analyses of cell spatial organization, GraphCompass promises to be a valuable asset in advancing our understanding of tissue function in health and disease. UNLABELLED:  .

2024 Scientific Article in ACS Omega ACS Omega 9, 30256-30269 (2024)

Inhibition of the early-stage cross-amyloid aggregation of amyloid-β and IAPP via EGCG: Insights from molecular dynamics simulations.

Amyloid-β (Aβ) and islet amyloid polypeptide (IAPP) are small peptides that have the potential to not only self-assemble but also cross-assemble and form cytotoxic amyloid aggregates. Recently, we experimentally investigated the nature of Aβ-IAPP coaggregation and its inhibition by small polyphenolic molecules. Notably, we found that epigallocatechin gallate (EGCG) had the ability to reduce heteroaggregate formation. However, the precise molecular mechanism behind the reduction of heteroaggregates remains unclear. In this study, the dimerization processes of Aβ40 and IAPP peptides with and without EGCG were characterized by the enhanced sampling technique. Our results showed that these amyloid peptides exhibited a tendency to form a stable heterodimer, which represented the first step toward coaggregation. Furthermore, we also found that the EGCG regulated the dimerization process. In the presence of EGCG, well-tempered metadynamics simulation indicated a notable shift in the bound state toward a greater center of mass (COM) distance. Additionally, the presence of EGCG led to a significant increase in the free energy barrier height (∼15kBT) along the COM distance, and we observed a transition state between the bound and unbound states. Our findings also unveiled that the EGCG formed a greater number of hydrogen bonds with Aβ40, effectively obstructing the dimer formation. In addition, we carried out microseconds of all-atom conventional molecular dynamics (cMD) simulations to investigate the formation of both hetero- and homo-oligomer states by these peptides. MD simulations illustrated that EGCG played a significant role in preventing oligomer formation by reducing the content of β-sheets in the peptide. Collectively, our results offered valuable insight into the mechanism of cross-amyloid aggregation between Aβ40 and IAPP and the inhibition effect of EGCG on the heteroaggregation process.

2024 Review in Nature Methods Nat. Methods 21, 1153-1165 (2024)

Deep 3D histology powered by tissue clearing, omics and AI.

To comprehensively understand tissue and organism physiology and pathophysiology, it is essential to create complete three-dimensional (3D) cellular maps. These maps require structural data, such as the 3D configuration and positioning of tissues and cells, and molecular data on the constitution of each cell, spanning from the DNA sequence to protein expression. While single-cell transcriptomics is illuminating the cellular and molecular diversity across species and tissues, the 3D spatial context of these molecular data is often overlooked. Here, I discuss emerging 3D tissue histology techniques that add the missing third spatial dimension to biomedical research. Through innovations in tissue-clearing chemistry, labeling and volumetric imaging that enhance 3D reconstructions and their synergy with molecular techniques, these technologies will provide detailed blueprints of entire organs or organisms at the cellular level. Machine learning, especially deep learning, will be essential for extracting meaningful insights from the vast data. Further development of integrated structural, molecular and computational methods will unlock the full potential of next-generation 3D histology.

2024 Scientific Article in Photoacoustics Photoacoustics 38:100628 (2024)

Non-invasive optoacoustic imaging of dermal microcirculatory revascularization in diet-induced obese mice undergoing exercise intervention.

Microcirculatory dysfunction has been observed in the dermal white adipose tissue (dWAT) and subcutaneous white adipose tissue (scWAT) of obese humans and has been proposed as an early prediction marker for cardio-metabolic disease progression. In-vivo visualization and longitudinal monitoring of microvascular remodeling in these tissues remains challenging. We compare the performance of two optoacoustic imaging methods, i.e. multi-spectral optoacoustic tomography (MSOT) and raster-scanning optoacoustic mesoscopy (RSOM) in visualizing lipid and hemoglobin contrast in scWAT and dWAT in a mouse model of diet-induced obesity (DIO) undergoing voluntary wheel running intervention for 32 weeks. MSOT visualized lipid and hemoglobin contrast in murine fat depots in a quantitative manner even at early stages of DIO. We show for the first time to our knowledge that RSOM allows precise visualization of the dWAT microvasculature and provides quantitative readouts of skin layer thickness and vascular density in dWAT and dermis. Combination of MSOT and RSOM resolved exercise-induced morphological changes in microvasculature density, tissue oxygen saturation, lipid and blood volume content in dWAT and scWAT. The combination of MSOT and RSOM may allow precise monitoring of microcirculatory dysfunction and intervention response in dWAT and scWAT in a mouse model for DIO. Our findings have laid out the foundation for future clinical studies using optoacoustic-derived vascular readouts from adipose tissues as a biomarker for monitoring microcirculatory function in metabolic disease.

2024 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 43, 2061-2073 (2024)

Synthetic optical coherence tomography angiographs for detailed retinal vessel segmentation without human annotations.

Optical coherence tomography angiography (OCTA) is a non-invasive imaging modality that can acquire high-resolution volumes of the retinal vasculature and aid the diagnosis of ocular, neurological and cardiac diseases. Segmenting the visible blood vessels is a common first step when extracting quantitative biomarkers from these images. Classical segmentation algorithms based on thresholding are strongly affected by image artifacts and limited signal-to-noise ratio. The use of modern, deep learning-based segmentation methods has been inhibited by a lack of large datasets with detailed annotations of the blood vessels. To address this issue, recent work has employed transfer learning, where a segmentation network is trained on synthetic OCTA images and is then applied to real data. However, the previously proposed simulations fail to faithfully model the retinal vasculature and do not provide effective domain adaptation. Because of this, current methods are unable to fully segment the retinal vasculature, in particular the smallest capillaries. In this work, we present a lightweight simulation of the retinal vascular network based on space colonization for faster and more realistic OCTA synthesis. We then introduce three contrast adaptation pipelines to decrease the domain gap between real and artificial images. We demonstrate the superior segmentation performance of our approach in extensive quantitative and qualitative experiments on three public datasets that compare our method to traditional computer vision algorithms and supervised training using human annotations. Finally, we make our entire pipeline publicly available, including the source code, pretrained models, and a large dataset of synthetic OCTA images.

2024 Scientific Article in Journal of Controlled Release J. Control. Release 372, 522-530 (2024)

Tuning the photophysical properties of cyanine by barbiturate functionalization and nanoformulation for efficient optoacoustics- guided phototherapy.

Cyanine derivatives are organic dyes widely used for optical imaging. However, their potential in longitudinal optoacoustic imaging and photothermal therapy remains limited due to challenges such as poor chemical stability, poor photostability, and low photothermal conversion. In this study, we present a new structural modification for cyanine dyes by introducing a strongly electron-withdrawing group (barbiturate), resulting in a new series of barbiturate-cyanine dyes (BC810, BC885, and BC1010) with suppressed fluorescence and enhanced stability. Furthermore, the introduction of BC1010 into block copolymers (PEG114-b-PCL60) induces aggregation-caused quenching, further boosting the photothermal performance. The photophysical properties of nanoparticles (BC1010-NPs) include their remarkably broad absorption range from 900 to 1200 nm for optoacoustic imaging, allowing imaging applications in NIR-I and NIR-II windows. The combined effect of these strategies, including improved photostability, enhanced nonradiative relaxation, and aggregation-caused quenching, enables the detection of optoacoustic signals with high sensitivity and effective photothermal treatment of in vivo tumor models when BC1010-NPs are administered before irradiation with a 1064 nm laser. This research introduces a barbiturate-functionalized cyanine derivative with optimal properties for efficient optoacoustics-guided theranostic applications. This new compound holds significant potential for biomedical use, facilitating advancements in optoacoustic-guided diagnostic and therapeutic approaches.

2024 Review in Current Opinion in Biotechnology Curr. Opin. Biotechnol. 88:103149 (2024)

Multimodal techniques and strategies for chemical and metabolic imaging at the single-cell level.

Single-cell chemical and metabolic imaging technologies provide unprecedented insights into individual cell dynamics, advancing our understanding of cellular processes, molecular interactions, and metabolic activities. Advances in fluorescence, Raman, optoacoustic (photoacoustic), or mass spectrometry methods have paved the way to characterize metabolites, signaling molecules, and other moieties within individual cells. These modalities can also lead to single-cell imaging capabilities by targeting endogenous cell contrast or by employing exogenous contrast generation techniques, including contrast agents that target specific cell structure or function. In this review, we present key developments, summarize recent applications in single-cell interrogation and imaging, and illustrate their advantages, limitations, and outlook.

2024 Scientific Article in Angewandte Chemie - Internationale Edition Angew. Chem.-Int. Edit.:e202405636 (2024)

Merged molecular switches excel as optoacoustic dyes: Azobenzene-cyanines are loud and photostable NIR imaging agents.

Optoacoustic (or photoacoustic) imaging promises micron-resolution noninvasive bioimaging with much deeper penetration (>cm) than fluorescence. However, optoacoustic imaging of enzyme activity would require loud, photostable, NIR-absorbing molecular contrast agents: which remain unknown. Most organic molecular contrast agents are repurposed fluorophores, with severe shortcomings of photoinstability or phototoxicity under optoacoustic imaging, as consequences of their slow S1→S0 electronic relaxation. We now report that known fluorophores can be rationally modified to reach ultrafast S1→S0 rates, without much extra molecular complexity, simply by merging them with molecular switches. Here, we merge azobenzene switches to cyanine dyes to give ultrafast relaxation (<10 ps, >100-fold faster). Without even adapting instrument settings, these azohemicyanines display outstanding improvements in signal longevity (>1000-fold increase of photostability) and signal loudness (here: >3-fold even at time zero). We show why this simple but unexplored design strategy can still offer stronger performance in the future, and can also increase the spatial resolution and the quantitative linearity of photoacoustic response over extended longitudinal imaging. By bringing the world of molecular switches and rotors to bear on problems facing optoacoustic agents, this practical strategy will help to unleash the full potential of optoacoustic imaging in fundamental studies and translational uses.

2024 Scientific Article in Nature Methods Nat. Methods, DOI: 10.1038/s41592-024-02245-2 (2024)

Virtual reality-empowered deep-learning analysis of brain cells.

Automated detection of specific cells in three-dimensional datasets such as whole-brain light-sheet image stacks is challenging. Here, we present DELiVR, a virtual reality-trained deep-learning pipeline for detecting c-Fos+ cells as markers for neuronal activity in cleared mouse brains. Virtual reality annotation substantially accelerated training data generation, enabling DELiVR to outperform state-of-the-art cell-segmenting approaches. Our pipeline is available in a user-friendly Docker container that runs with a standalone Fiji plugin. DELiVR features a comprehensive toolkit for data visualization and can be customized to other cell types of interest, as we did here for microglia somata, using Fiji for dataset-specific training. We applied DELiVR to investigate cancer-related brain activity, unveiling an activation pattern that distinguishes weight-stable cancer from cancers associated with weight loss. Overall, DELiVR is a robust deep-learning tool that does not require advanced coding skills to analyze whole-brain imaging data in health and disease.

2024 Scientific Article in Astronomy and Astrophysics Astron. Astrophys. 684:A155 (2024)

The first spatio-spectral Bayesian imaging of SN1006 in X-rays.

Supernovae (SNs) are an important source of energy in the interstellar medium. Young remnants of supernovae (SNRs) exhibit peak emission in the X-ray region, making them interesting objects for X-ray observations. In particular, the supernova remnant SN1006 is of great interest due to its historical record, proximity, and brightness. Thus, it has been studied with a number of X-ray telescopes. Improving X-ray imaging of this and other remnants is an important but challenging task, as it often requires multiple observations with different instrument responses to image the entire object. Here, we use Chandra observations to demonstrate the capabilities of Bayesian image reconstruction using information field theory (IFT). Our objective is to reconstruct denoised, deconvolved, and spatio-spectral resolved images from X-ray observations and to decompose the emission into different morphologies, namely, diffuse and point-like. Further, we aim to fuse data from different detectors and pointings into a mosaic and quantify the uncertainty of our result. By utilizing prior knowledge on the spatial and spectral correlation structure of the diffuse emission and point sources, this method allows for the effective decomposition of the signal into these two components. In order to accelerate the imaging process, we introduced a multi-step approach, in which the spatial reconstruction obtained for a single energy range is used to derive an informed starting point for the full spatio-spectral reconstruction. We applied this method to 11 Chandra observations of SN1006 from 2008 and 2012, providing a detailed, denoised, and decomposed view of the remnant. In particular, the separated view of the diffuse emission ought to provide new insights into the complex, small-scale structures in the center of the remnant and at the shock front profiles. For example, our analysis reveals sharp X-ray flux increases by up to two orders of magnitude at the shock fronts of SN1006.

2024 Letter to the Editor in Cell Discovery Cell Discov. 10:42 (2024)

Engineered, nucleocytoplasmic shuttling Cas13d enables highly efficient cytosolic RNA targeting.

2024 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 43, 3710-3718 (2024)

Performance assessment and quality control of fluorescence molecular endoscopy with a multi-parametric rigid standard.

Fluorescence molecular endoscopy (FME) is emerging as a “red-flag” technique with potential to deliver earlier, faster, and more personalized detection of disease in the gastrointestinal tract, including cancer, and to gain insights into novel drug distribution, dose finding, and response prediction. However, to date, the performance of FME systems is assessed mainly by endoscopists during a procedure, leading to arbitrary, potentially biased, and heavily subjective assessment. This approach significantly affects the repeatability of the procedures and the interpretation or comparison of the acquired data, representing a major bottleneck towards the clinical translation of the technology. Herein, we propose a robust methodology for FME performance assessment and quality control that is based on a novel multi-parametric rigid standard. This standard enables the characterization of an FME system’s sensitivity through a single acquisition, performance comparison of multiple systems, and, for the first time, quality control of a system as a function of time and number of usages. We show the photostability of the standard experimentally and demonstrate how it can be used to characterize the performance of an FME system. Moreover, we showcase how the standard can be employed for quality control of a system. In this study, we find that the use of composite fluorescence standards before endoscopic procedures can ensure that an FME system meets the performance criteria and that components prone to performance degradation are replaced in time, avoiding disruption of clinical endoscopy logistics. This will help overcome a major barrier for the translation of FME into the clinics.

2024 Scientific Article in Nature metabolism Nat. Metab. 6, 678-686 (2024)

Non-invasive measurements of blood glucose levels by time-gating mid-infrared optoacoustic signals.

Non-invasive glucose monitoring (NIGM) represents an attractive alternative to finger pricking for blood glucose assessment and management of diabetes. Nevertheless, current NIGM techniques do not measure glucose concentrations in blood but rely on indirect bulk measurement of glucose in interstitial fluid, where glucose is diluted and glucose dynamics are different from those in the blood, which impairs NIGM accuracy. Here we introduce a new biosensor, termed depth-gated mid-infrared optoacoustic sensor (DIROS), which allows, for the first time, non-invasive glucose detection in blood-rich volumes in the skin. DIROS minimizes interference caused by the stratum corneum and other superficial skin layers by time-gating mid-infrared optoacoustic signals to enable depth-selective localization of glucose readings in skin. In measurements on the ears of (female) mice, DIROS displays improved accuracy over bulk-tissue glucose measurements. Our work demonstrates how signal localization can improve NIGM accuracy and positions DIROS as a holistic approach, with high translational potential, that addresses a key limitation of current NIGM methods.

2024 Editorial in Nature metabolism Nat. Metab. 6, 615–616 (2024)

Blood glucose concentration measurement without finger pricking.

A new sensor that detects optoacoustic signals generated by mid-infrared light enables measurement of glucose concentration from intracutaneous tissue rich in blood. This technology does not rely on glucose measurements in interstitial fluid or blood sampling and might yield the next generation of non-invasive glucose-sensing devices for improved diabetes management.

2024 Scientific Article in Nature Aging Nature Aging 4, 595-612 (2024)

Proteomics of mouse brain endothelium uncovers dysregulation of vesicular transport pathways during aging.

Age-related decline in brain endothelial cell (BEC) function contributes critically to neurological disease. Comprehensive atlases of the BEC transcriptome have become available, but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting-based mouse BEC enrichment protocol compatible with proteomics and resolved the profiles of protein abundance changes during aging. Unsupervised cluster analysis revealed a segregation of age-related protein dynamics with biological functions, including a downregulation of vesicle-mediated transport. We found a dysregulation of key regulators of endocytosis and receptor recycling (most prominently Arf6), macropinocytosis and lysosomal degradation. In gene deletion and overexpression experiments, Arf6 affected endocytosis pathways in endothelial cells. Our approach uncovered changes not picked up by transcriptomic studies, such as accumulation of vesicle cargo and receptor ligands, including Apoe. Proteomic analysis of BECs from Apoe-deficient mice revealed a signature of accelerated aging. Our findings provide a resource for analysing BEC function during aging.

2024 Scientific Article in Applied and Computational Harmonic Analysis Appl. Comput. Harmon. Anal. 71:101651 (2024)

Marcinkiewicz–Zygmund inequalities for scattered and random data on the q-sphere.

The recovery of multivariate functions and estimating their integrals from finitely many samples is one of the central tasks in modern approximation theory. Marcinkiewicz–Zygmund inequalities provide answers to both the recovery and the quadrature aspect. In this paper, we put ourselves on the q-dimensional sphere Sq, and investigate how well continuous Lp-norms of polynomials f of maximum degree n on the sphere Sq can be discretized by positively weighted Lp-sum of finitely many samples, and discuss the distortion between the continuous and discrete quantities, the number and distribution of the (deterministic or randomly chosen) sample points ξ1,…,ξN on Sq, the dimension q, and the degree n of the polynomials.

2024 Scientific Article in Computer Methods and Programs in Biomedicine Comput. Meth. Programs Biomed. 248:108107 (2024)

Circadian assessment of heart failure using explainable deep learning and novel multi-parameter polar images.

BACKGROUND AND OBJECTIVE: Heart failure (HF) is a multi-faceted and life-threatening syndrome that affects more than 64.3 million people worldwide. Current gold-standard screening technique, echocardiography, neglects cardiovascular information regulated by the circadian rhythm and does not incorporate knowledge from patient profiles. In this study, we propose a novel multi-parameter approach to assess heart failure using heart rate variability (HRV) and patient clinical information. METHODS: In this approach, features from 24-hour HRV and clinical information were combined as a single polar image and fed to a 2D deep learning model to infer the HF condition. The edges of the polar image correspond to the timely variation of different features, each of which carries information on the function of the heart, and internal illustrates color-coded patient clinical information. RESULTS: Under a leave-one-subject-out cross-validation scheme and using 7,575 polar images from a multi-center cohort (American and Greek) of 303 coronary artery disease patients (median age: 58 years [50-65], median body mass index (BMI): 27.28 kg/m2 [24.91-29.41]), the model yielded mean values for the area under the receiver operating characteristics curve (AUC), sensitivity, specificity, normalized Matthews correlation coefficient (NMCC), and accuracy of 0.883, 90.68%, 95.19%, 0.93, and 92.62%, respectively. Moreover, interpretation of the model showed proper attention to key hourly intervals and clinical information for each HF stage. CONCLUSIONS: The proposed approach could be a powerful early HF screening tool and a supplemental circadian enhancement to echocardiography which sets the basis for next-generation personalized healthcare.

2024 Scientific Article in European Radiology Eur. Radiol., DOI: 10.1007/s00330-024-10672-0 (2024)

Recommender-based bone tumour classification with radiographs-a link to the past.

OBJECTIVES: To develop an algorithm to link undiagnosed patients to previous patient histories based on radiographs, and simultaneous classification of multiple bone tumours to enable early and specific diagnosis. MATERIALS AND METHODS: For this retrospective study, data from 2000 to 2021 were curated from our database by two orthopaedic surgeons, a radiologist and a data scientist. Patients with complete clinical and pre-therapy radiographic data were eligible. To ensure feasibility, the ten most frequent primary tumour entities, confirmed histologically or by tumour board decision, were included. We implemented a ResNet and transformer model to establish baseline results. Our method extracts image features using deep learning and then clusters the k most similar images to the target image using a hash-based nearest-neighbour recommender approach that performs simultaneous classification by majority voting. The results were evaluated with precision-at-k, accuracy, precision and recall. Discrete parameters were described by incidence and percentage ratios. For continuous parameters, based on a normality test, respective statistical measures were calculated. RESULTS: Included were data from 809 patients (1792 radiographs; mean age 33.73 ± 18.65, range 3-89 years; 443 men), with Osteochondroma (28.31%) and Ewing sarcoma (1.11%) as the most and least common entities, respectively. The dataset was split into training (80%) and test subsets (20%). For k = 3, our model achieved the highest mean accuracy, precision and recall (92.86%, 92.86% and 34.08%), significantly outperforming state-of-the-art models (54.10%, 55.57%, 19.85% and 62.80%, 61.33%, 23.05%). CONCLUSION: Our novel approach surpasses current models in tumour classification and links to past patient data, leveraging expert insights. CLINICAL RELEVANCE STATEMENT: The proposed algorithm could serve as a vital support tool for clinicians and general practitioners with limited experience in bone tumour classification by identifying similar cases and classifying bone tumour entities. KEY POINTS: • Addressed accurate bone tumour classification using radiographic features. • Model achieved 92.86%, 92.86% and 34.08% mean accuracy, precision and recall, respectively, significantly surpassing state-of-the-art models. • Enhanced diagnosis by integrating prior expert patient assessments.

2024 Scientific Article in BMJ Open Respiratory Research BMJ Open Respir. Res. 11:e001762 (2024)

Protective role of the HSP90 inhibitor, STA-9090, in lungs of SARS-CoV-2-infected Syrian golden hamsters.

INTRODUCTION: The emergence of new SARS-CoV-2 variants, capable of escaping the humoral immunity acquired by the available vaccines, together with waning immunity and vaccine hesitancy, challenges the efficacy of the vaccination strategy in fighting COVID-19. Improved therapeutic strategies are urgently needed to better intervene particularly in severe cases of the disease. They should aim at controlling the hyperinflammatory state generated on infection, reducing lung tissue pathology and inhibiting viral replication. Previous research has pointed to a possible role for the chaperone HSP90 in SARS-CoV-2 replication and COVID-19 pathogenesis. Pharmacological intervention through HSP90 inhibitors was shown to be beneficial in the treatment of inflammatory diseases, infections and reducing replication of diverse viruses. METHODS: In this study, we investigated the effects of the potent HSP90 inhibitor Ganetespib (STA-9090) in vitro on alveolar epithelial cells and alveolar macrophages to characterise its effects on cell activation and viral replication. Additionally, the Syrian hamster animal model was used to evaluate its efficacy in controlling systemic inflammation and viral burden after infection. RESULTS: In vitro, STA-9090 reduced viral replication on alveolar epithelial cells in a dose-dependent manner and lowered significantly the expression of proinflammatory genes, in both alveolar epithelial cells and alveolar macrophages. In vivo, although no reduction in viral load was observed, administration of STA-9090 led to an overall improvement of the clinical condition of infected animals, with reduced oedema formation and lung tissue pathology. CONCLUSION: Altogether, we show that HSP90 inhibition could serve as a potential treatment option for moderate and severe cases of COVID-19.

2024 Scientific Article in Journal of Privacy and Confidentiality J. Priv. Confid. 14, DOI: 10.29012/jpc.820 (2024)

Differential privacy guarantees for analytics and machine learning on graphs: A survey of results.

We study differential privacy (DP) in the context of graph-structured data and discuss its formulations and applications to the publication of graphs and their associated statistics, graph generation methods, and machine learning on graph-based data, including graph neural networks (GNNs). Interpreting DP guarantees in the context of graphstructured data can be challenging, as individual data points are interconnected (often non-linearly or sparsely). This differentiates graph databases from tabular databases, which are usually used in DP, and complicates related concepts like the derivation of per-sample gradients in GNNs. The problem is exacerbated by an absence of a single, well-established formulation of DP in graph settings. A lack of prior systematisation work motivated us to study graph-based learning from a privacy perspective. In this work, we systematise different formulations of DP on graphs, and discuss challenges and promising applications, including the GNN domain. We compare and separate works into methods that privately estimate graph data (either by statistical analysis or using GNNs), and methods that aim at generating new graph data. We conclude our work with a discussion of open questions and potential directions for further research in this area.

2024 Scientific Article in Journal of Experimental and Clinical Cancer Research J. Exp. Clin. Cancer Res. 43:53 (2024)

PARP1-targeted fluorescence molecular endoscopy as novel tool for early detection of esophageal dysplasia and adenocarcinoma.

BACKGROUND: Esophageal cancer is one of the 10 most common cancers worldwide and its incidence is dramatically increasing. Despite some improvements, the current surveillance protocol with white light endoscopy and random untargeted biopsies collection (Seattle protocol) fails to diagnose dysplastic and cancerous lesions in up to 50% of patients. Therefore, new endoscopic imaging technologies in combination with tumor-specific molecular probes are needed to improve early detection. Herein, we investigated the use of the fluorescent Poly (ADP-ribose) Polymerase 1 (PARP1)-inhibitor PARPi-FL for early detection of dysplastic lesions in patient-derived organoids and transgenic mouse models, which closely mimic the transformation from non-malignant Barrett's Esophagus (BE) to invasive esophageal adenocarcinoma (EAC). METHODS: We determined PARP1 expression via immunohistochemistry (IHC) in human biospecimens and mouse tissues. We also assessed PARPi-FL uptake in patient- and mouse-derived organoids. Following intravenous injection of 75 nmol PARPi-FL/mouse in L2-IL1B (n = 4) and L2-IL1B/IL8Tg mice (n = 12), we conducted fluorescence molecular endoscopy (FME) and/or imaged whole excised stomachs to assess PARPi-FL accumulation in dysplastic lesions. L2-IL1B/IL8Tg mice (n = 3) and wild-type (WT) mice (n = 2) without PARPi-FL injection served as controls. The imaging results were validated by confocal microscopy and IHC of excised tissues. RESULTS: IHC on patient and murine tissue revealed similar patterns of increasing PARP1 expression in presence of dysplasia and cancer. In human and murine organoids, PARPi-FL localized to PARP1-expressing epithelial cell nuclei after 10 min of incubation. Injection of PARPi-FL in transgenic mouse models of BE resulted in the successful detection of lesions via FME, with a mean target-to-background ratio > 2 independently from the disease stage. The localization of PARPi-FL in the lesions was confirmed by imaging of the excised stomachs and confocal microscopy. Without PARPi-FL injection, identification of lesions via FME in transgenic mice was not possible. CONCLUSION: PARPi-FL imaging is a promising approach for clinically needed improved detection of dysplastic and malignant EAC lesions in patients with BE. Since PARPi-FL is currently evaluated in a phase 2 clinical trial for oral cancer detection after topical application, clinical translation for early detection of dysplasia and EAC in BE patients via FME screening appears feasible.

2024 Scientific Article in Science Advances Sci. Adv. 10:eadj7944 (2024)

Phase-shifting optothermal microscopy enables live-cell mid-infrared hyperspectral imaging of large cell populations at high confluency.

Rapid live-cell hyperspectral imaging at large fields of view (FOVs) and high cell confluency remains challenging for conventional vibrational spectroscopy-based microscopy technologies. At the same time, imaging at high cell confluency and large FOVs is important for proper cell function and statistical significance of measurements, respectively. Here, we introduce phase-shifting mid-infrared optothermal microscopy (PSOM), which interprets molecular-vibrational information as the optical path difference induced by mid-infrared absorption and can take snapshot vibrational images over broad excitation areas at high live-cell confluency. By means of phase-shifting, PSOM suppresses noise to a quarter of current optothermal microscopy modalities to allow capturing live-cell vibrational images at FOVs up to 50 times larger than state of the art. PSOM also reduces illumination power flux density (PFD) down to four orders of magnitude lower than other conventional vibrational microscopy methods, such as coherent anti-Stokes Raman scattering (CARS), thus considerably decreasing the risk of cell photodamage.

2024 Scientific Article in Nature Communications Nat. Commun. 15:2679 (2024)

Benchtop mesoSPIM: A next-generation open-source light-sheet microscope for cleared samples.

In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.

2024 Review in Hellenic Journal of Cardiology Hell. J. Cardiol. 78, 72-83 (2024)

Mitral valve repair of the anterior leaflet: Are we there yet?

Mitral regurgitation is one of the most prevalent valvulopathies with a disease burden that incurs significant healthcare costs globally. Surgical repair of the posterior mitral valve leaflet is a standard treatment but approaches for repairing the anterior mitral valve leaflet are not widely established. Since anterior leaflet involvement is less common and more difficult to repair, fewer studies have investigated its natural history and treatment options. In this review, we discuss surgical techniques for repairing the anterior leaflet and their outcomes including survival, reoperation, and recurrence of regurgitation. We show that most patients with mitral regurgitation from the anterior leaflet can be repaired with good outcomes if performed at centers with expertise. Additionally, equal consideration for early repair should be given to patients with mitral regurgitation from both anterior and posterior pathology. However, more studies to better evaluate the efficacy and safety of anterior mitral valve leaflet repair are needed.

2024 Scientific Article in Nature Methods Nat. Methods 21, 455-464 (2024)

Exonuclease-enhanced prime editors.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.

2024 Scientific Article in IEEE Transactions on Automation Science and Engineering IEEE Trans. Autom. Sci. Eng., DOI: 10.1109/TASE.2024.3351076 (2024)

Robot-assisted deep venous thrombosis ultrasound examination using virtual fixture.

Deep Venous Thrombosis (DVT) is a common vascular disease with blood clots inside deep veins, which may block blood flow or even cause a life-threatening pulmonary embolism. A typical exam for DVT using ultrasound (US) imaging is by pressing the target vein until its lumen is fully compressed. However, the compression exam is highly operator-dependent. To alleviate intra-and inter-variations, we present a robotic US system with a novel hybrid force motion control scheme ensuring position and force tracking accuracy, and soft landing of the probe onto the target surface. In addition, a path-based virtual fixture is proposed to realize easy human-robot interaction for repeat compression operation at the lesion location. To ensure the biometric measurements obtained in different examinations are comparable, the 6D scanning path is determined in a coarse-to-fine manner using both an external RGBD camera and US images. The RGBD camera is first used to extract a rough scanning path on the object. Then, the segmented vascular lumen from US images are used to optimize the scanning path to ensure the visibility of the target object. To generate a continuous scan path for developing virtual fixtures, an arc-length based path fitting model considering both position and orientation is proposed. Finally, the whole system is evaluated on a human-like arm phantom with an uneven surface. The code (https://github.com/dianyeHuang/RobDVTUS) and intuitive demonstration video (https://www.youtube.com/ watch?v=3xFyqU1rV8c) can be publicly accessed. Note to Practitioners—Robotic ultrasound (US) systems have attracted attention for various applications in the past decades. However, the existing studies are not mature and intelligent enough for some challenging applications, such as DVT exam, which requires rich contact interaction between patients and clinicians. To tackle with this challenge, this study presents a novel human-centric robotic DVT exam program using the technique of virtual fixture. The coarse-to-fine path planning module ensures the repeatability of US acquisitions carried out at different times. During DVT exam, the proposed continuous 6D path virtual fixture can guide clinicians to freely move the probe along the scan path while limiting the probe motion in other directions. In order to perform the compress-release exam, a decoupled position/force controller is developed to precisely generate the contact force conveyed by clinicians and to restrict the probe motion along the probe centerline. We believe such a robot-assisted system is a promising solution to take both advantages of robots about the accuracy and repeatability and human operators about the advanced physiological knowledge.

2024 Scientific Article in British Journal of Cancer BJC Br. J. Cancer 130, 1036-1045 (2024)

Metabolic heterogeneity affects trastuzumab response and survival in HER2-positive advanced gastric cancer.

BACKGROUND: Trastuzumab is the only first-line treatment targeted against the human epidermal growth factor receptor 2 (HER2) approved for patients with HER2-positive advanced gastric cancer. The impact of metabolic heterogeneity on trastuzumab treatment efficacy remains unclear. METHODS: Spatial metabolomics via high mass resolution imaging mass spectrometry was performed in pretherapeutic biopsies of patients with HER2-positive advanced gastric cancer in a prospective multicentre observational study. The mass spectra, representing the metabolic heterogeneity within tumour areas, were grouped by K-means clustering algorithm. Simpson's diversity index was applied to compare the metabolic heterogeneity level of individual patients. RESULTS: Clustering analysis revealed metabolic heterogeneity in HER2-positive gastric cancer patients and uncovered nine tumour subpopulations. High metabolic heterogeneity was shown as a factor indicating sensitivity to trastuzumab (p = 0.008) and favourable prognosis at trend level. Two of the nine tumour subpopulations associated with favourable prognosis and trastuzumab sensitivity, and one subpopulation associated with poor prognosis and trastuzumab resistance. CONCLUSIONS: This work revealed that tumour metabolic heterogeneity associated with prognosis and trastuzumab response based on tissue metabolomics of HER2-positive gastric cancer. Tumour metabolic subpopulations may provide an association with trastuzumab therapy efficacy. CLINICAL TRIAL REGISTRATION: The patient cohort was conducted from a multicentre observational study (VARIANZ;NCT02305043).

2024 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 43, 2074-2085 (2024)

Machine learning analysis of human skin by optoacoustic mesoscopy for automated extraction of psoriasis and aging biomarkers.

Ultra-wideband raster-scan optoacoustic mesoscopy (RSOM) is a novel modality that has demonstrated unprecedented ability to visualize epidermal and dermal structures in-vivo. However, an automatic and quantitative analysis of three-dimensional RSOM datasets remains unexplored. In this work we present our framework: Deep Learning RSOM Analysis Pipeline (DeepRAP), to analyze and quantify morphological skin features recorded by RSOM and extract imaging biomarkers for disease characterization. DeepRAP uses a multi-network segmentation strategy based on convolutional neural networks with transfer learning. This strategy enabled the automatic recognition of skin layers and subsequent segmentation of dermal microvasculature with an accuracy equivalent to human assessment. DeepRAP was validated against manual segmentation on 25 psoriasis patients under treatment and our biomarker extraction was shown to characterize disease severity and progression well with a strong correlation to physician evaluation and histology. In a unique validation experiment, we applied DeepRAP in a time series sequence of occlusion-induced hyperemia from 10 healthy volunteers. We observe how the biomarkers decrease and recover during the occlusion and release process, demonstrating accurate performance and reproducibility of DeepRAP. Furthermore, we analyzed a cohort of 75 volunteers and defined a relationship between aging and microvascular features in-vivo. More precisely, this study revealed that fine microvascular features in the dermal layer have the strongest correlation to age. The ability of our newly developed framework to enable the rapid study of human skin morphology and microvasculature in-vivo promises to replace biopsy studies, increasing the translational potential of RSOM.

2024 Scientific Article in Radiation Oncology Radiat. Oncol. 19:7 (2024)

The radiation- and chemo-sensitizing capacity of diclofenac can be predicted by a decreased lactate metabolism and stress response.

BACKGROUND: An enhanced aerobic glycolysis ("Warburg effect") associated with an increase in lactic acid in the tumor microenvironment contributes to tumor aggressiveness and resistance to radiation and chemotherapy. We investigated the radiation- and chemo-sensitizing effects of the nonsteroidal anti-inflammatory drug (NSAID) diclofenac in different cancer cell types. METHODS: The effects of a non-lethal concentration of diclofenac was investigated on c-MYC and Lactate Dehydrogenase (LDH) protein expression/activity and the Heat shock Protein (HSP)/stress response in human colorectal (LS174T, LoVo), lung (A549), breast (MDA-MB-231) and pancreatic (COLO357) carcinoma cells. Radiation- and chemo-sensitization of diclofenac was determined using clonogenic cell survival assays and a murine xenograft tumor model. RESULTS: A non-lethal concentration of diclofenac decreases c-MYC protein expression and LDH activity, reduces cytosolic Heat Shock Factor 1 (HSF1), Hsp70 and Hsp27 levels and membrane Hsp70 positivity in LS174T and LoVo colorectal cancer cells, but not in A549 lung carcinoma cells, MDA-MB-231 breast cancer cells and COLO357 pancreatic adenocarcinoma cells. The impaired lactate metabolism and stress response in diclofenac-sensitive colorectal cancer cells was associated with a significantly increased sensitivity to radiation and 5Fluorouracil in vitro, and in a human colorectal cancer xenograft mouse model diclofenac causes radiosensitization. CONCLUSION: These findings suggest that a decrease in the LDH activity and/or stress response upon diclofenac treatment predicts its radiation/chemo-sensitizing capacity.

2024 Scientific Article in Photoacoustics Photoacoustics 35:100582 (2024)

Quality control in clinical raster-scan optoacoustic mesoscopy.

Optoacoustic (photoacoustic) mesoscopy bridges the gap between optoacoustic microscopy and macroscopy and enables high-resolution visualization deeper than optical microscopy. Nevertheless, as images may be affected by motion and noise, it is critical to develop methodologies that offer standardization and quality control to ensure that high-quality datasets are reproducibly obtained from patient scans. Such development is particularly important for ensuring reliability in applying machine learning methods or for reliably measuring disease biomarkers. We propose herein a quality control scheme to assess the quality of data collected. A reference scan of a suture phantom is performed to characterize the system noise level before each raster-scan optoacoustic mesoscopy (RSOM) measurement. Using the recorded RSOM data, we develop a method that estimates the amount of motion in the raw data. These motion metrics are employed to classify the quality of raw data collected and derive a quality assessment index (QASIN) for each raw measurement. Using simulations, we propose a selection criterion of images with sufficient QASIN, leading to the compilation of RSOM datasets with consistent quality. Using 160 RSOM measurements from healthy volunteers, we show that RSOM images that were selected using QASIN were of higher quality and fidelity compared to non-selected images. We discuss how this quality control scheme can enable the standardization of RSOM images for clinical and biomedical applications.

2024 Scientific Article in Nature Communications Nat. Commun. 15:101 (2024)

Exact inversion of partially coherent dynamical electron scattering for picometric structure retrieval.

The greatly nonlinear diffraction of high-energy electron probes focused to subatomic diameters frustrates the direct inversion of ptychographic data sets to decipher the atomic structure. Several iterative algorithms have been proposed to yield atomically-resolved phase distributions within slices of a 3D specimen, corresponding to the scattering centers of the electron wave. By pixelwise phase retrieval, current approaches do not only involve orders of magnitude more free parameters than necessary, but also neglect essential details of scattering physics such as the atomistic nature of the specimen and thermal effects. Here, we introduce a parametrized, fully differentiable scheme employing neural network concepts which allows the inversion of ptychographic data by means of entirely physical quantities. Omnipresent thermal diffuse scattering in thick specimens is treated accurately using frozen phonons, and atom types, positions and partial coherence are accounted for in the inverse model as relativistic scattering theory demands. Our approach exploits 4D experimental data collected in an aberration-corrected momentum-resolved scanning transmission electron microscopy setup. Atom positions in a 20 nm thick PbZr0.2Ti0.8O3 ferroelectric are measured with picometer precision, including the discrimination of different atom types and positions in mixed columns.

2024 Editorial in Nature Reviews - Cancer Nat. Rev. Cancer 24:1 (2024)

wildDISCO: HD whole-body imaging.

In this Tools of the Trade article, Hongcheng Mai describes the development of wildDISCO, an approach for whole-body immunolabelling, optical clearing and imaging in mice.

2023 Scientific Article in npj Imaging npj Imaging 1:3 (2023)

Fast histological assessment of adipose tissue inflammation by label-free mid-infrared optoacoustic microscopy.

2023 Editorial in Nature Biotechnology Nat. Biotechnol., DOI: 10.1038/s41587-023-01864-y (2023)

Breaking boundaries in whole-body imaging and disease understanding with wildDISCO.

2023 Scientific Article in IEEE Transactions on Pattern Analysis and Machine Intelligence IEEE Trans. Pattern Anal. Mach. Intell. 45, 7308-7318 (2023)

Differentially private graph neural networks for whole-graph classification.

Graph Neural Networks (GNNs) have established themselves as state-of-the-art for many machine learning applications such as the analysis of social and medical networks. Several among these datasets contain privacy-sensitive data. Machine learning with differential privacy is a promising technique to allow deriving insight from sensitive data while offering formal guarantees of privacy protection. However, the differentially private training of GNNs has so far remained under-explored due to the challenges presented by the intrinsic structural connectivity of graphs. In this work, we introduce a framework for differential private graph-level classification. Our method is applicable to graph deep learning on multi-graph datasets and relies on differentially private stochastic gradient descent (DP-SGD). We show results on a variety of datasets and evaluate the impact of different GNN architectures and training hyperparameters on model performance for differentially private graph classification, as well as the scalability of the method on a large medical dataset. Our experiments show that DP-SGD can be applied to graph classification tasks with reasonable utility losses. Furthermore, we apply explainability techniques to assess whether similar representations are learned in the private and non-private settings. Our results can also function as robust baselines for future work in this area.

2023 Scientific Article in SIAM Journal on Imaging Sciences SIAM J. Imaging Sci. 16, 1308-1337 (2023)

Image recovery for blind polychromatic ptychography.

Ptychography is a lensless imaging technique, which considers reconstruction from a set of far-field diffraction patterns obtained by illuminating small overlapping regions of the specimen. In many cases, the distribution of light inside the illuminated region is unknown and has to be estimated along with the object of interest. This problem is referred to as blind ptychography. While in ptychography the illumination is commonly assumed to have a point spectrum, in this paper we consider an alternative scenario with a nontrivial light spectrum known as blind polychromatic ptychography. First, we show that nonblind polychromatic ptychography can be seen as a recovery from quadratic measurements. Then, a reconstruction from such measurements can be performed by a variant of the Amplitude Flow algorithm, which has guaranteed sublinear convergence to a critical point. Second, we address recovery from blind polychromatic ptychographic measurements by devising an alternating minimization version of Amplitude Flow and showing that it converges to a critical point at a sublinear rate.

2023 Scientific Article in Astronomy and Astrophysics Astron. Astrophys. 680:30 (2023)

Multicomponent imaging of the Fermi gamma-ray sky in the spatio-spectral domain.

The gamma-ray sky as seen by the Large Area Telescope (LAT) on board the Fermi satellite is a superposition of emissions from many processes. To study them, a rich toolkit of analysis methods for gamma-ray observations has been developed, most of which rely on emission templates to model foreground emissions. Here, we aim to complement these methods by presenting a template-free spatio-spectral imaging approach for the gamma-ray sky, based on a phenomenological modeling of its emission components. It is formulated in a Bayesian variational inference framework and allows a simultaneous reconstruction and decomposition of the sky into multiple emission components, enabled by a self-consistent inference of their spatial and spectral correlation structures. Additionally, we formulated the extension of our imaging approach to template-informed imaging, which includes adding emission templates to our component models while retaining the "data-drivenness"of the reconstruction. We demonstrate the performance of the presented approach on the ten-year Fermi LAT data set. With both template-free and template-informed imaging, we achieve a high quality of fit and show a good agreement of our diffuse emission reconstructions with the current diffuse emission model published by the Fermi Collaboration. We quantitatively analyze the obtained data-driven reconstructions and critically evaluate the performance of our models, highlighting strengths, weaknesses, and potential improvements. All reconstructions have been released as data products.

2023 Review in Medical Physics Med. Phys., DOI: 10.1002/mp.16849 (2023)

AAPM Task Group Report 311: Guidance for performance evaluation of fluorescence-guided surgery systems.

The last decade has seen a large growth in fluorescence-guided surgery (FGS) imaging and interventions. With the increasing number of clinical specialties implementing FGS, the range of systems with radically different physical designs, image processing approaches, and performance requirements is expanding. This variety of systems makes it nearly impossible to specify uniform performance goals, yet at the same time, utilization of different devices in new clinical procedures and trials indicates some need for common knowledge bases and a quality assessment paradigm to ensure that effective translation and use occurs. It is feasible to identify key fundamental image quality characteristics and corresponding objective test methods that should be determined such that there are consistent conventions across a variety of FGS devices. This report outlines test methods, tissue simulating phantoms and suggested guidelines, as well as personnel needs and professional knowledge bases that can be established. This report frames the issues with guidance and feedback from related societies and agencies having vested interest in the outcome, coming from an independent scientific group formed from academics and international federal agencies for the establishment of these professional guidelines.

2023 Scientific Article in Brain Pathology Brain Pathol.:e13222 (2023)

Structural changes of CA1 pyramidal neurons after stroke in the contralesional hippocampus.

Significant progress has been made with regard to understanding how the adult brain responds after a stroke. However, a large number of patients continue to suffer lifelong disabilities without adequate treatment. In the present study, we have analyzed possible microanatomical alterations in the contralesional hippocampus from the ischemic stroke mouse model tMCAo 12-14 weeks after transient middle cerebral artery occlusion. After individually injecting Lucifer yellow into pyramidal neurons from the CA1 field of the hippocampus, we performed a detailed three-dimensional analysis of the neuronal complexity, dendritic spine density, and morphology. We found that, in both apical (stratum radiatum) and basal (stratum oriens) arbors, CA1 pyramidal neurons in the contralesional hippocampus of tMCAo mice have a significantly higher neuronal complexity, as well as reduced spine density and alterations in spine volume and spine length. Our results show that when the ipsilateral hippocampus is dramatically damaged, the contralesional hippocampus exhibits several statistically significant selective alterations. However, these alterations are not as significant as expected, which may help to explain the recovery of hippocampal function after stroke. Further anatomical and physiological studies are necessary to better understand the modifications in the "intact" contralesional lesioned brain regions, which are probably fundamental to recover functions after stroke.

2023 Scientific Article in Nature biomedical engineering Nat. Bio. Eng. 7, 1667-1682 (2023)

Dermal features derived from optoacoustic tomograms via machine learning correlate microangiopathy phenotypes with diabetes stage.

Skin microangiopathy has been associated with diabetes. Here we show that skin-microangiopathy phenotypes in humans can be correlated with diabetes stage via morphophysiological cutaneous features extracted from raster-scan optoacoustic mesoscopy (RSOM) images of skin on the leg. We obtained 199 RSOM images from 115 participants (40 healthy and 75 with diabetes), and used machine learning to segment skin layers and microvasculature to identify clinically explainable features pertaining to different depths and scales of detail that provided the highest predictive power. Features in the dermal layer at the scale of detail of 0.1-1 mm (such as the number of junction-to-junction branches) were highly sensitive to diabetes stage. A 'microangiopathy score' compiling the 32 most-relevant features predicted the presence of diabetes with an area under the receiver operating characteristic curve of 0.84. The analysis of morphophysiological cutaneous features via RSOM may allow for the discovery of diabetes biomarkers in the skin and for the monitoring of diabetes status.

2023 Scientific Article in Journal of Nuclear Medicine J. Nucl. Med. 64, 803-808 (2023)

Detection of early esophageal neoplastic barrett lesions with quantified fluorescence molecular endoscopy using Cetuximab-800CW.

Esophageal adenocarcinoma causes 6% of cancer-related deaths worldwide. Near-infrared fluorescence molecular endoscopy (NIR-FME) uses a tracer that targets overexpressed proteins. In this study, we aimed to investigate the feasibility of an epidermal growth factor receptor (EGFR)–targeted tracer, cetuximab-800CW, to improve detection of early-stage esophageal adenocarcinoma. Methods: We validated EGFR expression in 73 esophageal tissue sections. Subsequently, we topically administered cetuximab-800CW and performed high-definition white-light endoscopy (HD-WLE), narrow-band imaging, and NIR-FME in 15 patients with Barrett esophagus (BE). Intrinsic fluorescence values were quantified using multidiameter single-fiber reflectance and single-fiber fluorescence spectroscopy. Back-table imaging, histopathologic examination, and EGFR immunohistochemistry on biopsy samples collected during NIR-FME procedures were performed and compared with in vivo imaging results. Results: Immunohistochemical preanalysis showed high EGFR expression in 67% of dysplastic tissue sections. NIR-FME visualized all 12 HD-WLE–visible lesions and 5 HD-WLE–invisible dysplastic lesions, with increased fluorescence signal in visible dysplastic BE lesions compared with nondysplastic BE as shown by multidiameter single-fiber reflectance/single-fiber fluorescence, reflecting a target-to-background ratio of 1.5. Invisible dysplastic lesions also showed increased fluorescence, with a target-to-background ratio of 1.67. Immunohistochemistry analysis showed EGFR overexpression in 16 of 17 (94%) dysplastic BE lesions, which all showed fluorescence signal. Conclusion: This study has shown that NIR-FME using cetuximab-800CW can improve detection of dysplastic lesions missed by HD-WLE and narrow-band imaging.

2023 Scientific Article in Advanced functional materials Adv. Func. Mat., DOI: 10.1002/adfm.202301820 (2023)

Fully biogenic near-infrared phosphors: Phycobiliproteins and  cellulose at force toward highly efficient and stable bio-hybrid light-emitting diodes.

Stable/efficient low-energy emitters for photon down-conversion in bio-hybrid light-emitting diodes (Bio-HLEDs) are still challenging, as the archetypal fluorescent protein (FP) mCherry has led to the best deep-red Bio-HLEDs with poor stabilities: 3 h (on-chip)/160 h (remote). Capitalizing on the excellent refolding under temperature/pH/chemical stress, high brightness, and high compatibility with polysaccharides of phycobiliproteins (smURFP), first-class low-energy emitting Bio-HLEDs are achieved. They outperform those with mCherry regardless of using reference polyethylene oxide (on-chip: 24 h vs. 3 h) and new biopolymer hydroxypropyl cellulose (HPC; on-chip: 44 h vs. 3 h) coatings. Fine optimization of smURFP-HPC-coatings leads to stable record devices (on-chip: 2600 h/108 days) compared to champion devices with perylene diimides (on-chip: <700 h) and artificial FPs (on-chip: 35 h). Finally, spectroscopy/computational/thermal assays confirm that device degradation is related to the photo-induced reduction of biliverdin to bilirubin. Overall, this study pinpoints a new family of biogenic emitters toward superior protein-based lighting.

2023 Scientific Article in Proceedings of SPIE Proc. SPIE 12392:1239202 (2023)

Functional live-cell mid-infrared microscopy and spectroscopy by optoacoustic and optothermal detection.

Mid-infrared (mid-IR) spectroscopy and optoacoustic/optothermal (OA/OT) imaging are perfectly complementary technologies to each other. Vibrational molecular excitations by mid-IR absorption are utterly de-excited in the form of heat while efficient OA/OT signal generation primarily depends on heat deposition. This synergy allows overcoming the (otherwise) persistent limitations of traditional mid-IR spectroscopy and imaging in live-cell/fresh-tissue applications—i.e., sample opacity due to water absorption. Combination of mid-IR excitation and OA/OT detection has resulted in new tools for label-free live-cell, tissues, and in vivo metabolic research. Here we discuss basic principles on mid-IR detection for spectroscopy and imaging as well as the most recent developments on mid-IR OA and OT microscopy that overcome the limitations of conventional vibrational spectroscopy for biosensing and label-free metabolic microscopy.

2023 Review in Frontiers in Cardiovascular Medicine Front. Cardiovasc. Med. 10:1210032 (2023)

Optoacoustic biomarkers of lipids, hemorrhage and inflammation in carotid atherosclerosis.

Imaging plays a critical role in exploring the pathophysiology and enabling the diagnostics and therapy assessment in carotid artery disease. Ultrasonography, computed tomography, magnetic resonance imaging and nuclear medicine techniques have been used to extract of known characteristics of plaque vulnerability, such as inflammation, intraplaque hemorrhage and high lipid content. Despite the plethora of available techniques, there is still a need for new modalities to better characterize the plaque and provide novel biomarkers that might help to detect the vulnerable plaque early enough and before a stroke occurs. Optoacoustics, by providing a multiscale characterization of the morphology and pathophysiology of the plaque could offer such an option. By visualizing endogenous (e.g., hemoglobin, lipids) and exogenous (e.g., injected dyes) chromophores, optoacoustic technologies have shown great capability in imaging lipids, hemoglobin and inflammation in different applications and settings. Herein, we provide an overview of the main optoacoustic systems and scales of detail that enable imaging of carotid plaques in vitro, in small animals and humans. Finally, we discuss the limitations of this novel set of techniques while investigating their potential to enable a deeper understanding of carotid plaque pathophysiology and possibly improve the diagnostics in future patients with carotid artery disease.

2023 Scientific Article in Nature Communications Nat. Commun. 14:7674 (2023)

A reversible state of hypometabolism in a human cellular model of sporadic Parkinson's disease.

Sporadic Parkinson's Disease (sPD) is a progressive neurodegenerative disorder caused by multiple genetic and environmental factors. Mitochondrial dysfunction is one contributing factor, but its role at different stages of disease progression is not fully understood. Here, we showed that neural precursor cells and dopaminergic neurons derived from induced pluripotent stem cells (hiPSCs) from sPD patients exhibited a hypometabolism. Further analysis based on transcriptomics, proteomics, and metabolomics identified the citric acid cycle, specifically the α-ketoglutarate dehydrogenase complex (OGDHC), as bottleneck in sPD metabolism. A follow-up study of the patients approximately 10 years after initial biopsy demonstrated a correlation between OGDHC activity in our cellular model and the disease progression. In addition, the alterations in cellular metabolism observed in our cellular model were restored by interfering with the enhanced SHH signal transduction in sPD. Thus, inhibiting overactive SHH signaling may have potential as neuroprotective therapy during early stages of sPD.

2023 Scientific Article in Gastroenterology Gastroenterology 165, 187-200.e7 (2023)

ARBM101 (Methanobactin SB2) drains excess liver copper via biliary excretion in Wilson's disease rats.

Background & Aims: Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents—methanobactins (MBs)—for efficient liver copper depletion in WD rats as well as their safety and effect duration. Methods: Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. Results: We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. Conclusions: ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

2023 Scientific Article in Laser & Photonics Reviews Laser Photon. Rev. 17:2300443 (2023)

Fast 3D optoacoustic mesoscopy of neuromelanin through entire human midbrain organoids at single-cell resolution.

Parkinson's disease is a devastating neurodegenerative disorder characterized by loss of neuromelanin-containing dopaminergic neurons. Novel 3D culture systems like human midbrain-like organoids (hMOs) enable new research avenues for patient-specific therapies, but cannot reach their full potential unless rapid optical imaging of entire organoids is enabled. Currently, hMOs have to undergo tissue clearing processes before imaging to overcome light scattering. Since tissue clearing is a lengthy chemical procedure, large ensemble studies and pharmacological longitudinal studies, which require live cultures, are impossible. To address this obstacle, raster scanning optoacoustic mesoscopy (RSOM) is considered for imaging intact hMOs. RSOM is an optical imaging technique that leverages the optoacoustic effect to overcome the need of tissue clearing. Moreover, by using tomographic principles, large specimens can be imaged within minutes. The results confirm that RSOM can image the neuromelanin distribution in complete hMOs at a single-cell resolution. Whole hMO volumes of standard size can be imaged in 4 min. Comparison with bright-field microscopy and histology confirms the ground truth of the RSOM images. This work opens several research opportunities regarding neuromelanin in hMOs with potential to boost research in Parkinson disease.

2023 Review in Archives of Computational Methods in Engineering Arch. Comp. Met. Engineering 31, 615-622 (2023)

Deep learning approaches applied to image classification of renal tumors: A systematic review.

Renal cancer is one of the ten most common cancers in the population that affects 65,000 new patients a year. Nowadays, to predict pathologies or classify tumors, deep learning (DL) methods are effective in addition to extracting high-performance features and dealing with segmentation tasks. This review has focused on the different studies related to the application of DL techniques for the detection or segmentation of renal tumors in patients. From the bibliographic search carried out, a total of 33 records were identified in Scopus, PubMed and Web of Science. The results derived from the systematic review give a detailed description of the research objectives, the types of images used for analysis, the data sets used, whether the database used is public or private, and the number of patients involved in the studies. The first paper where DL is applied compared to other types of tumors was in 2019 which is relatively recent. Public collection and sharing of data sets are of utmost importance to increase research in this field as many studies use private databases. We can conclude that future research will identify many benefits, such as unnecessary incisions for patients and more accurate diagnoses. As research in this field grows, the amount of open data is expected to increase.

2023 Scientific Article in Nature machine intelligence Nat. Mach. Intell. 5, 1130–1141 (2023)

A deep neural network for real-time optoacoustic image reconstruction with adjustable speed of sound.

Multispectral optoacoustic tomography is a high-resolution functional imaging modality that can non-invasively access a broad range of pathophysiological phenomena. Real-time imaging would enable translation of multispectral optoacoustic tomography into clinical imaging, visualize dynamic pathophysiological changes associated with disease progression and enable in situ diagnoses. Model-based reconstruction affords state-of-the-art optoacoustic images but cannot be used for real-time imaging. On the other hand, deep learning enables fast reconstruction of optoacoustic images, but the lack of experimental ground-truth training data leads to reduced image quality for in vivo scans. In this work we achieve accurate optoacoustic image reconstruction in 31 ms per image for arbitrary (experimental) input data by expressing model-based reconstruction with a deep neural network. The proposed deep learning framework, DeepMB, generalizes to experimental test data through training on optoacoustic signals synthesized from real-world images and ground truth optoacoustic images generated by model-based reconstruction. Based on qualitative and quantitative evaluation on a diverse dataset of in vivo images, we show that DeepMB reconstructs images approximately 1,000-times faster than the iterative model-based reference method while affording near-identical image qualities. Accurate and real-time image reconstructions with DeepMB can enable full access to the high-resolution and multispectral contrast of handheld optoacoustic tomography, thus adoption into clinical routines.

2023 Scientific Article in Laser & Photonics Reviews Laser Photon. Rev. 17:2200846 (2023)

Beyond early development: Observing Zebrafish over 6 weeks with hybrid optical and optoacoustic imaging.

Zebrafish are common model organisms in developmental biology, but have recently emerged as imaging targets of research in cancer, tissue regeneration, metabolic disorders, functional genomics, and phenotype-based drug discovery. Conventionally, zebrafish are studied during the first few days of development using optical microscopy methods. However, optical methods are not suited for imaging at later stages, since the fish become opaque. To address needs to visualize beyond the first days of development, a novel multimodality system for observing zebrafish from larval stage to adulthood is developed. Using a hybrid platform for concurrent selective plane illumination microscopy (SPIM) and optoacoustic mesoscopy, fish (ex vivo) at stages of development up to 47 days at a similar object size-to-resolution ratio are imaged. Using multiple wavelength illumination over the visible and short-wavelength infrared regions, it is demonstrated that the optoacoustic method can follow GFP-based contrast used in SPIM, enabling molecular imaging interrogation in adult fish. Moreover, the optoacoustic modality reveals zebrafish features based on optical contrast absent in SPIM, including contrast from endogenous blood, water, and lipids. It is discussed how the hybrid system method can enable the study of zebrafish in a wider range of applications and over time-scales not possible currently when using optical microscopy.

2023 Scientific Article in Cancer Nanotechnology Cancer Nanotechnol. 14:36 (2023)

Bacterial outer membrane vesicles as cationic dye carriers for optoacoustics-guided phototherapy of cancer.

Background: Cationic dyes are widely used as biomarkers for optical imaging. However, most of these are hydrophobic and cannot be employed in vivo without chemical conjugation or modification. Herein, we report for the first time the use of bacterial outer membrane vesicles (OMVs) as nanocarriers of cationic dyes for cancer theranostics. Results: We demonstrate that cationic dyes (IR780, Cy7, and Cy7.5) form stable complexes with negatively charged bacterial-OMVs, improving the dyes’ in vivo circulation and optoacoustic properties. Such OMV-Dye complexes are biodegradable and safe for in vivo applications. Importantly, this method of cationic dye loading is faster and easier than synthetic chemistry approaches, and the efficient tumor accumulation of OMV-Dyes enables sensitive tumor detection using optoacoustic technology. As a proof-of-concept, we generated OMV-IR780 for optoacoustics-guided in vivo tumor phototherapy in a mouse model. Conclusions: Our results demonstrate cationic dye-bound OMVs as promising novel nanoagents for tumor theranostics.

2023 Scientific Article in ACS Photonics ACS Photonics 10, 559–570 (2023)

Optoacoustic endoscopy of the gastrointestinal tract.

Optical endoscopy is the most commonly applied procedure for inspecting the gastrointestinal (GI) tract, and it is based on different approaches and designs, from using a flexible optical scope to swallowing a small camera capsule that obtains photographs as it advances through the digestive tract. Despite its wide use in GI diagnostics and theranostics, optical visualization only allows a superficial inspection of the wall lining (mucosa), therefore limiting the ability to obtain information from deeper GI layers. In the quest for developing methods to visualize under the mucosal layers, we review herein progress with optoacoustic endoscopy, a technique that captures optical contrast in high resolution deep inside tissues, enabling imaging beneath the surface of the mucosa. Optoacoustic endoscopy combines imaging of optical contrast with the resolution and depth penetration afforded by ultrasonography, thus merging highly advantageous characteristics for clinical applications. We review progress and the current status of the technology, its key endoscopic competitors, and challenges for clinical application. We further offer a perspective regarding future directions and the overall application potential of the technique to complement the current state-of-the-art.

2023 Scientific Article in Gut Microbes Gut Microbes 15:2271597 (2023)

Ascorbate deficiency increases progression of shigellosis in guinea pigs and mice infection models.

Shigella spp. are the causative agents of bacterial dysentery and shigellosis, mainly in children living in developing countries. The study of Shigella entire life cycle in vivo and the evaluation of vaccine candidates' protective efficacy have been hampered by the lack of a suitable animal model of infection. None of the studies evaluated so far (rabbit, guinea pig, mouse) allowed the recapitulation of full shigellosis symptoms upon Shigella oral challenge. Historical reports have suggested that dysentery and scurvy are both metabolic diseases associated with ascorbate deficiency. Mammals, which are susceptible to Shigella infection (humans, non-human primates and guinea pigs) are among the few species unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate deficiency, but not scurvy, in guinea pigs to investigate whether poor vitamin C status increases the progression of shigellosis. Moderate ascorbate deficiency increased shigellosis symptom severity during an extended period of time (up to 48 h) in all strains tested (Shigella sonnei, Shigella flexneri 5a, and 2a). At late time points, an important influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although Shigella was able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. Moreover, we found that ascorbate deficiency also increased Shigella penetration into the colon epithelium layer in a Gulo-/- mouse infection model. The use of these new rodent models of shigellosis opens new doors for the study of both Shigella infection strategies and immune responses to Shigella infection.

2023 Scientific Article in npj Precision Oncology npj Precis. Oncol. 7:114 (2023)

Spatial metabolomics identifies distinct tumor-specific and stroma-specific subtypes in patients with lung squamous cell carcinoma.

Molecular subtyping of lung squamous cell carcinoma (LUSC) has been performed at the genomic, transcriptomic, and proteomic level. However, LUSC stratification based on tissue metabolomics is still lacking. Combining high-mass-resolution imaging mass spectrometry with consensus clustering, four tumor- and four stroma-specific subtypes with distinct metabolite patterns were identified in 330 LUSC patients. The first tumor subtype T1 negatively correlated with DNA damage and immunological features including CD3, CD8, and PD-L1. The same features positively correlated with the tumor subtype T2. Tumor subtype T4 was associated with high PD-L1 expression. Compared with the status of subtypes T1 and T4, patients with subtype T3 had improved prognosis, and T3 was an independent prognostic factor with regard to UICC stage. Similarly, stroma subtypes were linked to distinct immunological features and metabolic pathways. Stroma subtype S4 had a better prognosis than S2. Subsequently, analyses based on an independent LUSC cohort treated by neoadjuvant therapy revealed that the S2 stroma subtype was associated with chemotherapy resistance. Clinically relevant patient subtypes as determined by tissue-based spatial metabolomics are a valuable addition to existing molecular classification systems. Metabolic differences among the subtypes and their associations with immunological features may contribute to the improvement of personalized therapy.

2023 Scientific Article in ACS Nano ACS Nano 17, 21056-21072 (2023)

Nanoparticle-exposure-triggered virus reactivation induces lung emphysema in mice.

Nanoparticles (NPs) released from engineered materials or combustion processes as well as persistent herpesvirus infection are omnipresent and are associated with chronic lung diseases. Previously, we showed that pulmonary exposure of a single dose of soot-like carbonaceous NPs (CNPs) or fiber-shaped double-walled carbon nanotubes (DWCNTs) induced an increase of lytic virus protein expression in mouse lungs latently infected with murine γ-herpesvirus 68 (MHV-68), with a similar pattern to acute infection suggesting virus reactivation. Here we investigate the effects of a more relevant repeated NP exposure on lung disease development as well as herpesvirus reactivation mechanistically and suggest an avenue for therapeutic prevention. In the MHV-68 mouse model, progressive lung inflammation and emphysema-like injury were detected 1 week after repetitive CNP and DWCNT exposure. NPs reactivated the latent herpesvirus mainly in CD11b+ macrophages in the lungs. In vitro, in persistently MHV-68 infected bone marrow-derived macrophages, ERK1/2, JNK, and p38 MAPK were rapidly activated after CNP and DWCNT exposure, followed by viral gene expression and increased viral titer but without generating a pro-inflammatory signature. Pharmacological inhibition of p38 activation abrogated CNP- but not DWCNT-triggered virus reactivation in vitro, and inhibitor pretreatment of latently infected mice attenuated CNP-exposure-induced pulmonary MHV-68 reactivation. Our findings suggest a crucial contribution of particle-exposure-triggered herpesvirus reactivation for nanomaterial exposure or air pollution related lung emphysema development, and pharmacological p38 inhibition might serve as a protective target to alleviate air pollution related chronic lung disease exacerbations. Because of the required precondition of latent infection described here, the use of single hit models might have severe limitations when assessing the respiratory toxicity of nanoparticle exposure.

2023 Scientific Article in Current Biology Curr. Biol. 33, 4538-4547.e5 (2023)

Ecosystem consequences of invertebrate decline.

Human activities cause substantial changes in biodiversity.1,2 Despite ongoing concern about the implications of invertebrate decline,3,4,5,6,7 few empirical studies have examined the ecosystem consequences of invertebrate biomass loss. Here, we test the responses of six ecosystem services informed by 30 above- and belowground ecosystem variables to three levels of aboveground (i.e., vegetation associated) invertebrate community biomass (100%, 36%, and 0% of ambient biomass) in experimental grassland mesocosms in a controlled Ecotron facility. In line with recent reports on invertebrate biomass loss over the last decade, our 36% biomass treatment also represented a decrease in invertebrate abundance (-70%) and richness (-44%). Moreover, we simulated the pronounced change in invertebrate biomass and turnover in community composition across the season. We found that the loss of invertebrate biomass decreases ecosystem multifunctionality, including two critical ecosystem services, aboveground pest control and belowground decomposition, while harvested plant biomass increases, likely because less energy was channeled up the food chain. Moreover, communities and ecosystem functions become decoupled with a lower biomass of invertebrates. Our study shows that invertebrate loss threatens the integrity of grasslands by decoupling ecosystem processes and decreasing ecosystem-service supply.

2023 Scientific Article in Biomedical Optics Express Biomed. Opt. Express 14, 5499-5511 (2023)

Using reflectometry to minimize the dependence of fluorescence intensity on optical absorption and scattering.

The total diffuse reflectance RT and the effective attenuation coefficient µeff of an optically diffuse medium map uniquely onto its absorption and reduced scattering coefficients. Using this premise, we developed a methodology where RT and the slope of the logarithmic spatially resolved reflectance, a quantity related to µeff, are the inputs of a look-up table to correct the dependence of fluorescent signals on the media's optical properties. This methodology does not require an estimation of the medium's optical property, avoiding elaborate simulations and their errors to offer accurate and fast corrections. The experimental demonstration of our method yielded a mean relative error in fluorophore concentrations of less than 4% over a wide range of optical property variations. We discuss how the method developed can be employed to improve image fidelity and fluorochrome quantification in fluorescence molecular imaging clinical applications.

2023 Scientific Article in Scientific Reports Sci. Rep. 13:19542 (2023)

Overdriven laser diode optoacoustic microscopy.

Laser diodes are small and inexpensive but don't afford the pulse energy and beam profile required for optoacoustic (photoacoustic) microscopy. Using two novel modulation concepts, i.e. overdriving continuous-wave laser diodes (CWLD) and frequency-wavelength multiplexing (FWM) based on illumination pulse-trains, we demonstrate concurrent multi-wavelength optoacoustic microscopy with signal-to-noise ratios of > 17 dB, < 2 µm resolution at repetition rates of 1 MHz. This unprecedented performance based on an adaptable trigger engine allowed us to contrast FWM to wavelength alternating acquisition using identical optical components. We showcase this concept's superiority over conventional optoacoustic microscopes by visualizing vascular oxygenation dynamics and circulating tumor cells in mice. This work positions laser diodes as a technology allowing affordable, tunable, and miniaturizable optoacoustic microscopy.

2023 Scientific Article in Lancet Diabetes and Endocrinology Lancet Diabet. Endocrinol. 11, 798-810 (2023)

Mechanisms of weight loss-induced remission in people with prediabetes: A post-hoc analysis of the randomised, controlled, multicentre Prediabetes Lifestyle Intervention Study (PLIS).

BACKGROUND: Remission of type 2 diabetes can occur as a result of weight loss and is characterised by liver fat and pancreas fat reduction and recovered insulin secretion. In this analysis, we aimed to investigate the mechanisms of weight loss- induced remission in people with prediabetes. METHODS: In this prespecified post-hoc analysis, weight loss-induced resolution of prediabetes in the randomised, controlled, multicentre Prediabetes Lifestyle Intervention Study (PLIS) was assessed, and the results were validated against participants from the Diabetes Prevention Program (DPP) study. For PLIS, between March 1, 2012, and Aug 31, 2016, participants were recruited from eight clinical study centres (including seven university hospitals) in Germany and randomly assigned to receive either a control intervention, a standard lifestyle intervention (ie, DPP-based intervention), or an intensified lifestyle intervention for 12 months. For DPP, participants were recruited from 23 clinical study centres in the USA between July 31, 1996, and May 18, 1999, and randomly assigned to receive either a standard lifestyle intervention, metformin, or placebo. In both PLIS and DPP, only participants who were randomly assigned to receive lifestyle intervention or placebo and who lost at least 5% of their bodyweight were included in this analysis. Responders were defined as people who returned to normal fasting plasma glucose (FPG; <5·6 mmol/L), normal glucose tolerance (<7·8 mmol/L), and HbA1c less than 39 mmol/mol after 12 months of lifestyle intervention or placebo or control intervention. Non-responders were defined as people who had FPG, 2 h glucose, or HbA1c more than these thresholds. The main outcomes for this analysis were insulin sensitivity, insulin secretion, visceral adipose tissue (VAT), and intrahepatic lipid content (IHL) and were evaluated via linear mixed models. FINDINGS: Of 1160 participants recruited to PLIS, 298 (25·7%) had weight loss of 5% or more of their bodyweight at baseline. 128 (43%) of 298 participants were responders and 170 (57%) were non-responders. Responders were younger than non-responders (mean age 55·6 years [SD 9·9] vs 60·4 years [8·6]; p<0·0001). The DPP validation cohort included 683 participants who lost at least 5% of their bodyweight at baseline. Of these, 132 (19%) were responders and 551 (81%) were non-responders. In PLIS, BMI reduction was similar between responders and non-responders (responders mean at baseline 32·4 kg/m2 [SD 5·6] to mean at 12 months 29·0 kg/m2 [4·9] vs non-responders 32·1 kg/m2 [5·9] to 29·2 kg/m2 [5·4]; p=0·86). However, whole-body insulin sensitivity increased more in responders than in non-responders (mean at baseline 291 mL/[min × m2], SD 60 to mean at 12 months 378 mL/[min × m2], 56 vs 278 mL/[min × m2], 62, to 323 mL/[min × m2], 66; p<0·0001), whereas insulin secretion did not differ within groups over time or between groups (responders mean at baseline 175 pmol/mmol [SD 64] to mean at 12 months 163·7 pmol/mmol [60·6] vs non-responders 158·0 pmol/mmol [55·6] to 154·1 pmol/mmol [56·2]; p=0·46). IHL decreased in both groups, without a difference between groups (responders mean at baseline 10·1% [SD 8·7] to mean at 12 months 3·5% [3·9] vs non-responders 10·3% [8·1] to 4·2% [4·2]; p=0·34); however, VAT decreased more in responders than in non-responders (mean at baseline 6·2 L [SD 2·9] to mean at 12 months 4·1 L [2·3] vs 5·7 L [2·3] to 4·5 L [2·2]; p=0·0003). Responders had a 73% lower risk of developing type 2 diabetes than non-responders in the 2 years after the intervention ended. INTERPRETATION: By contrast to remission of type 2 diabetes, resolution of prediabetes was characterised by an improvement in insulin sensitivity and reduced VAT. Because return to normal glucose regulation (NGR) prevents development of type 2 diabetes, we propose the concept of remission of prediabetes in analogy to type 2 diabetes. We suggest that remission of prediabetes should be the primary therapeutic aim in individuals with prediabetes. FUNDING: German Federal Ministry for Education and Research via the German Center for Diabetes Research; the Ministry of Science, Research and the Arts Baden-Württemberg; the Helmholtz Association and Helmholtz Munich; the Cluster of Excellence Controlling Microbes to Fight Infections; and the German Research Foundation.

2023 Scientific Article in eLife eLife 12:23 (2023)

Spontaneous body wall contractions stabilize the fluid microenvironment that shapes host-microbe associations.

The freshwater polyp Hydra is a popular biological model system; however, we still do not understand one of its most salient behaviors, the generation of spontaneous body wall contractions. Here, by applying experimental fluid dynamics analysis and mathematical modeling, we provide functional evidence that spontaneous contractions of body walls enhance the transport of chemical compounds from and to the tissue surface where symbiotic bacteria reside. Experimentally, a reduction in the frequency of spontaneous body wall contractions is associated with a changed composition of the colonizing microbiota. Together, our findings suggest that spontaneous body wall contractions create an important fluid transport mechanism that (1) may shape and stabilize specific host-microbe associations and (2) create fluid microhabitats that may modulate the spatial distribution of the colonizing microbes. This mechanism may be more broadly applicable to animal-microbe interactions since research has shown that rhythmic spontaneous contractions in the gastrointestinal tracts are essential for maintaining normal microbiota.

2023 Scientific Article in Science Advances Sci. Adv. 9:eadi8986 (2023)

Deciphering sources of PET signals in the tumor microenvironment of glioblastoma at cellular resolution.

Various cellular sources hamper interpretation of positron emission tomography (PET) biomarkers in the tumor microenvironment (TME). We developed an approach of immunomagnetic cell sorting after in vivo radiotracer injection (scRadiotracing) with three-dimensional (3D) histology to dissect the cellular allocation of PET signals in the TME. In mice with implanted glioblastoma, translocator protein (TSPO) radiotracer uptake per tumor cell was higher compared to tumor-associated microglia/macrophages (TAMs), validated by protein levels. Translation of in vitro scRadiotracing to patients with glioma immediately after tumor resection confirmed higher single-cell TSPO tracer uptake of tumor cells compared to immune cells. Across species, cellular radiotracer uptake explained the heterogeneity of individual TSPO-PET signals. In consideration of cellular tracer uptake and cell type abundance, tumor cells were the main contributor to TSPO enrichment in glioblastoma; however, proteomics identified potential PET targets highly specific for TAMs. Combining cellular tracer uptake measures with 3D histology facilitates precise allocation of PET signals and serves to validate emerging novel TAM-specific radioligands.

2023 Scientific Article in New Journal of Physics New J. Phys. 25:043010 (2023)

Spatially-resolved dynamic sampling of different phasic magnetic resonances of nanoparticle ensembles in a magnetotactic bacterium Magnetospirillum magnetotacticum.

Nanoscaled magnetic particle ensembles are promising building blocks for realizing magnon based binary logic. Element-specific real-space monitoring of magnetic resonance modes with sampling rates in the GHz regime is imperative for the experimental verification of future complex magnonic devices. Here we present the observation of different phasic magnetic resonance modes using the element-specific technique of time-resolved scanning transmission x-ray microscopy within a chain of dipolarly coupled Fe3O4 nanoparticles (40-50 nm particle size) inside a single cell of a magnetotactic bacterium Magnetospirillum magnetotacticum. The particles are probed with 25 nm resolution at the Fe L3 x-ray absorption edge in response to a microwave excitation of 4.07 GHz. A plethora of resonance modes is observed within multiple particle segments oscillating in- and out-of-phase, well resembled by micromagnetic simulations.

2023 Scientific Article in Nature Communications Nat. Commun. 14, 16:5799 (2023)

Translating genomic tools to Raman spectroscopy analysis enables high-dimensional tissue characterization on molecular resolution.

Spatial transcriptomics of histological sections have revolutionized research in life sciences and enabled unprecedented insights into genetic processes involved in tissue reorganization. However, in contrast to genomic analysis, the actual biomolecular composition of the sample has fallen behind, leaving a gap of potentially highly valuable information. Raman microspectroscopy provides untargeted spatiomolecular information at high resolution, capable of filling this gap. In this study we demonstrate spatially resolved Raman “spectromics” to reveal homogeneity, heterogeneity and dynamics of cell matrix on molecular levels by repurposing state-of-the-art bioinformatic analysis tools commonly used for transcriptomic analyses. By exploring sections of murine myocardial infarction and cardiac hypertrophy, we identify myocardial subclusters when spatially approaching the pathology, and define the surrounding metabolic and cellular (immune-) landscape. Our innovative, label-free, non-invasive “spectromics” approach could therefore open perspectives for a profound characterization of histological samples, while additionally allowing the combination with consecutive downstream analyses of the very same specimen.

2023 Scientific Article in Stem Cell Reports Stem Cell Rep. 18, 1972-1986 (2023)

Single-cell characterization of neovascularization using hiPSC-derived endothelial cells in a 3D microenvironment.

The formation of vascular structures is fundamental for in vitro tissue engineering. Vascularization can enable the nutrient supply within larger structures and increase transplantation efficiency. We differentiated human induced pluripotent stem cells toward endothelial cells in 3D suspension culture. To investigate in vitro neovascularization and various 3D microenvironmental approaches, we designed a comprehensive single-cell transcriptomic study. Time-resolved single-cell transcriptomics of the endothelial and co-evolving mural cells gave insights into cell type development, stability, and plasticity. Transfer to a 3D hydrogel microenvironment induced neovascularization and facilitated tracing of migrating, coalescing, and tubulogenic endothelial cell states. During maturation, we monitored two pericyte subtypes evolving mural cells. Profiling cell-cell interactions between pericytes and endothelial cells revealed angiogenic signals during tubulogenesis. In silico discovered ligands were tested for their capability to attract endothelial cells. Our data, analyses, and results provide an in vitro roadmap to guide vascularization in future tissue engineering.

2023 Review in Journal of Cardiovascular Development and Disease J. Cardiovasc. Dev. Dis. 10:24 (2023)

Clinical and translational imaging and sensing of diabetic microangiopathy: A narrative review.

Microvascular changes in diabetes affect the function of several critical organs, such as the kidneys, heart, brain, eye, and skin, among others. The possibility of detecting such changes early enough in order to take appropriate actions renders the development of appropriate tools and techniques an imperative need. To this end, several sensing and imaging techniques have been developed or employed in the assessment of microangiopathy in patients with diabetes. Herein, we present such techniques; we provide insights into their principles of operation while discussing the characteristics that make them appropriate for such use. Finally, apart from already established techniques, we present novel ones with great translational potential, such as optoacoustic technologies, which are expected to enter clinical practice in the foreseeable future.

2023 Scientific Article in Neuroradiology Neuroradiology 65, 1631-1648 (2023)

Impact of cannabis use on brain metabolism using 31P and 1H magnetic resonance spectroscopy.

Purpose: This prospective cross-sectional study investigated the influence of regular cannabis use on brain metabolism in young cannabis users by using combined proton and phosphorus magnetic resonance spectroscopy. Methods: The study was performed in 45 young cannabis users aged 18–30, who had been using cannabis on a regular basis over a period of at least 2 years and in 47 age-matched controls. We acquired 31P MRS data in different brain regions at 3T with a double-resonant 1H/31P head coil, anatomic images, and 1H MRS data with a standard 20-channel 1H head coil. Absolute concentration values of proton metabolites were obtained via calibration from tissue water as an internal reference, whereas a standard solution of 75 mmol/l KH2PO4 was used as an external reference for the calibration of phosphorus signals. Results: We found an overall but not statistically significant lower concentration level of several proton and phosphorus metabolites in cannabis users compared to non-users. In particular, energy-related phosphates such as adenosine triphosphate (ATP) and inorganic phosphate (Pi) were reduced in all regions under investigation. Phosphocreatine (PCr) showed lowered values mainly in the left basal ganglia and the left frontal white matter. Conclusion: The results suggest that the increased risk of functional brain disorders observed in long-term cannabis users could be caused by an impairment of the energy metabolism of the brain, but this needs to be verified in future studies.

2023 Scientific Article in Strahlentherapie und Onkologie : Journal of Radiation Oncology, Biology, Physics Strahlenther. Onkol. 199, 1214-1224 (2023)

Chronic inflammatory effects of in vivo irradiation of the murine heart on endothelial cells mimic mechanisms involved in atherosclerosis.

Purpose: Radiotherapy is a major pillar in the treatment of solid tumors including breast cancer. However, epidemiological studies have revealed an increase in cardiac diseases approximately a decade after exposure of the thorax to ionizing irradiation, which might be related to vascular inflammation. Therefore, chronic inflammatory effects were examined in primary heart and lung endothelial cells (ECs) of mice after local heart irradiation. Methods: Long-lasting effects on primary ECs of the heart and lung were studied 20–50 weeks after local irradiation of the heart of mice (8 and 16 Gy) in vivo by multiparameter flow cytometry using antibodies directed against cell surface markers related to proliferation, stemness, lipid metabolism, and inflammation, and compared to those induced by occlusion of the left anterior descending coronary artery. Results: In vivo irradiation of the complete heart caused long-lasting persistent upregulation of inflammatory (HCAM, ICAM‑1, VCAM-1), proliferation (CD105), and lipid (CD36) markers on primary heart ECs and an upregulation of ICAM‑1 and VCAM‑1 on primary ECs of the partially irradiated lung lobe. An artificially induced heart infarction induces similar effects with respect to inflammatory markers, albeit in a shorter time period. Conclusion: The long-lasting upregulation of prominent inflammatory markers on primary heart and lung ECs suggests that local heart irradiation induces chronic inflammation in the microvasculature of the heart and partially irradiated lung that leads to cardiac injury which might be related to altered lipid metabolism in the heart.

2023 Scientific Article in Light: Science & Applications Light Sci. Appl. 12:231 (2023)

Opening a window to skin biomarkers for diabetes stage with optoacoustic mesoscopy.

Being the largest and most accessible organ of the human body, the skin could offer a window to diabetes-related complications on the microvasculature. However, skin microvasculature is typically assessed by histological analysis, which is not suited for applications to large populations or longitudinal studies. We introduce ultra-wideband raster-scan optoacoustic mesoscopy (RSOM) for precise, non-invasive assessment of diabetes-related changes in the dermal microvasculature and skin micro-anatomy, resolved with unprecedented sensitivity and detail without the need for contrast agents. Providing unique imaging contrast, we explored a possible role for RSOM as an investigational tool in diabetes healthcare and offer the first comprehensive study investigating the relationship between different diabetes complications and microvascular features in vivo. We applied RSOM to scan the pretibial area of 95 participants with diabetes mellitus and 48 age-matched volunteers without diabetes, grouped according to disease complications, and extracted six label-free optoacoustic biomarkers of human skin, including dermal microvasculature density and epidermal parameters, based on a novel image-processing pipeline. We then correlated these biomarkers to disease severity and found statistically significant effects on microvasculature parameters as a function of diabetes complications. We discuss how label-free RSOM biomarkers can lead to a quantitative assessment of the systemic effects of diabetes and its complications, complementing the qualitative assessment allowed by current clinical metrics, possibly leading to a precise scoring system that captures the gradual evolution of the disease.

2023 Scientific Article in Hypertension Hypertension 80, 1555-1567 (2023)

Primary aldosteronism: Spatial multi-omics mapping of genotype-dependent heterogeneity and tumor expansion of aldosterone-producing adenomas.

BACKGROUND: Primary aldosteronism is frequently caused by an adrenocortical aldosterone-producing adenoma (APA) carrying a somatic mutation that drives aldosterone overproduction. APAs with a mutation in KCNJ5 (APA-KCNJ5MUT) are characterized by heterogeneous CYP11B2 (aldosterone synthase) expression, a particular cellular composition and larger tumor diameter than those with wild-type KCNJ5 (APA-KCNJ5WT). We exploited these differences to decipher the roles of transcriptome and metabolome reprogramming in tumor pathogenesis. METHODS: Consecutive adrenal cryosections (7 APAs and 7 paired adjacent adrenal cortex) were analyzed by spatial transcriptomics (10x Genomics platform) and metabolomics (in situ matrix-assisted laser desorption/ionization mass spectrometry imaging) co-integrated with CYP11B2 immunohistochemistry. RESULTS: We identified intratumoral transcriptional heterogeneity that delineated functionally distinct biological pathways. Common transcriptomic signatures were established across all APA specimens which encompassed 2 distinct transcriptional profiles in CYP11B2-immunopositive regions (CYP11B2-type 1 or 2). The CYP11B2-type 1 signature was characterized by zona glomerulosa gene markers and was detected in both APA-KCNJ5MUT and APA-KCNJ5WT. The CYP11B2-type 2 signature displayed markers of the zona fasciculata or reticularis and predominated in APA-KCNJ5MUT. Metabolites that promote oxidative stress and cell death accumulated in APA-KCNJ5WT. In contrast, antioxidant metabolites were abundant in APA-KCNJ5MUT. Finally, APA-like cell subpopulations-negative for CYP11B2 gene expression-were identified in adrenocortical tissue adjacent to APAs suggesting the existence of tumor precursor states. CONCLUSIONS: Our findings provide insight into intra- and intertumoral transcriptional heterogeneity and support a role for prooxidant versus antioxidant systems in APA pathogenesis highlighting genotype-dependent capacities for tumor expansion.

2023 Scientific Article in ACS Nano ACS Nano 17, 16396-16411 (2023)

Patterns of carbon-bound exogenous compounds impact disease pathophysiology in lung cancer subtypes in different ways.

Carbon-bound exogenous compounds, such as polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines, aromatic amines, and organohalogens, are known to affect both tumor characteristics and patient outcomes in lung squamous cell carcinoma (LUSC); however, the roles of these compounds in lung adenocarcinoma (LUAD) remain unclear. We analyzed 11 carbon-bound exogenous compounds in LUAD and LUSC samples using in situ high mass-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging and performed a cluster analysis to compare the patterns of carbon-bound exogenous compounds between these two lung cancer subtypes. Correlation analyses were conducted to investigate associations among exogenous compounds, endogenous metabolites, and clinical data, including patient survival outcomes and smoking behaviors. Additionally, we examined differences in exogenous compound patterns between normal and tumor tissues. Our analyses revealed that PAHs, aromatic amines, and organohalogens were more abundant in LUAD than in LUSC, whereas the tobacco-specific nitrosamine nicotine-derived nitrosamine ketone was more abundant in LUSC. Patients with LUAD and LUSC could be separated according to carbon-bound exogenous compound patterns detected in the tumor compartment. The same compounds had differential impacts on patient outcomes, depending on the cancer subtype. Correlation and network analyses indicated substantial differences between LUAD and LUSC metabolomes, associated with substantial differences in the patterns of the carbon-bound exogenous compounds. These data suggest that the contributions of these carcinogenic compounds to cancer biology may differ according to the cancer subtypes.

2023 Scientific Article in JCI insight JCI insight 8:18 (2023)

Metabolic heterogeneity in adrenocortical carcinoma impacts patient outcomes.

Spatially resolved metabolomics enables the investigation of tumoral metabolites in situ. Inter- and intratumor heterogeneity are key factors associated with patient outcomes. Adrenocortical carcinoma (ACC) is an exceedingly rare tumor associated with poor survival. Its clinical prognosis is highly variable, but the contributions of tumor metabolic heterogeneity have not been investigated thus far to our knowledge. An in-depth understanding of tumor heterogeneity requires molecular feature-based identification of tumor subpopulations associated with tumor aggressiveness. Here, using spatial metabolomics by high-mass resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry imaging, we assessed metabolic heterogeneity by de novo discovery of metabolic subpopulations and Simpson's diversity index. After identification of tumor subpopulations in 72 patients with ACC, we additionally performed a comparison with 25 tissue sections of normal adrenal cortex to identify their common and unique metabolic subpopulations. We observed variability of ACC tumor heterogeneity and correlation of high metabolic heterogeneity with worse clinical outcome. Moreover, we identified tumor subpopulations that served as independent prognostic factors and, furthermore, discovered 4 associated anticancer drug action pathways. Our research may facilitate comprehensive understanding of the biological implications of tumor subpopulations in ACC and showed that metabolic heterogeneity might impact chemotherapy.

2023 Scientific Article in Scientific Reports Sci. Rep. 13:12594 (2023)

A pilot radiometabolomics integration study for the characterization of renal oncocytic neoplasia.

Differentiating benign renal oncocytic tumors and malignant renal cell carcinoma (RCC) on imaging and histopathology is a critical problem that presents an everyday clinical challenge. This manuscript aims to demonstrate a novel methodology integrating metabolomics with radiomics features (RF) to differentiate between benign oncocytic neoplasia and malignant renal tumors. For this purpose, thirty-three renal tumors (14 renal oncocytic tumors and 19 RCC) were prospectively collected and histopathologically characterised. Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) was used to extract metabolomics data, while RF were extracted from CT scans of the same tumors. Statistical integration was used to generate multilevel network communities of -omics features. Metabolites and RF critical for the differentiation between the two groups (delta centrality > 0.1) were used for pathway enrichment analysis and machine learning classifier (XGboost) development. Receiver operating characteristics (ROC) curves and areas under the curve (AUC) were used to assess classifier performance. Radiometabolomics analysis demonstrated differential network node configuration between benign and malignant renal tumors. Fourteen nodes (6 RF and 8 metabolites) were crucial in distinguishing between the two groups. The combined radiometabolomics model achieved an AUC of 86.4%, whereas metabolomics-only and radiomics-only classifiers achieved AUC of 72.7% and 68.2%, respectively. Analysis of significant metabolite nodes identified three distinct tumour clusters (malignant, benign, and mixed) and differentially enriched metabolic pathways. In conclusion, radiometabolomics integration has been presented as an approach to evaluate disease entities. In our case study, the method identified RF and metabolites important in differentiating between benign oncocytic neoplasia and malignant renal tumors, highlighting pathways differentially expressed between the two groups. Key metabolites and RF identified by radiometabolomics can be used to improve the identification and differentiation between renal neoplasms.

2023 Scientific Article in Pathogens Pathogens 12:21 (2023)

Newly designed poxviral promoters to improve immunogenicity and efficacy of MVA-NP candidate vaccines against lethal influenza virus infection in mice.

Influenza, a respiratory disease mainly caused by influenza A and B, viruses of the Orthomyxoviridae, is still a burden on our society's health and economic system. Influenza A viruses (IAV) circulate in mammalian and avian populations, causing seasonal outbreaks with high numbers of cases. Due to the high variability in seasonal IAV triggered by antigenic drift, annual vaccination is necessary, highlighting the need for a more broadly protective vaccine against IAV. The safety tested Modified Vaccinia virus Ankara (MVA) is licensed as a third-generation vaccine against smallpox and serves as a potent vector system for the development of new candidate vaccines against different pathogens. Here, we generated and characterized recombinant MVA candidate vaccines that deliver the highly conserved internal nucleoprotein (NP) of IAV under the transcriptional control of five newly designed chimeric poxviral promoters to further increase the immunogenic properties of the recombinant viruses (MVA-NP). Infections of avian cell cultures with the recombinant MVA-NPs demonstrated efficient synthesis of the IAV-NP which was expressed under the control of the five new promoters. Prime-boost or single shot immunizations in C57BL/6 mice readily induced circulating serum antibodies' binding to recombinant IAV-NP and the robust activation of IAV-NP-specific CD8+ T cell responses. Moreover, the MVA-NP candidate vaccines protected C57BL/6 mice against lethal respiratory infection with mouse-adapted IAV (A/Puerto Rico/8/1934/H1N1). Thus, further studies are warranted to evaluate the immunogenicity and efficacy of these recombinant MVA-NP vaccines in other IAV challenge models in more detail.

2023 Scientific Article in eLife eLife 12:34 (2023)

Succinate mediates inflammation-induced adrenocortical dysfunction.

The hypothalamus-pituitary-adrenal (HPA) axis is activated in response to inflammation leading to increased production of anti-inflammatory glucocorticoids by the adrenal cortex, thereby representing an endogenous feedback loop. However, severe inflammation reduces the responsiveness of the adrenal gland to adrenocorticotropic hormone (ACTH), although the underlying mechanisms are poorly understood. Here, we show by transcriptomic, proteomic, and metabolomic analyses that LPS-induced systemic inflammation triggers profound metabolic changes in steroidogenic adrenocortical cells, including downregulation of the TCA cycle and oxidative phosphorylation, in mice. Inflammation disrupts the TCA cycle at the level of succinate dehydrogenase (SDH), leading to succinate accumulation and disturbed steroidogenesis. Mechanistically, IL-1β reduces SDHB expression through upregulation of DNA methyltransferase 1 (DNMT1) and methylation of the SDHB promoter. Consequently, increased succinate levels impair oxidative phosphorylation and ATP synthesis and enhance ROS production, leading to reduced steroidogenesis. Together, we demonstrate that the IL-1β-DNMT1-SDHB-succinate axis disrupts steroidogenesis. Our findings not only provide a mechanistic explanation for adrenal dysfunction in severe inflammation, but also offer a potential target for therapeutic intervention.

2023 Scientific Article in Cellular and Molecular Gastroenterology and Hepatology Cell. Mol. Gast. Hept. 16, 201-221 (2023)

Hepatitis B virus targets lipid transport pathways to infect hepatocytes.

BACKGROUND & AIMS: A single hepatitis B virus (HBV) particle is sufficient to establish chronic infection of the liver after intravenous injection, suggesting that the virus targets hepatocytes via a highly efficient transport pathway. We therefore investigated whether HBV uses a physiological liver-directed pathway that supports specific host-cell targeting in vivo. METHODS: We established the ex vivo perfusion of intact human liver tissue that recapitulates the liver physiology to investigate HBV liver targeting. This model allowed us to investigate virus-host cell interactions in a cellular microenvironment mimicking the in vivo situation. RESULTS: HBV was rapidly sequestered by liver macrophages within 1 hour after a virus pulse perfusion but was detected in hepatocytes only after 16 hours. We found that HBV associates with lipoproteins in serum and within machrophages. Electron and immunofluorescence microscopy corroborated a co-localization in recycling endosomes within peripheral and liver macrophages. Recycling endosomes accumulated HBV and cholesterol, followed by transport of HBV back to the cell surface along the cholesterol efflux pathway. To reach hepatocytes as final target cells, HBV was able to utilize the hepatocyte-directed cholesterol transport machinery of macrophages. CONCLUSIONS: Our results propose that by binding to liver targeted lipoproteins and using the reverse cholesterol transport pathway of macrophages, HBV hijacks the physiological lipid transport pathways to the liver to most efficiently reach its target organ. This may involve transinfection of liver macrophages and result in deposition of HBV in the perisinusoidal space from where HBV can bind its receptor on hepatocytes.

2023 Scientific Article in Nature Biotechnology Nat. Biotechnol. 41, 1734–1745 (2023)

Genetically encoded barcodes for correlative volume electron microscopy.

While genetically encoded reporters are common for fluorescence microscopy, equivalent multiplexable gene reporters for electron microscopy (EM) are still scarce. Here, by installing a variable number of fixation-stable metal-interacting moieties in the lumen of encapsulin nanocompartments of different sizes, we developed a suite of spherically symmetric and concentric barcodes (EMcapsulins) that are readable by standard EM techniques. Six classes of EMcapsulins could be automatically segmented and differentiated. The coding capacity was further increased by arranging several EMcapsulins into distinct patterns via a set of rigid spacers of variable length. Fluorescent EMcapsulins were expressed to monitor subcellular structures in light and EM. Neuronal expression in Drosophila and mouse brains enabled the automatic identification of genetically defined cells in EM. EMcapsulins are compatible with transmission EM, scanning EM and focused ion beam scanning EM. The expandable palette of genetically controlled EM-readable barcodes can augment anatomical EM images with multiplexed gene expression maps.

2023 Scientific Article in Cardiovascular Diabetology Cardiovasc. Diabetol. 22:217 (2023)

GIP receptor agonism improves dyslipidemia and atherosclerosis independently of body weight loss in preclinical mouse model for cardio-metabolic disease.

BACKGROUND: Agonism at the receptor for the glucose-dependent insulinotropic polypeptide (GIPR) is a key component of the novel unimolecular GIPR:GLP-1R co-agonists, which are among the most promising drugs in clinical development for the treatment of obesity and type 2 diabetes. The therapeutic effect of chronic GIPR agonism to treat dyslipidemia and thus to reduce the cardiovascular disease risk independently of body weight loss has not been explored yet. METHODS: After 8 weeks on western diet, LDL receptor knockout (LDLR-/-) male mice were treated with daily subcutaneous injections of long-acting acylated GIP analog (acyl-GIP; 10nmol/kg body weight) for 28 days. Body weight, food intake, whole-body composition were monitored throughout the study. Fasting blood glucose and intraperitoneal glucose tolerance test (ipGTT) were determined on day 21 of the study. Circulating lipid levels, lipoprotein profiles and atherosclerotic lesion size was assessed at the end of the study. Acyl-GIP effects on fat depots were determined by histology and transcriptomics. RESULTS: Herein we found that treatment with acyl-GIP reduced dyslipidemia and atherogenesis in male LDLR-/- mice. Acyl-GIP administration resulted in smaller adipocytes within the inguinal fat depot and RNAseq analysis of the latter revealed that acyl-GIP may improve dyslipidemia by directly modulating lipid metabolism in this fat depot. CONCLUSIONS: This study identified an unanticipated efficacy of chronic GIPR agonism to improve dyslipidemia and cardiovascular disease independently of body weight loss, indicating that treatment with acyl-GIP may be a novel approach to alleviate cardiometabolic disease.

2023 Scientific Article in EMBO Reports EMBO Rep. 24:e55981 (2023)

Fasting-sensitive SUMO-switch on Prox1 controls hepatic cholesterol metabolism.

Accumulation of excess nutrients hampers proper liver function and is linked to nonalcoholic fatty liver disease (NAFLD) in obesity. However, the signals responsible for an impaired adaptation of hepatocytes to obesogenic dietary cues remain still largely unknown. Post-translational modification by the small ubiquitin-like modifier (SUMO) allows for a dynamic regulation of numerous processes including transcriptional reprogramming. We demonstrate that specific SUMOylation of transcription factor Prox1 represents a nutrient-sensitive determinant of hepatic fasting metabolism. Prox1 is highly SUMOylated on lysine 556 in the liver of ad libitum and refed mice, while this modification is abolished upon fasting. In the context of diet-induced obesity, Prox1 SUMOylation becomes less sensitive to fasting cues. The hepatocyte-selective knock-in of a SUMOylation-deficient Prox1 mutant into mice fed a high-fat/high-fructose diet leads to a reduction of systemic cholesterol levels, associated with the induction of liver bile acid detoxifying pathways during fasting. The generation of tools to maintain the nutrient-sensitive SUMO-switch on Prox1 may thus contribute to the development of "fasting-based" approaches for the preservation of metabolic health.

2023 Scientific Article in European Journal of Nuclear Medicine and Molecular Imaging Eur. J. Nucl. Med. Mol. Imaging 50, 2736-2750 (2023)

Towards in vivo characterization of thyroid nodules suspicious for malignancy using multispectral optoacoustic tomography.

PURPOSE: Patient-tailored management of thyroid nodules requires improved risk of malignancy stratification by accurate preoperative nodule assessment, aiming to personalize decisions concerning diagnostics and treatment. Here, we perform an exploratory pilot study to identify possible patterns on multispectral optoacoustic tomography (MSOT) for thyroid malignancy stratification. For the first time, we directly correlate MSOT images with histopathology data on a detailed level. METHODS: We use recently enhanced data processing and image reconstruction methods for MSOT to provide next-level image quality by means of improved spatial resolution and spectral contrast. We examine optoacoustic features in thyroid nodules associated with vascular patterns and correlate these directly with reference histopathology. RESULTS: Our methods show the ability to resolve blood vessels with diameters of 250 μm at depths of up to 2 cm. The vessel diameters derived on MSOT showed an excellent correlation (R2-score of 0.9426) with the vessel diameters on histopathology. Subsequently, we identify features of malignancy observable in MSOT, such as intranodular microvascularity and extrathyroidal extension verified by histopathology. Despite these promising features in selected patients, we could not determine statistically relevant differences between benign and malignant thyroid nodules based on mean oxygen saturation in thyroid nodules. Thus, we illustrate general imaging artifacts of the whole field of optoacoustic imaging that reduce image fidelity and distort spectral contrast, which impedes quantification of chromophore presence based on mean concentrations. CONCLUSION: We recommend examining optoacoustic features in addition to chromophore quantification to rank malignancy risk. We present optoacoustic images of thyroid nodules with the highest spatial resolution and spectral contrast to date, directly correlated to histopathology, pushing the clinical translation of MSOT.

2023 Scientific Article in Journal of Biomedical Optics J. Biomed. Opt. 28:046001 (2023)

Accounting for blood attenuation in intravascular near-infrared fluorescence-ultrasound imaging using a fluorophore-coated guidewire.

SIGNIFICANCE: Intravascular near-infrared fluorescence (NIRF) imaging aims to improve the inspection of vascular pathology using fluorescent agents with specificity to vascular disease biomarkers. The method has been developed to operate in tandem with an anatomical modality, such as intravascular ultrasound (IVUS), and complements anatomical readings with pathophysiological contrast, enhancing the information obtained from the hybrid examination. AIM: However, attenuation of NIRF signals by blood challenges NIRF quantification. We propose a new method for attenuation correction in NIRF intravascular imaging based on a fluorophore-coated guidewire that is used as a reference for the fluorescence measurement and provides a real-time measurement of blood attenuation during the NIRF examination. APPROACH: We examine the performance of the method in a porcine coronary artery ex vivo and phantoms using a 3.2F NIRF-IVUS catheter. RESULTS: We demonstrate marked improvement over uncorrected signals of up to 4.5-fold and errors of < 11 % for target signals acquired at distances up to 1 mm from the catheter system employed. CONCLUSIONS: The method offers a potential means of improving the accuracy of intravascular NIRF imaging under in vivo conditions.

2023 Scientific Article in Advanced science Adv. Sci. 10:e2301322 (2023)

Spotlight on nerves: Portable multispectral optoacoustic imaging of peripheral nerve vascularization and morphology.

Various morphological and functional parameters of peripheral nerves and their vascular supply are indicative of pathological changes due to injury or disease. Based on recent improvements in optoacoustic image quality, the ability of multispectral optoacoustic tomography, to investigate the vascular environment and morphology of peripheral nerves is explored in vivo in a pilot study on healthy volunteers in tandem with ultrasound imaging (OPUS). The unique ability of optoacoustic imaging to visualize the vasa nervorum by observing intraneural vessels in healthy nerves is showcased in vivo for the first time. In addition, it is demonstrated that the label-free spectral optoacoustic contrast of the perfused connective tissue of peripheral nerves can be linked to the endogenous contrast of hemoglobin and collagen. Metrics are introduced to analyze the composition of tissue based on its optoacoustic contrast and show that the high-resolution spectral contrast reveals specific differences between nervous tissue and reference tissue in the nerve's surrounding. How this showcased extraction of peripheral nerve characteristics using multispectral optoacoustic and ultrasound imaging could offer new insights into the pathophysiology of nerve damage and neuropathies, for example, in the context of diabetes is discussed.

2023 Scientific Article in Photoacoustics Photoacoustics 30:100468 (2023)

Skeletal muscle optoacoustics reveals patterns of circulatory function and oxygen metabolism during exercise.

Imaging skeletal muscle function and metabolism, as reported by local hemodynamics and oxygen kinetics, can elucidate muscle performance, severity of an underlying disease or outcome of a treatment. Herein, we used multispectral optoacoustic tomography (MSOT) to image hemodynamics and oxygen kinetics within muscle during exercise. Four healthy volunteers underwent three different hand-grip exercise challenges (60s isometric, 120s intermittent isometric and 60s isotonic). During isometric contraction, MSOT showed a decrease of HbO2, Hb and total blood volume (TBV), followed by a prominent increase after the end of contraction. Corresponding hemodynamic behaviors were recorded during the intermittent isometric and isotonic exercises. A more detailed analysis of MSOT readouts revealed insights into arteriovenous oxygen differences and muscle oxygen consumption during all exercise schemes. These results demonstrate an excellent capability of visualizing both circulatory function and oxygen metabolism within skeletal muscle under exercise, with great potential implications for muscle research, including relevant disease diagnostics.

2023 Scientific Article in Diagnostics Diagnostics 13:21 (2023)

Detection of tumour-targeted IRDye800CW tracer with commercially available laparoscopic surgical systems.

(1) Introduction: Near-infrared fluorescence (NIRF) combined with tumour-targeted tracers, such as bevacizumab-800CW, could aid surgical decision-making. This study explored the use of IRDye800CW, conjugated to bevacizumab, with four commercially available NIRF laparoscopes optimised for indocyanine green (ICG). (2) Methods: A (lymph node) phantom was made from a calibration device for NIRF and tissue-mimicking material. Serial dilutions of bevacizumab-800CW were made and ICG functioned as a reference. System settings, working distance, and thickness of tissue-mimicking material were varied to assess visibility of the fluorescence signal and tissue penetration. Tests were performed with four laparoscopes: VISERA ELITE II, Olympus; IMAGE1 S™ 4U Rubina, KARL STORZ; ENDOCAM Logic 4K platform, Richard Wolf; da Vinci Xi, Intuitive Surgical. (3) Results: The lowest visible bevacizumab-800CW concentration ranged between 13-850 nM (8-512 times diluted stock solution) for all laparoscopes, but the tracer was not visible through 0.8 cm of tissue in all systems. In contrast, ICG was still visible at a concentration of 0.4 nM (16,384 times diluted) and through 1.6-2.4 cm of tissue. Visibility and tissue penetration generally improved with a reduced working distance and manually adjusted system settings. (4) Conclusion: Depending on the application, bevacizumab-800CW might be sufficiently visible with current laparoscopes, but optimisation would widen applicability of tumour-targeted IRDye800CW tracers.

2023 Scientific Article in Photoacoustics Photoacoustics 31:100513 (2023)

Raster-scanning optoacoustic mesoscopy biomarkers for atopic dermatitis skin lesions.

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease worldwide. Its severity is assessed using scores that rely on visual observation of the affected body surface area, the morphology of the lesions and subjective symptoms, like pruritus or insomnia. Ideally, such scores should be complemented by objective and accurate measurements of disease severity to standardize disease scoring in routine care and clinical trials. Recently, it was shown that raster-scanning optoacoustic mesoscopy (RSOM) can provide detailed three-dimensional images of skin inflammation processes that capture the most relevant features of their pathology. Moreover, precise RSOM biomarkers of inflammation have been identified for psoriasis. However, the objectivity and validity of such biomarkers in repeated measurements have not yet been assessed for AD. Here, we report the results of a study on the repeatability of RSOM inflammation biomarkers in AD to estimate their precision. Optoacoustic imaging analysis revealed morphological inflammation biomarkers with precision well beyond standard clinical severity metrics. Our findings suggest that optoacoustic mesoscopy may be a good choice for quantitative evaluations of AD that are inaccessible by other methods. This could potentially enable the optimization of disease scoring and drug development.

2023 Editorial in Frontiers in Cardiovascular Medicine Front. Cardiovasc. Med. 10:1178101 (2023)

Editorial: Research topic for frontiers in cardiovascular medicine: non-invasive sensing and imaging techniques for cardiometabolic diseases.

2023 Review in Journal of Clinical Medicine J. Clin. Med. 12:13 (2023)

Edge-to-edge transcatheter mitral valve repair using PASCAL vs. MitraClip: A systematic review and meta-analysis.

BACKGROUND: Transcatheter edge-to-edge repair (TEER) of the mitral valve (MV) can be performed using the PASCAL or MitraClip devices. Few studies offer a head-to-head outcome comparison of these two devices. MATERIAL AND METHODS: PubMed, EMBASE, Cochrane Library, Clinicaltrials.gov and WHO's International Clinical Trials Registry Platform, from 1 January 2000 until 1 March 2023, were searched. Study protocol details were registered in the International Prospective Register of Systematic Reviews (PROSPERO ID: CRD42023405400). Randomized Controlled Trials and observational studies reporting head-to-head clinical comparison of PASCAL and MitraClip devices were eligible for selection. Patients with severe functional or degenerative mitral regurgitation (MR) who had undergone TEER of the MV with either PASCAL or MitraClip devices were included in the meta-analysis. Data from six studies (five observational and one randomized clinical trial) were extracted and analyzed. The main outcomes were a reduction in MR to 2+ or less, improvement of New York Heart Association (NYHA) and 30-day all-cause mortality. Peri-procedural mortality, success rate and adverse events were also compared. RESULTS: Data from 785 and 796 patients that underwent TEER using PASCAL and MitraClip, respectively, were analyzed. Thirty-day all-cause mortality (Risk ratio [RR] = 1.51, 95% CI 0.79-2.89), MR reduction to maximum 2+ (RR = 1.00, 95% CI 0.98-1.02) and NYHA improvement (RR = 0.98, 95% CI 0.84-1.15) were similar in both device groups. Both devices had high and similar success rates (96.9% and 96.7% for the PASCAL and MitraClip group, respectively, p value = 0.91). MR reduction to 1+ or less at discharge was similar in both device groups (RR = 1.06, 95% CI 0.95-1.19). Composite peri-procedural and in-hospital mortality was 0.64% and 1.66% in the PASCAL and MitraClip groups, respectively (p value = 0.094). Rates of peri-procedural cerebrovascular accidents were 0.26% in PASCAL and 1.01% in MitraClip (p value = 0.108). CONCLUSIONS: Both PASCAL and MitraClip devices have high success and low complication rates for TEER of the MV. PASCAL was not inferior to MitraClip in reducing the MR level at discharge.

2023 Scientific Article in Physics in Medicine and Biology Phys. Med. Biol. 68:19 (2023)

Range verification of a clinical proton beam in an abdominal phantom by co-registration of ionoacoustics and ultrasound.

Objective.The range uncertainty in proton radiotherapy is a limiting factor to achieve optimum dose conformity to the tumour volume. Ionoacoustics is a promising approach forin siturange verification, which would allow to reduce the size of the irradiated volume relative to the tumour volume. The energy deposition of a pulsed proton beam leads to an acoustic pressure wave (ionoacoustics), the detection of which allows conclusion about the distance between the Bragg peak and the acoustic detector. This information can be transferred into a co-registered ultrasound image, marking the Bragg peak position relative to the surrounding anatomy.Approach.A CIRS 3D abdominal phantom was irradiated with 126 MeV protons at a clinical proton therapy centre. Acoustic signals were recorded on the beam axis distal to the Bragg peak with a Cetacean C305X hydrophone. The ionoacoustic measurements were processed with a correlation filter using simulated filter templates. The hydrophone was rigidly attached to an ultrasound device (Interson GP-C01) recording ultrasound images of the irradiated region.Main results.The time of flight obtained from ionoacoustic measurements were transferred to an ultrasound image by means of an optoacoustic calibration measurement. The Bragg peak position was marked in the ultrasound image with a statistical uncertainty ofσ= 0.5 mm of 24 individual measurements depositing 1.2 Gy at the Bragg peak. The difference between the evaluated Bragg peak position and the one obtained from irradiation planning (1.0 mm) is smaller than the typical range uncertainty (≈4 mm) at the given penetration depth (10 cm).Significance.The measurements show that it is possible to determine the Bragg peak position of a clinical proton beam with submillimetre precision and transfer the information to an ultrasound image of the irradiated region. The dose required for this is smaller than that used for a typical irradiation fraction.

2023 Review in Molecular Imaging and Biology Mol. Imaging Biol. 25, 799-814 (2023)

Neurotoxin-derived optical probes for biological and medical imaging.

The superb specificity and potency of biological toxins targeting various ion channels and receptors are of major interest for the delivery of therapeutics to distinct cell types and subcellular compartments. Fused with reporter proteins or labelled with fluorophores and nanocomposites, animal toxins and their detoxified variants also offer expanding opportunities for visualisation of a range of molecular processes and functions in preclinical models, as well as clinical studies. This article presents state-of-the-art optical probes derived from neurotoxins targeting ion channels, with discussions of their applications in basic and translational biomedical research. It describes the design and production of probes and reviews their applications with advantages and limitations, with prospects for future improvements. Given the advances in imaging tools and expanding research areas benefiting from the use of optical probes, described here resources should assist the discovery process and facilitate high-precision interrogation and therapeutic interventions.

2023 Review in FEBS Letters FEBS Lett. 597, 1319-1344 (2023)

It's a two-way street: Photoswitching and reversible changes of the protein matrix in photoswitchable fluorescent proteins and bacteriophytochromes.

Chromophore-bearing proteins that are (reversibly) altered after light illumination are major functional components of nature. They gained considerable attention in the last decades since the dynamic interactions of the chromophore and protein matrix can be used to control downstream effects altering the functionality of proteins, cells, or complete organisms with light (optogenetics). Additionally, the photophysical effects can be employed to add capabilities to optical imaging. For example, light can be used to reversibly switch the signal on or off (e.g., fluorescence). In this article, we review chromophore and protein matrix interactions, focusing on photoswitching fluorescent proteins of the GFP family (RSFPs) and natively photoswitching bacteriophytochromes (BphPs). This review aims to provide an in-depth understanding of the dynamic interplay between photoswitching photophysics and the protein matrix and a thorough discussion on how this connection has been harnessed for the development of optogenetic and imaging tools.

2023 Scientific Article in Cells Cells 12:19 (2023)

Primary ciliary dyskinesia patient-specific hiPSC-derived airway epithelium in air-liquid interface culture recapitulates disease specific phenotypes in vitro.

Primary ciliary dyskinesia (PCD) is a rare heterogenic genetic disorder associated with perturbed biogenesis or function of motile cilia. Motile cilia dysfunction results in diminished mucociliary clearance (MCC) of pathogens in the respiratory tract and chronic airway inflammation and infections successively causing progressive lung damage. Current approaches to treat PCD are symptomatic, only, indicating an urgent need for curative therapeutic options. Here, we developed an in vitro model for PCD based on human induced pluripotent stem cell (hiPSC)-derived airway epithelium in Air-Liquid-Interface cultures. Applying transmission electron microscopy, immunofluorescence staining, ciliary beat frequency, and mucociliary transport measurements, we could demonstrate that ciliated respiratory epithelia cells derived from two PCD patient-specific hiPSC lines carrying mutations in DNAH5 and NME5, respectively, recapitulate the respective diseased phenotype on a molecular, structural and functional level.

2023 Scientific Article in Materials Today Bio Mater. Today Bio 21:100713 (2023)

Breathing on chip: Dynamic flow and stretch accelerate mucociliary maturation of airway epithelium in vitro

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying the effects of dynamic medium and airflow as well as stretch on human primary airway epithelial cell differentiation and maturation, including mucociliary clearance, using an organ-on-chip device. Perfused epithelial cell cultures displayed accelerated maturation and polarization of mucociliary clearance, and changes in specific cell-types when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in an increase in polarization of mucociliary clearance towards the applied flow, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and maturation and that their fine-tuned application could generate models of specific epithelial pathologies, including mucociliary (dys)function.

2023 Scientific Article in Stroke Stroke 54, 1636-1644 (2023)

Spatial proteomics analysis of soft and stiff regions in human acute arterial thrombus.

BACKGROUND: The soft regions of a thrombus tend to be more susceptible to r-tPA (recombinant tissue-type plasminogen activator)-mediated thrombolysis and are more easily removed by mechanical thrombectomy than the stiff counterpart. This study aimed to understand the molecular pathological differences between the soft and stiff regions of human arterial thrombus. METHODS: We developed a spatial proteomic workflow combining proteomics with laser-captured microdissection to analyze human arterial thrombi with Masson trichrome staining to identify stiff and soft regions from 2 independent cohorts of patients with acute myocardial or cerebral infarction. Dysregulated proteins in a C57BL6/J male mouse model of arterial thrombosis were identified by pathway enrichment and pairwise analyses from the common gene ontology enrichment and dysregulated proteins between carotid and coronary arterial thrombi, and validated by immunohistochemistry. RESULTS: Spatial proteomics of the coronary arterial thrombi collected from 7 patients with myocardial infarct revealed 7 common dysregulated proteins in 2 cohorts of patients, and upregulation of TGF-β1 (transforming growth factor β1) was the most prominent fibrosis-related protein. Inhibition of TGF-β1 resulted in delayed arterial thrombosis and accelerated blood flow restoration in mouse model. We further expanded the spatial proteomic workflow to the carotid artery thrombi collected from 11 patients with cerebral infarction. Pairwise proteomic analysis of stiff and soft regions between carotid and coronary arterial thrombi further revealed 5 common gene ontology clusters including features of platelet activation, and a common dysregulated protein COL1A1 (collagen type 1 alpha 1) that was reported to be influenced by TGF-β1. We also verified the expression in human and mice carotid arterial thrombi. CONCLUSIONS: This study demonstrates the spatially distinct composition of proteins in the stiff and soft regions of human arterial thrombi, and suggests that TGF-β1 is a key therapeutic target for promoting arterial thrombolysis.

2023 Scientific Article in Bioactive Materials Bioact. Mater. 29, 196-213 (2023)

FUS-mediated HypEVs: Neuroprotective effects against ischemic stroke.

Few studies have investigated the properties and protein composition of small extracellular vesicles (sEVs) derived from neurons under hypoxic conditions. Presently, the extent of the involvement of these plentiful sEVs in the onset and progression of ischemic stroke remains an unresolved question. Our study systematically identified the characteristics of sEVs derived from neurons under hypoxic conditions (HypEVs) by physical characterization, sEV absorption, proteomics and transcriptomics analysis. The effects of HypEVs on neurites, cell survival, and neuron structure were assessed in vitro and in vivo by neural complexity tests, magnetic resonance imaging (MRI), Golgi staining, and Western blotting of synaptic plasticity-related proteins and apoptotic proteins. Knockdown of Fused in Sarcoma (FUS) small interfering RNA (siRNA) was used to validate FUS-mediated HypEV neuroprotection and mitochondrial mRNA release. Hypoxia promoted the secretion of sEVs, and HypEVs were more easily taken up and utilized by recipient cells. The MRI results illustrated that the cerebral infarction volume was reduced by 45% with the application of HypEVs, in comparison to the non- HypEV treatment group. Mechanistically, the FUS protein is necessary for the uptake and neuroprotection of HypEVs against ischemic stroke as well as carrying a large amount of mitochondrial mRNA in HypEVs. However, FUS knockdown attenuated the neuroprotective rescue capabilities of HypEVs. Our comprehensive dataset clearly illustrates that FUS-mediated HypEVs deliver exceptional neuroprotective effects against ischemic stroke, primarily through the maintenance of neurite integrity and the reduction of mitochondria-associated apoptosis.

2023 Scientific Article in Nature Biotechnology Nat. Biotechnol., DOI: 10.1038/s41587-023-01846-0 (2023)

Whole-body cellular mapping in mouse using standard IgG antibodies.

Whole-body imaging techniques play a vital role in exploring the interplay of physiological systems in maintaining health and driving disease. We introduce wildDISCO, a new approach for whole-body immunolabeling, optical clearing and imaging in mice, circumventing the need for transgenic reporter animals or nanobody labeling and so overcoming existing technical limitations. We identified heptakis(2,6-di-O-methyl)-β-cyclodextrin as a potent enhancer of cholesterol extraction and membrane permeabilization, enabling deep, homogeneous penetration of standard antibodies without aggregation. WildDISCO facilitates imaging of peripheral nervous systems, lymphatic vessels and immune cells in whole mice at cellular resolution by labeling diverse endogenous proteins. Additionally, we examined rare proliferating cells and the effects of biological perturbations, as demonstrated in germ-free mice. We applied wildDISCO to map tertiary lymphoid structures in the context of breast cancer, considering both primary tumor and metastases throughout the mouse body. An atlas of high-resolution images showcasing mouse nervous, lymphatic and vascular systems is accessible at http://discotechnologies.org/wildDISCO/atlas/index.php .

2023 Scientific Article in Cell Cell 186, 3706-3725.e29 (2023)

Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

2023 Scientific Article in Progress in Neurobiology Prog. Neurobiol. 229:102512 (2023)

The state of brain activity modulates cerebrospinal fluid transport.

Earlier studies based on 2-photon imaging have shown that glymphatic cerebrospinal fluid (CSF) transport is regulated by the sleep-wake cycle. To examine this association, we used 3DISCO whole-body tissue clearing to map CSF tracer distribution in awake, sleeping and ketamine-xylazine anesthetized mice. The results of our analysis showed that CSF tracers entered the brain to a significantly larger extent in natural sleep or ketamine-xylazine anesthesia than in wakefulness. Furthermore, awake mice showed preferential transport of CSF tracers in the rostro-caudal direction towards the cervical and spinal cord lymphatic vessels, and hence to venous circulation and excretion by the kidneys. The study extends the current literature by showing that CSF dynamics on the whole-body scale is controlled by the state of brain activity.

2023 Scientific Article in Science Science 381, 285-290 (2023)

Immune-mediated denervation of the pineal gland underlies sleep disturbance in cardiac disease.

Disruption of the physiologic sleep-wake cycle and low melatonin levels frequently accompany cardiac disease, yet the underlying mechanism has remained enigmatic. Immunostaining of sympathetic axons in optically cleared pineal glands from humans and mice with cardiac disease revealed their substantial denervation compared with controls. Spatial, single-cell, nuclear, and bulk RNA sequencing traced this defect back to the superior cervical ganglia (SCG), which responded to cardiac disease with accumulation of inflammatory macrophages, fibrosis, and the selective loss of pineal gland-innervating neurons. Depletion of macrophages in the SCG prevented disease-associated denervation of the pineal gland and restored physiological melatonin secretion. Our data identify the mechanism by which diurnal rhythmicity in cardiac disease is disturbed and suggest a target for therapeutic intervention.

2023 Scientific Article in Molecules Molecules 28:17 (2023)

Licochalcone A inhibits prostaglandin E2 by targeting the MAPK pathway in LPS activated primary microglia.

Neuroinflammation and oxidative stress are conditions leading to neurological and neuropsychiatric disorders. Natural compounds exerting anti-inflammatory and anti-oxidative effects, such as Licochalcone A, a bioactive flavonoid present in a traditional Chinese herb (licorice), might be beneficial for the treatment of those disorders. Therefore, this study aimed to investigate the anti-inflammatory and anti-oxidative effects of Licochalcone A in LPS-activated primary rat microglia. Licochalcone A dose-dependently prevented LPS-induced PGE2 release by inhibiting the arachidonic acid (AA)/cylcooxygenase (COX) pathway decreasing phospholipase A2, COX-1, and COX-2 protein levels. Furthermore, LPS-induced levels of the cytokines IL-6 and TNFα were reduced by Licochalcone A, which also inhibited the phosphorylation and, thus, activation of the mitogen-activated protein kinases (MAPK) p38 MAPK and Erk 1/2. With the reduction of 8-iso-PGF2α, a sensitive marker for oxidative stress, anti-oxidative effects of Licochalcone A were demonstrated. Our data demonstrate that Licochalcone A can affect microglial activation by interfering in important inflammatory pathways. These in vitro findings further demonstrate the potential value of Licochalcone A as a therapeutic option for the prevention of microglial dysfunction related to neuroinflammatory diseases. Future research should continue to investigate the effects of Licochalcone A in different disease models with a focus on its anti-oxidative and anti-neuroinflammatory properties.

2023 Scientific Article in Nature Communications Nat. Commun. 14:709 (2023)

Adipocyte-derived extracellular vesicles increase insulin secretion through transport of insulinotropic protein cargo.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.

2023 Review in Frontiers in Cardiovascular Medicine Front. Cardiovasc. Med. 9:949454 (2023)

Artificial intelligence in atherosclerotic disease: Applications and trends.

Atherosclerotic cardiovascular disease (ASCVD) is the most common cause of death globally. Increasing amounts of highly diverse ASCVD data are becoming available and artificial intelligence (AI) techniques now bear the promise of utilizing them to improve diagnosis, advance understanding of disease pathogenesis, enable outcome prediction, assist with clinical decision making and promote precision medicine approaches. Machine learning (ML) algorithms in particular, are already employed in cardiovascular imaging applications to facilitate automated disease detection and experts believe that ML will transform the field in the coming years. Current review first describes the key concepts of AI applications from a clinical standpoint. We then provide a focused overview of current AI applications in four main ASCVD domains: coronary artery disease (CAD), peripheral arterial disease (PAD), abdominal aortic aneurysm (AAA), and carotid artery disease. For each domain, applications are presented with refer to the primary imaging modality used [e.g., computed tomography (CT) or invasive angiography] and the key aim of the applied AI approaches, which include disease detection, phenotyping, outcome prediction, and assistance with clinical decision making. We conclude with the strengths and limitations of AI applications and provide future perspectives.

2023 Scientific Article in Photoacoustics Photoacoustics 29:100454 (2023)

Non-invasive multispectral optoacoustic tomography resolves intrahepatic lipids in patients with hepatic steatosis.

Hepatic steatosis is characterized by intrahepatic lipid accumulation and may lead to irreversible liver damage if untreated. Here, we investigate whether multispectral optoacoustic tomography (MSOT) can offer label-free detection of liver lipid content to enable non-invasive characterization of hepatic steatosis by analyzing the spectral region around 930 nm, where lipids characteristically absorb. In a pilot study, we apply MSOT to measure liver and surrounding tissues in five patients with liver steatosis and five healthy volunteers, revealing significantly higher absorptions at 930 nm in the patients, while no significant difference was observed in the subcutaneous adipose tissue of the two groups. We further corroborated the human observations with corresponding MSOT measurements in high fat diet (HFD) - and regular chow diet (CD)-fed mice. This study introduces MSOT as a potential non-invasive and portable technique for detecting/monitoring hepatic steatosis in clinical settings, providing justification for larger studies.

2023 Scientific Article in British Journal of Cancer BJC Br. J. Cancer 128, 1369-1376 (2023)

Predicting the HER2 status in oesophageal cancer from tissue microarrays using convolutional neural networks.

BACKGROUND: Fast and accurate diagnostics are key for personalised medicine. Particularly in cancer, precise diagnosis is a prerequisite for targeted therapies, which can prolong lives. In this work, we focus on the automatic identification of gastroesophageal adenocarcinoma (GEA) patients that qualify for a personalised therapy targeting epidermal growth factor receptor 2 (HER2). We present a deep-learning method for scoring microscopy images of GEA for the presence of HER2 overexpression. METHODS: Our method is based on convolutional neural networks (CNNs) trained on a rich dataset of 1602 patient samples and tested on an independent set of 307 patient samples. We additionally verified the CNN's generalisation capabilities with an independent dataset with 653 samples from a separate clinical centre. We incorporated an attention mechanism in the network architecture to identify the tissue regions, which are important for the prediction outcome. Our solution allows for direct automated detection of HER2 in immunohistochemistry-stained tissue slides without the need for manual assessment and additional costly in situ hybridisation (ISH) tests. RESULTS: We show accuracy of 0.94, precision of 0.97, and recall of 0.95. Importantly, our approach offers accurate predictions in cases that pathologists cannot resolve and that require additional ISH testing. We confirmed our findings in an independent dataset collected in a different clinical centre. The attention-based CNN exploits morphological information in microscopy images and is superior to a predictive model based on the staining intensity only. CONCLUSIONS: We demonstrate that our approach not only automates an important diagnostic process for GEA patients but also paves the way for the discovery of new morphological features that were previously unknown for GEA pathology.

2023 Review in Nature Protocols Nat. Protoc. 18, 1197-1242 (2023)

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

2023 Scientific Article in Clinical Chemistry Clin. Chem. 69, 149-159 (2023)

In situ metabolomics of cortisol-producing adenomas.

BACKGROUND: Recent advances in omics techniques have allowed detailed genetic characterization of cortisol-producing adrenal adenoma (CPA). In contrast, the pathophysiology of CPAs has not been elucidated in detail on the level of tumor metabolic alterations. METHODS: The current study conducted a comprehensive mass spectrometry imaging (MSI) map of CPAs in relation to clinical phenotypes and immunohistochemical profiles of steroidogenic enzymes. The study cohort comprised 46 patients with adrenal tumors including CPAs (n = 35) and nonfunctional adenomas (n = 11). RESULTS: Severity of cortisol hypersecretion was significantly correlated with 29 metabolites (adjusted P < 0.05). Adrenal androgens derived from the classic androgen pathway were inversely correlated with both cortisol secretion (rs = -0.41, adjusted P = 0.035) and CYP11B1 expression (rs = -0.77, adjusted P = 2.00E-08). The extent of cortisol excess and tumor CYP11B1 expression further correlated with serotonin (rs = 0.48 and 0.62, adjusted P = 0.008 and 2.41E-05). Tumor size was found to be correlated with abundance of 13 fatty acids (adjusted P < 0.05) and negatively associated with 9 polyunsaturated fatty acids including phosphatidic acid 38:8 (rs = -0.56, adjusted P = 0.009). CONCLUSIONS: MSI reveals novel metabolic links between endocrine function and tumorigenesis, which will further support the understanding of CPA pathophysiology.

2023 Scientific Article in Experimental Eye Research Exp. Eye Res. 226:109346 (2023)

Identification of ocular regulatory functions of core histone variant H3.2.

The posttranscriptional modifications (PTM) of the Histone H3 family play an important role in ocular system differentiation. However, there has been no study on the nature of specific Histone H3 subtype carrying these modifications. Fortuitously, we had previously identified a dominant small-eye mutant Aey69 mouse with a mutation in the H3.2 encoding Hist2h3c1 gene (Vetrivel et al., 2019). In continuation, in the present study, the role of Histone H3.2 with relation to the microphtalmic Aey69 has been elaborated. Foremost, a transgenic mouse line expressing the fusion protein H3.2-GFP was generated using Crispr/Cas9. The approach was intended to confer a unique tag to the Hist2h3c1 gene which is similar in sequence and encoded protein structure to other histones. The GFP tag was then used for ChIP Seq analysis of the genes regulated by H3.2. The approach revealed ocular specific H3.2 targets including Ephrin family genes. Altered enrichment of H3.2 was found in the mutant Aey69 mouse, specifically around the ligand Efna5 and the receptor Ephb2. The effect of this altered enrichment on Ephrin signaling was further analyzed by QPCR and immunohistochemistry. This study identifies Hist2h3c1 encoded H3.2 as an important epigenetic player in ocular development. By binding to specific regions of ocular developmental factors Histone H3.2 facilitates the function of these genes for successful early ocular development.

2023 Scientific Article in Monatshefte für Mathematik Monatsh. Math. 200, 23–42 (2023)

How many Fourier coefficients are needed?

We are looking at families of functions or measures on the torus which are specified by a finite number of parameters N. The task, for a given family, is to look at a small number of Fourier coefficients of the object, at a set of locations that is predetermined and may depend only on N, and determine the object. We look at (a) the indicator functions of at most N intervals of the torus and (b) at sums of at most N complex point masses on the multidimensional torus. In the first case we reprove a theorem of Courtney which says that the Fourier coefficients at the locations 0 , 1 , … , N are sufficient to determine the function (the intervals). In the second case we produce a set of locations of size O(Nlog d-1N) which suffices to determine the measure.

2023 Scientific Article in Neural regeneration research Neural Regen. Res. 18, 652-656 (2023)

Hypoxic pre-conditioned adipose-derived stem/progenitor cells embedded in fibrin conduits promote peripheral nerve regeneration in a sciatic nerve graft model.

Recent results emphasize the supportive effects of adipose-derived multipotent stem/progenitor cells (ADSPCs) in peripheral nerve recovery. Cultivation under hypoxia is considered to enhance the release of the regenerative potential of ADSPCs. This study aimed to examine whether peripheral nerve regeneration in a rat model of autologous sciatic nerve graft benefits from an additional custom-made fibrin conduit seeded with hypoxic pre-conditioned (2% oxygen for 72 hours) autologous ADSPCs (n = 9). This treatment mode was compared with three others: fibrin conduit seeded with ADSPCs cultivated under normoxic conditions (n = 9); non-cell-carrying conduit (n = 9); and nerve autograft only (n = 9). A 16-week follow-up included functional testing (sciatic functional index and static sciatic index) as well as postmortem muscle mass analyses and morphometric nerve evaluations (histology, g-ratio, axon density, and diameter). At 8 weeks, the hypoxic pre-conditioned group achieved significantly higher sciatic functional index/static sciatic index scores than the other three groups, indicating faster functional regeneration. Furthermore, histologic evaluation showed significantly increased axon outgrowth/branching, axon density, remyelination, and a reduced relative connective tissue area. Hypoxic pre-conditioned ADSPCs seeded in fibrin conduits are a promising adjunct to current nerve autografts. Further studies are needed to understand the underlying cellular mechanism and to investigate a potential application in clinical practice.

2022 Scientific Article in Applied Physics Letters Appl. Phys. Lett. 121:213502 (2022)

Contribution of multiple plasmon scattering in low-angle electron diffraction investigated by energy-filtered atomically resolved 4D-STEM.

We report the influence of multiple plasmon losses on the dynamical diffraction of high-energy electrons, in a scanning transmission electron microscopy (STEM) study. Using an experimental setup enabling energy-filtered momentum-resolved STEM, it is shown that the successive excitation of up to five plasmons within the imaged material results in a subsequent and significant redistribution of low-angle intensity in diffraction space. An empirical approach, based on the convolution with a Lorentzian kernel, is shown to reliably model this redistribution in dependence of the energy-loss. Our study demonstrates that both the significant impact of inelastic scattering in low-angle diffraction at elevated specimen thickness and a rather straightforward model can be applied to mimic multiple plasmon scattering, which otherwise is currently not within reach for multislice simulations due to computational complexity.

2022 Scientific Article in Oncology Oncology 101, 126-133 (2022)

MALDI mass spectrometry imaging-Diagnostic pathways and metabolites for renal tumor entities.

Background: Correct tumor subtyping of primary renal tumors is essential for treatment decision in daily routine. Most of the tumors can be classified based on morphology alone. Nevertheless, some diagnoses are difficult, and further investigations are needed for correct tumor subtyping. Besides histochemical investigations, high-mass-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can detect new diagnostic biomarkers and hence improve the diagnostic. Patients and Methods: Formalin-fixed paraffin embedded tissue specimens from clear cell renal cell carcinoma (ccRCC, n = 552), papillary renal cell carcinoma (pRCC, n = 122), chromophobe renal cell carcinoma (chRCC, n = 108), and renal oncocytoma (rO, n = 71) were analyzed by high-mass-resolution MALDI fourier-transform ion cyclotron resonance (FT-ICR) MSI. The SPACiAL pipeline was executed for automated co-registration of histological and molecular features. Pathway enrichment and pathway topology analysis were performed to determine significant differences between RCC subtypes. Results: We discriminated the four histological subtypes (ccRCC, pRCC, chRCC, and rO) and established the subtype-specific pathways and metabolic profiles. rO showed an enrichment of pentose phosphate, taurine and hypotaurine, glycerophospholipid, amino sugar and nucleotide sugar, fructose and mannose, glycine, serine, and threonine pathways. ChRCC is defined by enriched pathways including the amino sugar and nucleotide sugar, fructose and mannose, glycerophospholipid, taurine and hypotaurine, glycine, serine, and threonine pathways. Pyrimidine, amino sugar and nucleotide sugar, glycerophospholipids, and glutathione pathways are enriched in ccRCC. Furthermore, we detected enriched phosphatidylinositol and glycerophospholipid pathways in pRCC. Conclusion: In summary, we performed a classification system with a mean accuracy in tumor discrimination of 85.13%. Furthermore, we detected tumor-specific biomarkers for the four most common primary renal tumors by MALDI-MSI. This method is a useful tool in differential diagnosis and biomarker detection.

2022 Scientific Article in Medical Image Analysis Med. Image Anal. 84:102680 (2022)

The Liver Tumor Segmentation Benchmark (LiTS).

In this work, we report the set-up and results of the Liver Tumor Segmentation Benchmark (LiTS), which was organized in conjunction with the IEEE International Symposium on Biomedical Imaging (ISBI) 2017 and the International Conferences on Medical Image Computing and Computer-Assisted Intervention (MICCAI) 2017 and 2018. The image dataset is diverse and contains primary and secondary tumors with varied sizes and appearances with various lesion-to-background levels (hyper-/hypo-dense), created in collaboration with seven hospitals and research institutions. Seventy-five submitted liver and liver tumor segmentation algorithms were trained on a set of 131 computed tomography (CT) volumes and were tested on 70 unseen test images acquired from different patients. We found that not a single algorithm performed best for both liver and liver tumors in the three events. The best liver segmentation algorithm achieved a Dice score of 0.963, whereas, for tumor segmentation, the best algorithms achieved Dices scores of 0.674 (ISBI 2017), 0.702 (MICCAI 2017), and 0.739 (MICCAI 2018). Retrospectively, we performed additional analysis on liver tumor detection and revealed that not all top-performing segmentation algorithms worked well for tumor detection. The best liver tumor detection method achieved a lesion-wise recall of 0.458 (ISBI 2017), 0.515 (MICCAI 2017), and 0.554 (MICCAI 2018), indicating the need for further research. LiTS remains an active benchmark and resource for research, e.g., contributing the liver-related segmentation tasks in http://medicaldecathlon.com/. In addition, both data and online evaluation are accessible via https://competitions.codalab.org/competitions/17094.

2022 Scientific Article in Small Small 19:e2205318 (2022)

Folate receptor-mediated delivery of Cas9 RNP for enhanced immune checkpoint disruption in cancer cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.

2022 Scientific Article in BioSpektrum BioSpektrum 28, 711-715 (2022)

Schallschalter: Photoschaltbare Reporter und Sensoren in der Optoakustik.

Optoacoustic imaging offers a unique combination of observation volume and achievable resolution in vivo. However, the use of transgene labeling agents has been impractical because their signal is low compared to the background of the tissue. Thus, detection was limited to large numbers of cells. We tackle this problem by using switchable agents — switching creates a signal modulation which allows to separate the labeled cells from the constant background — making it virtually invisible.

2022 Scientific Article in Frontiers in Oncology Front. Oncol. 12:925542 (2022)

Proton beam range verification by means of ionoacoustic measurements at clinically relevant doses using a correlation-based evaluation.

Purpose: The Bragg peak located at the end of the ion beam range is one of the main advantages of ion beam therapy compared to X-Ray radiotherapy. However, verifying the exact position of the Bragg peak within the patient online is a major challenge. The goal of this work was to achieve submillimeter proton beam range verification for pulsed proton beams of an energy of up to 220 MeV using ionoacoustics for a clinically relevant dose deposition of typically 2 Gy per fraction by i) using optimal proton beam characteristics for ionoacoustic signal generation and ii) improved signal detection by correlating the signal with simulated filter templates. Methods: A water tank was irradiated with a preclinical 20 MeV proton beam using different pulse durations ranging from 50 ns up to 1 μs in order to maximise the signal-to-noise ratio (SNR) of ionoacoustic signals. The ionoacoustic signals were measured using a piezo-electric ultrasound transducer in the MHz frequency range. The signals were filtered using a cross correlation-based signal processing algorithm utilizing simulated templates, which enhances the SNR of the recorded signals. The range of the protons is evaluated by extracting the time of flight (ToF) of the ionoacoustic signals and compared to simulations from a Monte Carlo dose engine (FLUKA). Results: Optimised SNR of 28.0 ± 10.6 is obtained at a beam current of 4.5 μA and a pulse duration of 130 ns at a total peak dose deposition of 0.5 Gy. Evaluated ranges coincide with Monte Carlo simulations better than 0.1 mm at an absolute range of 4.21 mm. Higher beam energies require longer proton pulse durations for optimised signal generation. Using the correlation-based post-processing filter a SNR of 17.8 ± 5.5 is obtained for 220 MeV protons at a total peak dose deposition of 1.3 Gy. For this clinically relevant dose deposition and proton beam energy, submillimeter range verification was achieved at an absolute range of 303 mm in water. Conclusion: Optimal proton pulse durations ensure an ideal trade-off between maximising the ionoacoustic amplitude and minimising dose deposition. In combination with a correlation-based post-processing evaluation algorithm, a reasonable SNR can be achieved at low dose levels putting clinical applications for online proton or ion beam range verification into reach.

2022 Scientific Article in IEEE Transactions on Computational Imaging IEEE Trans. Comput. Imaging 8, 996-1011 (2022)

Inverse multislice ptychography by layer-wise optimisation and sparse matrix decomposition.

We propose algorithms based on an optimisation method for inverse multislice ptychography in, e.g. electron microscopy. The multislice method is widely used to model the interaction between relativistic electrons and thick specimens. Since only the intensity of diffraction patterns can be recorded, the challenge in applying inverse multislice ptychography is to uniquely reconstruct the electrostatic potential in each slice up to some ambiguities. In this conceptual study, we show that a unique separation of atomic layers for simulated data is possible when considering a low acceleration voltage. We also introduce an adaptation for estimating the illuminating probe. For the sake of practical application, we finally present slice reconstructions using experimental 4D scanning transmission electron microscopy (STEM) data.

2022 Nature Nature 612:E7 (2022)

Publisher Correction: Stroke genetics informs drug discovery and risk prediction across ancestries.

2022 Scientific Article in Frontiers in Medicine Front. Med. 9:992993 (2022)

18 kDa translocator protein positron emission tomography facilitates early and robust tumor detection in the immunocompetent SB28 glioblastoma mouse model.

Introduction: The 18 kDa translocator protein (TSPO) receives growing interest as a biomarker in glioblastoma. Mouse models can serve as an important tool for the investigation of biomarkers in glioblastoma, but several glioblastoma models indicated only low TSPO-PET signals in contrast to high TSPO-PET signals of human glioblastoma. Thus, we aimed to investigate TSPO-PET imaging in the syngeneic immunocompetent SB28 mouse model, which is thought to closely represent the tumor microenvironment (TME) of human glioblastoma. Methods: Dynamic TSPO-PET/CT imaging was performed for 60 min after injection of 13.6 ± 4.2 MBq [18F]GE-180. Contrast enhanced CT (ceCT) was acquired prior to PET and served for assessment of tumor volumes and attenuation correction. SB28 and sham mice were imaged at an early (week-1; n = 6 SB28, n = 6 sham) and a late time-point (week-3; n = 8 SB28, n = 9 sham) after inoculation. Standard of truth ex vivo tumor volumes were obtained for SB28 mice at the late time-point. Tracer kinetics were analyzed for the lesion site and the carotid arteries to establish an image derived input function (IDIF). TSPO-PET and ceCT lesion volumes were compared with ex vivo volumes by calculation of root-mean-square-errors (RMSE). Volumes of distribution (VTmax/mean) in the lesion were calculated using carotid IDIF and standardized uptake values (SUVmax/mean) were obtained for a 40–60 min time frame. Results: Higher uptake rate constants (K1) were observed for week-1 SB28 tumor lesions when compared to week-3 SB28 tumor lesions. Highest agreement between TSPO-PET lesion volumes and ex vivo tumor volumes was achieved with a 50% maximum threshold (RMSE-VT: 39.7%; RMSE-SUV: 34.4%), similar to the agreement of ceCT tumor volumes (RMSE: 30.1%). Lesions of SB28 mice had higher PET signal when compared to sham mice at week-1 (VTmax 6.6 ± 2.9 vs. 3.9 ± 0.8, p = 0.035; SUVmax 2.3 ± 0.5 vs. 1.2 ± 0.1, p < 0.001) and PET signals remained at a similar level at week-3 (VTmax 5.0 ± 1.6 vs. 2.7 ± 0.8, p = 0.029; SUVmax 1.9 ± 0.5 vs. 1.2 ± 0.2, p = 0.0012). VTmax correlated with SUVmax (R2 = 0.532, p < 0.001). Conclusion: TSPO-PET imaging of immunocompetent SB28 mice facilitates early detection of tumor signals over sham lesions. SB28 tumors mirror high TSPO-PET signals of human glioblastoma and could serve as a valuable translational model to study TSPO as an imaging biomarker.

2022 Scientific Article in JCI insight JCI insight 7:e162138 (2022)

Spatial metabolomics reveals upregulation of several pyrophosphate-producing pathways in cortical bone of Hyp mice.

Patients with the renal phosphate-wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI-based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency-induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.

2022 Review in Biosensors Biosensors 12:901 (2022)

Image quality improvement techniques and assessment adequacy in clinical optoacoustic imaging: A systematic review.

Optoacoustic imaging relies on the detection of optically induced acoustic waves to offer new possibilities in morphological and functional imaging. As the modality matures towards clinical application, research efforts aim to address multifactorial limitations that negatively impact the resulting image quality. In an endeavor to obtain a clear view on the limitations and their effects, as well as the status of this progressive refinement process, we conduct an extensive search for optoacoustic image quality improvement approaches that have been evaluated with humans in vivo, thus focusing on clinically relevant outcomes. We query six databases (PubMed, Scopus, Web of Science, IEEE Xplore, ACM Digital Library, and Google Scholar) for articles published from 1 January 2010 to 31 October 2021, and identify 45 relevant research works through a systematic screening process. We review the identified approaches, describing their primary objectives, targeted limitations, and key technical implementation details. Moreover, considering comprehensive and objective quality assessment as an essential prerequisite for the adoption of such approaches in clinical practice, we subject 36 of the 45 papers to a further in-depth analysis of the reported quality evaluation procedures, and elicit a set of criteria with the intent to capture key evaluation aspects. Through a comparative criteria-wise rating process, we seek research efforts that exhibit excellence in quality assessment of their proposed methods, and discuss features that distinguish them from works with similar objectives. Additionally, informed by the rating results, we highlight areas with improvement potential, and extract recommendations for designing quality assessment pipelines capable of providing rich evidence.

2022 Scientific Article in Nature Cell Biology Nat. Cell Biol. 24, 1666-1676 (2022)

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs.

Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.

2022 Scientific Article in Journal of Biomedical Optics J. Biomed. Opt. 27:106004 (2022)

Deep learning methods hold promise for light fluence compensation in three-dimensional optoacoustic imaging.

SIGNIFICANCE: Quantitative optoacoustic imaging (QOAI) continues to be a challenge due to the influence of nonlinear optical fluence distribution, which distorts the optoacoustic image representation. Nonlinear optical fluence correction in OA imaging is highly ill-posed, leading to the inaccurate recovery of optical absorption maps. This work aims to recover the optical absorption maps using deep learning (DL) approach by correcting for the fluence effect. AIM: Different DL models were compared and investigated to enable optical absorption coefficient recovery at a particular wavelength in a nonhomogeneous foreground and background medium. APPROACH: Data-driven models were trained with two-dimensional (2D) Blood vessel and three-dimensional (3D) numerical breast phantom with highly heterogeneous/realistic structures to correct for the nonlinear optical fluence distribution. The trained DL models such as U-Net, Fully Dense (FD) U-Net, Y-Net, FD Y-Net, Deep residual U-Net (Deep ResU-Net), and generative adversarial network (GAN) were tested to evaluate the performance of optical absorption coefficient recovery (or fluence compensation) with in-silico and in-vivo datasets. RESULTS: The results indicated that FD U-Net-based deconvolution improves by about 10% over reconstructed optoacoustic images in terms of peak-signal-to-noise ratio. Further, it was observed that DL models can indeed highlight deep-seated structures with higher contrast due to fluence compensation. Importantly, the DL models were found to be about 17 times faster than solving diffusion equation for fluence correction. CONCLUSIONS: The DL methods were able to compensate for nonlinear optical fluence distribution more effectively and improve the optoacoustic image quality.

2022 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 41, 3182-3193 (2022)

Deep-learning-based electrical noise removal enables high spectral optoacoustic contrast in deep tissue.

Image contrast in multispectral optoacoustic tomography (MSOT) can be severely reduced by electrical noise and interference in the acquired optoacoustic signals. Previously employed signal processing techniques have proven insufficient to remove the effects of electrical noise because they typically rely on simplified models and fail to capture complex characteristics of signal and noise. Moreover, they often involve time-consuming processing steps that are unsuited for real-time imaging applications. In this work, we develop and demonstrate a discriminative deep learning approach to separate electrical noise from optoacoustic signals prior to image reconstruction. The proposed deep learning algorithm is based on two key features. First, it learns spatiotemporal correlations in both noise and signal by using the entire optoacoustic sinogram as input. Second, it employs training on a large dataset of experimentally acquired pure noise and synthetic optoacoustic signals. We validated the ability of the trained model to accurately remove electrical noise on synthetic data and on optoacoustic images of a phantom and the human breast. We demonstrate significant enhancements of morphological and spectral optoacoustic images reaching 19% higher blood vessel contrast and localized spectral contrast at depths of more than 2 cm for images acquired in vivo. We discuss how the proposed denoising framework is applicable to clinical multispectral optoacoustic tomography and suitable for real-time operation.

2022 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 41, 3373-3384 (2022)

Disentangling the frequency content in optoacoustics.

Signals acquired by optoacoustic tomography systems have broadband frequency content that encodes information about structures on different physical scales. Concurrent processing and rendering of such broadband signals may result in images with poor contrast and fidelity due to a bias towards low frequency contributions from larger structures. This problem cannot be addressed by filtering different frequency bands and reconstructing them individually, as this procedure leads to artefacts due to its incompatibility with the entangled frequency content of signals generated by structures of different sizes. Here we introduce frequency-band model-based (fbMB) reconstruction to separate frequency-band-specific optoacoustic image components during image formation, thereby enabling structures of all sizes to be rendered with high fidelity. In order to disentangle the overlapping frequency content of image components, fbMB uses soft priors to achieve an optimal trade-off between localization of the components in frequency bands and their structural integrity. We demonstrate that fbMB produces optoacoustic images with improved contrast and fidelity, which reveal anatomical structures in in vivo images of mice in unprecedented detail. These enhancements further improve the accuracy of spectral unmixing in small vasculature. By offering a precise treatment of the frequency components of optoacoustic signals, fbMB improves the quality, accuracy, and quantification of optoacoustic images and provides a method of choice for optoacoustic reconstructions.

2022 Scientific Article in Acta Neuropathologica Communications Acta Neuropathol. Commun. 10:129 (2022)

Purkinje cell vulnerability induced by diffuse traumatic brain injury is linked to disruption of long-range neuronal circuits.

Cerebellar dysfunction is commonly observed following traumatic brain injury (TBI). While direct impact to the cerebellum by TBI is rare, cerebellar pathology may be caused by indirect injury via cortico-cerebellar pathways. To address the hypothesis that degeneration of Purkinje cells (PCs), which constitute the sole output from the cerebellum, is linked to long-range axonal injury and demyelination, we used the central fluid percussion injury (cFPI) model of widespread traumatic axonal injury in mice. Compared to controls, TBI resulted in early PC loss accompanied by alterations in the size of pinceau synapses and levels of non-phosphorylated neurofilament in PCs. A combination of vDISCO tissue clearing technique and immunohistochemistry for vesicular glutamate transporter type 2 show that diffuse TBI decreased mossy and climbing fiber synapses on PCs. At 2 days post-injury, numerous axonal varicosities were found in the cerebellum supported by fractional anisotropy measurements using 9.4 T MRI. The disruption and demyelination of the cortico-cerebellar circuits was associated with poor performance of brain-injured mice in the beam-walk test. Despite a lack of direct input from the injury site to the cerebellum, these findings argue for novel long-range mechanisms causing Purkinje cell injury that likely contribute to cerebellar dysfunction after TBI.

2022 Review in European heart journal - cardiovascular imaging Eur. Heart J. Cardiovasc. Imaging 24, e1-e16 (2022)

Intravascular molecular imaging: Translating pathophysiology of atherosclerosis into human disease conditions.

Progression of atherosclerotic plaque in coronary arteries is characterized by complex cellular and non-cellular molecular interactions. Within recent years, atherosclerosis has been recognized as inflammation-driven disease condition, where progressive stages are characterized by morphological changes in plaque composition but also relevant molecular processes resulting in increased plaque vulnerability. While existing intravascular imaging modalities are able to resolve key morphological features during plaque progression, they lack capability to characterize the molecular profile of advanced atherosclerotic plaque. Because hybrid imaging modalities may provide incremental information related to plaque biology, they are expected to provide synergistic effects in detecting high risk patients and lesions. The aim of this article is to review existing literature on intravascular molecular imaging approaches, and to provide clinically oriented proposals of their application. In addition, we assembled an overview of future developments in this field geared towards detection of patients at risk for cardiovascular events.

2022 Review in Advanced Drug Delivery Reviews Adv. Drug Deliv. Rev. 189:114506 (2022)

The sound of drug delivery: Optoacoustic imaging in pharmacology.

Optoacoustic (photoacoustic) imaging offers unique opportunities for visualizing biological function in vivo by achieving high-resolution images of optical contrast much deeper than any other optical technique. The method detects ultrasound waves that are generated inside tissue by thermo-elastic expansion, i.e., the conversion of light absorption by tissue structures to ultrasound when the tissue is illuminated by the light of varying intensity. Listening instead of looking to light offers the major advantage of image formation with a resolution that obeys ultrasonic diffraction and not photon diffusion laws. While the technique has been widely used to explore contrast from endogenous photo-absorbing molecules, such as hemoglobin or melanin, the use of exogenous agents can extend applications to a larger range of biological and possible clinical applications, such as image-guided surgery, disease monitoring, and the evaluation of drug delivery, biodistribution, and kinetics. This review summarizes recent developments in optoacoustic agents, and highlights new functions visualized and potent pharmacology applications enabled with the use of external contrast agents.

2022 Scientific Article in Nature Communications Nat. Commun. 13:4689 (2022)

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus.

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.

2022 Scientific Article in Nature Communications Nat. Commun. 13:4448 (2022)

Frequency wavelength multiplexed optoacoustic tomography.

Optoacoustics (OA) is overwhelmingly implemented in the Time Domain (TD) to achieve high signal-to-noise ratios by maximizing the excitation light energy transient. Implementations in the Frequency Domain (FD) have been proposed, but suffer from low signal-to-noise ratios and have not offered competitive advantages over time domain methods to reach high dissemination. It is therefore commonly believed that TD is the optimal way to perform optoacoustics. Here we introduce an optoacoustic concept based on pulse train illumination and frequency domain multiplexing and theoretically demonstrate the superior merits of the approach compared to the time domain. Then, using recent advances in laser diode illumination, we launch Frequency Wavelength Multiplexing Optoacoustic Tomography (FWMOT), at multiple wavelengths, and experimentally showcase how FWMOT optimizes the signal-to-noise ratios of spectral measurements over time-domain methods in phantoms and in vivo. We further find that FWMOT offers the fastest multi-spectral operation ever demonstrated in optoacoustics.

2022 Scientific Article in Lecture Notes in Computer Science Lect. Notes Comput. Sc. 12963 LNCS, 383-391 (2022)

FedCostWAvg: A new averaging for better federated learning.

We propose a simple new aggregation strategy for federated learning that won the MICCAI Federated Tumor Segmentation Challenge 2021 (FETS), the first ever challenge on Federated Learning in the Machine Learning community. Our method addresses the problem of how to aggregate multiple models that were trained on different data sets. Conceptually, we propose a new way to choose the weights when averaging the different models, thereby extending the current state of the art (FedAvg). Empirical validation demonstrates that our approach reaches a notable improvement in segmentation performance compared to FedAvg.

2022 Review in Nature Protocols Nat. Protoc. 17, 2188-2215 (2022)

Scalable tissue labeling and clearing of intact human organs.

Advances in tissue labeling and clearing methods include improvement of tissue transparency, better preservation of fluorescence signal, compatibility with immunostaining and large sample volumes. However, as existing methods share the common limitation that they can only be applied to human tissue slices, rendering intact human organs transparent remains a challenge. Here, we describe experimental details of the small-micelle-mediated human organ efficient clearing and labeling (SHANEL) pipeline, which can be applied for cellular mapping of intact human organs. We have successfully cleared multiple human organs, including kidney, pancreas, heart, lung, spleen and brain, as well as hard tissue like skull. We also describe an advanced volumetric imaging system using a commercial light-sheet fluorescence microscope that can accommodate most human organs and a pipeline for whole-organ imaging and visualization. The complete experimental process of labeling and clearing whole human organs takes months and the analysis process takes several weeks, depending on the organ types and sizes.

2022 Scientific Article in Molecular Metabolism Mol. Metab. 66:101616 (2022)

Validation of Mct8/Oatp1c1 dKO mice as a model organism for the Allan-Herndon-Dudley Syndrome.

OBJECTIVE: The Allan-Herndon-Dudley syndrome (AHDS) is a severe disease caused by dysfunctional central thyroid hormone transport due to functional loss of the monocarboxylate transporter 8 (MCT8). In this study, we assessed whether mice with concomitant deletion of the thyroid hormone transporters Mct8 and the organic anion transporting polypeptide (Oatp1c1) represent a valid preclinical model organism for the AHDS. METHODS: We generated and metabolically characterized a new CRISPR/Cas9 generated Mct8/Oatp1c1 double-knockout (dKO) mouse line for the clinical features observed in patients with AHDS. RESULTS: We show that Mct8/Oatp1c1 dKO mice mimic key hallmarks of the AHDS, including decreased life expectancy, central hypothyroidism, peripheral hyperthyroidism, impaired neuronal myelination, impaired motor abilities and enhanced peripheral thyroid hormone action in the liver, adipose tissue, skeletal muscle and bone. CONCLUSIONS: We conclude that Mct8/Oatp1c1 dKO mice are a valuable model organism for the preclinical evaluation of drugs designed to treat the AHDS.

2022 Scientific Article in Cell Chemical Biology Cell Chem. Bio. 29, 1434-1445.e7 (2022)

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

2022 Scientific Article in Nature metabolism Nat. Metab. 4, 1071-1083 (2022)

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice.

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography-mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance.

2022 Scientific Article in Nanophotonics Nanophotonics 11, 4637–4647 (2022)

Multifunctional croconaine nanoparticles for efficient optoacoustic imaging of deep tumors and photothermal therapy.

The proper design of near-infrared light-absorbing agents enables efficient optoacoustic imaging-guided phototherapy. In particular, several croconaine-based organic agents with excellent optical properties have been recently reported for this purpose. However, most of them absorb light below 800 nm, limiting deep-tissue imaging applications. To this end, we utilized a recently described novel croconaine derivative (CR880) to develop CR880-based nanoparticles (CR880-NPs) for effective in vivo delivery, deep tissue optoacoustic imaging and photothermal therapy applications. Radicals and strong π-πstacking in CR880 result in an 880 nm absorption peak with no blue-shift upon condensing to the solid phase. DSPE-PEG2000-formulated CR880-NPs exhibited high optoacoustic generation efficiency and photostability, and could be visualized in the tumors of three different mouse tumor models (breast, brain, and colon tumor) with high image contrast. The high photothermal conversion efficiency of CR880-NPs (∼58%) subsequently enabled efficient in vivo tumor elimination using a low energy laser, while remaining biocompatible and well-tolerated. This work introduces a promising novel agent for cancer theranostics of challenging deep-seated tumors.

2022 Scientific Article in EBioMedicine EBioMedicine 85:104296 (2022)

The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling.

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript.

2022 Scientific Article in European Journal of Cancer Eur. J. Cancer 176, 41-49 (2022)

DeepClassPathway: Molecular pathway aware classification using explainable deep learning.

OBJECTIVE: HPV-associated head and neck cancer is correlated with favorable prognosis; however, its underlying biology is not fully understood. We propose an explainable convolutional neural network (CNN) classifier, DeepClassPathway, that predicts HPV-status and allows patient-specific identification of molecular pathways driving classifier decisions. METHODS: The CNN was trained to classify HPV-status on transcriptome data from 264 (13% HPV-positive) and tested on 85 (25% HPV-positive) head and neck squamous carcinoma patients after transformation into 2D-treemaps representing molecular pathways. Grad-CAM saliency was used to quantify pathways contribution to individual CNN decisions. Model stability was assessed by shuffling pathways within 2D-images. RESULTS: The classification performance of the CNN-ensembles achieved ROC-AUC/PR-AUC of 0.96/0.90 for all treemap variants. Quantification of the averaged pathway saliency heatmaps consistently identified KRAS, spermatogenesis, bile acid metabolism, and inflammation signaling pathways as the four most informative for classifying HPV-positive patients and MYC targets, epithelial-mesenchymal transition, and protein secretion pathways for HPV-negative patients. CONCLUSION: We have developed and applied an explainable CNN classification approach to transcriptome data from an oncology cohort with typical sample size that allows classification while accounting for the importance of molecular pathways in individual-level decisions.

2022 Editorial in Journal of Biomedical Optics J. Biomed. Opt. 27:074701 (2022)

Special section guest editorial: Tissue phantoms to advance biomedical optical systems.

The editorial introduces the JBO Special Section on Tissue Phantoms to Advance Biomedical Optical Systems.

2022 Scientific Article in Journal of Biophotonics J. Biophotonics 15:e202200032 (2022)

Enabling the autofocus approach for parameter optimization in planar measurement geometry clinical optoacoustic imaging.

In optoacoustic (photoacoustic) tomography, several parameters related to tissue and detector features are needed for image formation, but they may not be known a priori. An autofocus (AF) algorithm is generally used to estimate these parameters. However, the algorithm works iteratively and is therefore impractical for clinical imaging with planar geometry systems due to the long reconstruction times. We have developed a fast autofocus (FAF) algorithm for 3D optoacoustic systems with planar geometry. Such an algorithm exploits the symmetries of the planar geometry and a virtual source concept to reduce the dimensionality of the parameter estimation problem. The dimensionality reduction makes FAF much simpler computationally than the conventional AF algorithm. We show that the FAF algorithm required about 5 sec to provide accurate estimates of the speed of sound in simulated data and experimental data obtained using an imaging system that is poised to enter the clinic. The applicability of FAF for estimating other image formation parameters is discussed. We expect the FAF algorithm to contribute decisively to the clinical use of optoacoustic tomography systems with planar geometry.

2022 Scientific Article in Cancers Cancers 14:3745 (2022)

Integration of p16/HPV DNA status with a 24-miRNA-defined molecular phenotype improves clinically relevant stratification of head and neck cancer patients.

Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD-L1 expression/PD-1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.

2022 Scientific Article in Laboratory Investigation Lab. Invest. 102, 1400-1405 (2022)

Simultaneous metabolite MALDI-MSI, whole exome and transcriptome analysis from formalin-fixed paraffin-embedded tissue sections.

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows spatial analysis of proteins, metabolites, or small molecules from tissue sections. Here, we present the simultaneous generation and analysis of MALDI-MSI, whole-exome sequencing (WES), and RNA-sequencing data from the same formalin-fixed paraffin-embedded (FFPE) tissue sections. Genomic DNA and total RNA were extracted from (i) untreated, (ii) hematoxylin-eosin (HE) stained, and (iii) MALDI-MSI-analyzed FFPE tissue sections from three head and neck squamous cell carcinomas. MALDI-MSI data were generated by a time-of-flight analyzer prior to preprocessing and visualization. WES data were generated using a low-input protocol followed by detection of single-nucleotide variants (SNVs), tumor mutational burden, and mutational signatures. The transcriptome was determined using 3’-RNA sequencing and was examined for similarities and differences between processing stages. All data met the commonly accepted quality criteria. Besides SNVs commonly identified between differently processed tissues, FFPE-typical artifactual variants were detected. Tumor mutational burden was in the same range for tissues from the same patient and mutational signatures were highly overlapping. Transcriptome profiles showed high levels of correlation. Our data demonstrate that simultaneous molecular profiling of MALDI-MSI-processed FFPE tissue sections at the transcriptome and exome levels is feasible and reliable.

2022 Scientific Article in International Journal of Biological Sciences Int. J. Biol. Sci. 18, 5230-5240 (2022)

Inhibition of APOE potentiates immune checkpoint therapy for cancer.

Checkpoint immunotherapy is capable of unleashing T cells for controlling tumor, whereas it is destroyed by immunosuppressive myeloid cell. Apoprotein E (APOE) refers to a ligand in terms of the members of low-density lipoprotein (LDL) receptor family for mediating Apoprotein B-involving atherogenic lipoprotein clearance. Besides, tumor-infiltration macrophage can express APOE. The present study reported Apoe-/- mice to exhibit higher resistance toward the development of three types of carcinomas as compared with mice with wild type and to have greater responses to αPD-1 (anti-PD-1) immunotherapy. Moreover, treatment by exploiting APOE inhibitor (COG 133TFA, αAPOE) was capable of curbing tumor development and fostering regression if in combination of αPD-1. According to single-cell RNA sequencing (scRNA-seq), Apoe deletion was correlated with the decline of C1QC+ and CCR2+ macrophage within tumor infiltration, and mass spectrometry results noticeably showed down-regulated the number of M2 macrophages as well. Furthermore, APOE expression in cancer patients resistant to αPD-1 treatment significantly exceeded that in the sensitive group. For this reason, APOE is likely to be targeted for modifying tumor macrophage infiltrate and augmenting checkpoint immunotherapy.

2022 Scientific Article in Photoacoustics Photoacoustics 26:100362 (2022)

Multispectral optoacoustic tomography for in vivo detection of lymph node metastases in oral cancer patients using an EGFR-targeted contrast agent and intrinsic tissue contrast: A proof-of-concept study.

Oral cancer patients undergo diagnostic surgeries to detect occult lymph node metastases missed by preoperative structural imaging techniques. Reducing these invasive procedures that are associated with considerable morbidity, requires better preoperative detection. Multispectral optoacoustic tomography (MSOT) is a rapidly evolving imaging technique that may improve preoperative detection of (early-stage) lymph node metastases, enabling the identification of molecular changes that often precede structural changes in tumorigenesis. Here, we characterize the optoacoustic properties of cetuximab-800CW, a tumor-specific fluorescent tracer showing several photophysical properties that benefit optoacoustic signal generation. In this first clinical proof-of-concept study, we explore its use as optoacoustic to differentiate between malignant and benign lymph nodes. We characterize the appearance of malignant lymph nodes and show differences in the distribution of intrinsic chromophores compared to benign lymph nodes. In addition, we suggest several approaches to improve the efficiency of follow-up studies.

2022 Review in Medical Sciences Med. Sci. 10:23 (2022)

Genital wound repair and scarring.

Skin wound repair has been the central focus of clinicians and scientists for almost a century. Insights into acute and chronic wound healing as well as scarring have influenced and ameliorated wound treatment. Our knowledge of normal skin notwithstanding, little is known of acute and chronic wound repair of genital skin. In contrast to extra-genital skin, hypertrophic scarring is uncommon in genital tissue. Chronic wound healing disorders of the genitals are mostly confined to mucosal tissue diseases. This article will provide insights into the differences between extra-genital and genital skin with regard to anatomy, physiology and aberrant wound repair. In light of fundamental differences between genital and normal skin, it is recommended that reconstructive and esthetic surgery should exclusively be performed by specialists with profound expertise in genital wound repair.

2022 Scientific Article in Cerebral Cortex Cereb. Cortex 33, 1074-1089 (2022)

Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.

At present, many studies support the notion that after stroke, remote regions connected to the infarcted area are also affected and may contribute to functional outcome. In the present study, we have analyzed possible microanatomical alterations in pyramidal neurons from the contralesional hemisphere after induced stroke. We performed intracellular injections of Lucifer yellow in pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere in an ischemic stroke mouse model. A detailed 3-dimensional analysis of the neuronal complexity and morphological alterations of dendritic spines was then performed. Our results demonstrate that pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere show selective changes in their dendritic arbors, namely, less dendritic complexity of the apical dendritic arbor-but no changes in the basal dendritic arbor. In addition, we found differences in spine morphology in both apical and basal dendrites comparing the contralesional hemisphere with the lesional hemisphere. Our results show that pyramidal neurons of remote areas connected to the infarct zone exhibit a series of selective changes in neuronal complexity and morphological distribution of dendritic spines, supporting the hypothesis that remote regions connected to the peri-infarcted area are also affected after stroke.

2022 Scientific Article in Acta Neuropathologica Communications Acta Neuropathol. Commun. 10:51 (2022)

Mapping of neuroinflammation-induced hypoxia in the spinal cord using optoacoustic imaging.

Recent studies suggest that metabolic changes and oxygen deficiency in the central nervous system play an important role in the pathophysiology of multiple sclerosis (MS). In our present study, we investigated the changes in oxygenation and analyzed the vascular perfusion of the spinal cord in a rodent model of MS. We performed multispectral optoacoustic tomography of the lumbar spinal cord before and after an oxygen enhancement challenge in mice with experimental autoimmune encephalomyelitis (EAE), a model for MS. In addition, mice were transcardially perfused with lectin to label the vasculature and their spinal columns were optically cleared, followed by light sheet fluorescence microscopy. To analyze the angioarchitecture of the intact spine, we used VesSAP, a novel deep learning-based framework. In EAE mice, the spinal cord had lower oxygen saturation and hemoglobin concentration compared to healthy mice, indicating compromised perfusion of the spinal cord. Oxygen administration reversed hypoxia in the spinal cord of EAE mice, although the ventral region remained hypoxic. Additionally, despite the increased vascular density, we report a reduction in length and complexity of the perfused vascular network in EAE. Taken together, these findings highlight a new aspect of neuroinflammatory pathology, revealing a significant degree of hypoxia in EAE in vivo that is accompanied by changes in spinal vascular perfusion. The study also introduces optoacoustic imaging as a tractable technique with the potential to further decipher the role of hypoxia in EAE and to monitor it in MS patients.

2022 Scientific Article in Oecologia Australis Oecologia Australis 26, 326-338 (2022)

Inorganic nitrogen stimulates methane oxidation in coastal lagoon sediments.

Methane (CH4) oxidation is a critical process to reduce CH4 emissions from aquatic environments to the atmosphere. Considering the continuous increase in nitrogen in rivers, lakes, and lagoons from human sources, we re-evaluated the still controversial potential effect of inorganic nitrogen on CH4 oxidation. Here, we approached three shallow coastal lagoons that represent great environmental heterogeneity and used slurry sediments as a model system. The addition of ammonium chloride (NH4 Cl) and potassium nitrate (KNO3) significantly stimulated CH4 oxidation in the sediments of all studied lagoons, indicating the potential limitation of nitrogen for the growth of CH4 oxidizing bacteria. Our findings contrast to some previous reports, where ammonium and nitrate inhibited CH4 oxidation in sediments. Indeed, our experiment was performed in a more realistic range in relation to natural concentrations of inorganic nitrogen in aquatic systems (0.5 to 1 mM) and was opposed to extreme concentrations previously used (2 to 50 mM). Our results point to the need to further assess the connection between nitrogen inputs and CH4 budgets in aquatic sediments, considering the potential fuel for CH4 oxidation that may affect the global greenhouse gas balance.

2022 Scientific Article in Cancers Cancers 14:1763 (2022)

MALDI mass spectrometry imaging—prognostic pathways and metabolites for renal cell carcinomas.

High mass resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a suitable method for biomarker detection for several tumor entities. Renal cell carcinoma (RCC) is the seventh most common cancer type and accounts for more than 80% of all renal tumors. Prognostic biomarkers for RCC are still missing. Therefore, we analyzed a large, multicenter cohort including the three most common RCC subtypes (clear cell RCC (ccRCC), papillary RCC (pRCC) and chromophobe RCC (chRCC)) by MALDI for prognostic biomarker detection. MALDI-Fourier-transform ion cyclotron resonance (FT-ICR)-MSI analysis was performed for renal carcinoma tissue sections from 782 patients. SPACiAL pipeline was integrated for automated co-registration of histological and molecular features. Kaplan–Meier analyses with overall survival as endpoint were executed to determine the metabolic features associated with clinical outcome. We detected several pathways and metabolites with prognostic power for RCC in general and also for different RCC subtypes.

2022 Scientific Article in Photoacoustics Photoacoustics 26:100354 (2022)

Label-free analytic histology of carotid atherosclerosis by mid-infrared optoacoustic microscopy.

Background and aims: Analysis of atherosclerotic plaque composition is a vital tool for unraveling the pathological metabolic processes that contribute to plaque growth. Methods: We visualize the constitution of human carotid plaques by mid-infrared optoacoustic microscopy (MiROM), a method for label-free analytic histology that requires minimal tissue preparation, rapidly yielding large field-of-view en-face images with a resolution of a few micrometers. We imaged endarterectomy specimens (n = 3, 12 sections total) at specific vibrational modes, targeting carbohydrates, lipids and proteins. Additionally, we recorded spectra at selected tissue locations. We identified correlations in the variability in this high-dimensional data set using non-negative matrix factorization (NMF). Results: We visualized high-risk plaque features with molecular assignment. Consistent NMF components relate to different dominant tissue constituents, dominated by lipids, proteins, and cholesterol and carbohydrates respectively. Conclusions: These results introduce MiROM as an innovative, stain-free, analytic histology technology for the biochemical characterization of complex human vascular pathology.

2022 Scientific Article in Histochemistry and Cell Biology Histochem. Cell Biol. 157, 595–605 (2022)

A simple preparation step to remove excess liquid lipids in white adipose tissue enabling improved detection of metabolites via MALDI-FTICR imaging MS.

Matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) imaging mass spectrometry (MS) is a powerful technology used to analyze metabolites in various tissues. However, it faces significant challenges in studying adipose tissues. Poor matrix distribution and crystallization caused by excess liquid lipids on the surface of tissue sections hamper m/z species detection, an adverse effect that particularly presents in lipid-rich white adipose tissue (WAT). In this study, we integrated a simple and low-cost preparation step into the existing MALDI-FTICR imaging MS pipeline. The new method—referred to as filter paper application—is characterized by an easy sample handling and high reproducibility. The aforementioned filter paper is placed onto the tissue prior to matrix application in order to remove the layer of excess liquid lipids. Consequently, MALDI-FTICR imaging MS detection was significantly improved, resulting in a higher number of detected m/z species and higher ion intensities. After analyzing various durations of filter paper application, 30 s was found to be optimal, resulting in the detection of more than 3700 m/z species. Apart from the most common lipids found in WAT, other molecules involved in various metabolic pathways were detected, including nucleotides, carbohydrates, and amino acids. Our study is the first to propose a solution to a specific limitation of MALDI-FTICR imaging MS in investigating lipid-rich WAT. The filter paper approach can be performed quickly and is particularly effective for achieving uniform matrix distribution on fresh frozen WAT while maintaining tissue integrity. It thus helps to gain insight into the metabolism in WAT.

2022 Scientific Article in BMC Cancer BMC Cancer 22:254 (2022)

Combining gene expression analysis of gastric cancer cell lines and tumor specimens to identify biomarkers for anti-HER therapies-the role of HAS2, SHB and HBEGF.

BACKGROUND: The standard treatment for patients with advanced HER2-positive gastric cancer is a combination of the antibody trastuzumab and platin-fluoropyrimidine chemotherapy. As some patients do not respond to trastuzumab therapy or develop resistance during treatment, the search for alternative treatment options and biomarkers to predict therapy response is the focus of research. We compared the efficacy of trastuzumab and other HER-targeting drugs such as cetuximab and afatinib. We also hypothesized that treatment-dependent regulation of a gene indicates its importance in response and that it can therefore be used as a biomarker for patient stratification. METHODS: A selection of gastric cancer cell lines (Hs746T, MKN1, MKN7 and NCI-N87) was treated with EGF, cetuximab, trastuzumab or afatinib for a period of 4 or 24 h. The effects of treatment on gene expression were measured by RNA sequencing and the resulting biomarker candidates were tested in an available cohort of gastric cancer patients from the VARIANZ trial or functionally analyzed in vitro. RESULTS: After treatment of the cell lines with afatinib, the highest number of regulated genes was observed, followed by cetuximab and trastuzumab. Although trastuzumab showed only relatively small effects on gene expression, BMF, HAS2 and SHB could be identified as candidate biomarkers for response to trastuzumab. Subsequent studies confirmed HAS2 and SHB as potential predictive markers for response to trastuzumab therapy in clinical samples from the VARIANZ trial. AREG, EREG and HBEGF were identified as candidate biomarkers for treatment with afatinib and cetuximab. Functional analysis confirmed that HBEGF is a resistance factor for cetuximab. CONCLUSION: By confirming HAS2, SHB and HBEGF as biomarkers for anti-HER therapies, we provide evidence that the regulation of gene expression after treatment can be used for biomarker discovery. TRIAL REGISTRATION: Clinical specimens of the VARIANZ study (NCT02305043) were used to test biomarker candidates.

2022 Scientific Article in Photoacoustics Photoacoustics 26, 100343 (2022)

Image processing improvements afford second-generation handheld optoacoustic imaging of breast cancer patients.

Background: Since the initial breast transillumination almost a century ago, breast cancer imaging using light has been considered in different implementations aiming to improve diagnostics, minimize the number of available biopsies, or monitor treatment. However, due to strong photon scattering, conventional optical imaging yields low resolution images, challenging quantification and interpretation. Optoacoustic imaging addresses the scattering limitation and yields high-resolution visualization of optical contrast, offering great potential value for breast cancer imaging. Nevertheless, the image quality of experimental systems remains limited due to a number of factors, including signal attenuation with depth and partial view angle and motion effects, particularly in multi-wavelength measurements. Methods: We developed data analytics methods to improve the accuracy of handheld optoacoustic breast cancer imaging, yielding second-generation optoacoustic imaging performance operating in tandem with ultrasonography. Results: We produced the most advanced images yet with handheld optoacoustic examinations of the human breast and breast cancer, in terms of resolution and contrast. Using these advances, we examined optoacoustic markers of malignancy, including vasculature abnormalities, hypoxia, and inflammation, on images obtained from breast cancer patients. Conclusions: We achieved a new level of quality for optoacoustic images from a handheld examination of the human breast, advancing the diagnostic and theranostic potential of the hybrid optoacoustic-ultrasound (OPUS) examination over routine ultrasonography.

2022 Scientific Article in International Journal of Molecular Sciences Int. J. Mol. Sci. 23:2169 (2022)

Micro-RNA and proteomic profiles of plasma-derived exosomes from irradiated mice reveal molecular changes preventing apoptosis in neonatal cerebellum.

Cell communication via exosomes is capable of influencing cell fate in stress situations such as exposure to ionizing radiation. In vitro and in vivo studies have shown that exosomes might play a role in out-of-target radiation effects by carrying molecular signaling mediators of radiation damage, as well as opposite protective functions resulting in resistance to radiotherapy. However, a global understanding of exosomes and their radiation-induced regulation, especially within the context of an intact mammalian organism, has been lacking. In this in vivo study, we demonstrate that, compared to sham-irradiated (SI) mice, a distinct pattern of proteins and miRNAs is found packaged into circulating plasma exosomes after whole-body and partial-body irradiation (WBI and PBI) with 2 Gy X-rays. A high number of deregulated proteins (59% of WBI and 67% of PBI) was found in the exosomes of irradiated mice. In total, 57 and 13 miRNAs were deregulated in WBI and PBI groups, respectively, suggesting that the miRNA cargo is influenced by the tissue volume exposed to radiation. In addition, five miRNAs (miR-99b-3p, miR-200a-3p, miR-200a, miR-182-5p, miR-182) were commonly overexpressed in the exosomes from the WBI and PBI groups. In this study, particular emphasis was also given to the determination of the in vivo effect of exosome transfer by intracranial injection in the highly radiosensitive neonatal cerebellum at postnatal day 3. In accordance with a major overall anti-apoptotic function of the commonly deregulated miRNAs, here, we report that exosomes from the plasma of irradiated mice, especially in the case of WBI, prevent radiation-induced apoptosis, thus holding promise for exosome-based future therapeutic applications against radiation injury.

2022 Scientific Article in Cancer Cell Cancer Cell 40, 639-655.e13 (2022)

Spatially resolved multi-omics deciphers bidirectional tumor-host interdependence in glioblastoma.

Glioblastomas are malignant tumors of the central nervous system hallmarked by subclonal diversity and dynamic adaptation amid developmental hierarchies. The source of dynamic reorganization within the spatial context of these tumors remains elusive. Here, we characterized glioblastomas by spatially resolved transcriptomics, metabolomics, and proteomics. By deciphering regionally shared transcriptional programs across patients, we infer that glioblastoma is organized by spatial segregation of lineage states and adapts to inflammatory and/or metabolic stimuli, reminiscent of the reactive transformation in mature astrocytes. Integration of metabolic imaging and imaging mass cytometry uncovered locoregional tumor-host interdependence, resulting in spatially exclusive adaptive transcriptional programs. Inferring copy-number alterations emphasizes a spatially cohesive organization of subclones associated with reactive transcriptional programs, confirming that environmental stress gives rise to selection pressure. A model of glioblastoma stem cells implanted into human and rodent neocortical tissue mimicking various environments confirmed that transcriptional states originate from dynamic adaptation to various environments.

2022 Scientific Article in IEEE Robotics and Automation Letters IEEE Robot. Autom. Lett. 7, 6638-6645 (2022)

VesNet-RL: Simulation-based reinforcement learning for real-world US probe navigation.

Ultrasound (US) is one of the most common medical imaging modalities since it is radiation-free, low-cost, and real-time. In freehand US examinations, sonographers often navigate a US probe to visualize standard examination planes with rich diagnostic information. However, reproducibility and stability of the resulting images often suffer from intra- and inter-operator variation. Reinforcement learning (RL), as an interaction-based learning method, has demonstrated its effectiveness in visual navigating tasks; however, RL is limited in terms of generalization. To address this challenge, we propose a simulation-based RL framework for real-world navigation of US probes towards the standard longitudinal views of vessels. A UNet is used to provide binary masks from US images; thereby, the RL agent trained on simulated binary vessel images can be applied in real scenarios without further training. To accurately characterize actual states, a multi-modality state representation structure is introduced to facilitate the understanding of environments. Moreover, considering the characteristics of vessels, a novel standard view recognition approach based on the minimum bounding rectangle is proposed to terminate the searching process. To evaluate the effectiveness of the proposed method, the trained policy is validated virtually on 3D volumes of a volunteer’s in-vivo carotid artery, and physically on custom-designed gel phantoms using robotic US. The results demonstrate that proposed approach can effectively and accurately navigate the probe towards the longitudinal view of vessels.

2022 Scientific Article in Annals of Translational Medicine Ann. Transl. Med. 10:625 (2022)

A 3D multi-modal intelligent intervention system using electromagnetic navigation for real-time positioning and ultrasound images: A prospective randomized controlled trial.

Background: Anesthesia, nerve block, therapeutic injections, and biopsies all require an acupuncture intervention. However, traditional two-dimensional (2D) ultrasound-guided needle puncture is often challenging and therefore requires the use of three-dimensional (3D) ultrasound images to accurately identify and evaluate the patient’s anatomical structure. Methods: In this study, a 3D multi-modal intelligent intervention system using electromagnetic navigation for real-time positioning and ultrasound images was described. A total of 190 cases requiring puncture were randomly divided into control (conventional 2D ultrasound instrument) and experimental (novel 3D ultrasound imedis9000) groups. The advantages and disadvantages of the two puncture methods were prospectively analyzed in the 190 cases, and the feasibility of electromagnetic navigation real-time positioning was compared to ultrasound imaging. Results: This study included 190 cases from two centers that required puncture treatment and were randomly assigned to the control (conventional 2D ultrasound instrument; n=95) or the experimental (novel 3D ultrasound imedis9000; n=95) groups. Percutaneous vascular puncture, percutaneous biopsy, percutaneous bile duct puncture, thoracic paravertebral nerve block, and sciatic nerve block operations were performed separately. The results indicated that the puncture time and number of trials in the experimental group were significantly lower than those in the control group. No significant difference was identified in the basic vital signs between the two groups before and after surgery. The success rate of the novel 3D ultrasound imedis9000 was 100%, and the success rate of the conventional 2D ultrasound instrument was 95.7%. Furthermore, the results also showed that the novel 3D ultrasound imedis9000 and the matching coaxial positioning channel puncture needle had low pain, good toughness and strength, and great convenience. Conclusions: The new 3D multi-modal intelligent intervention system using electromagnetic navigation real-time positioning and ultrasound images has significant advantages compared with conventional 2D ultrasound in terms of puncture time, number of trials, operation difficulty, and convenience, and is worthy of further promotion and use in clinics. Trial Registration: Beijing Municipal Drug Administration, 20190015.

2022 Scientific Article in Cancers Cancers 14:2462 (2022)

Validation of novel molecular imaging targets identified by functional genomic mRNA profiling to detect dysplasia in Barrett's Esophagus.

Barrett's esophagus (BE) is the precursor of esophageal adenocarcinoma (EAC). Dysplastic BE (DBE) has a higher progression risk to EAC compared to non-dysplastic BE (NDBE). However, the miss rates for the endoscopic detection of DBE remain high. Fluorescence molecular endoscopy (FME) can detect DBE and mucosal EAC by highlighting the tumor-specific expression of proteins. This study aimed to identify target proteins suitable for FME. Publicly available RNA expression profiles of EAC and NDBE were corrected by functional genomic mRNA (FGmRNA) profiling. Following a class comparison between FGmRNA profiles of EAC and NDBE, predicted, significantly upregulated genes in EAC were prioritized by a literature search. Protein expression of prioritized genes was validated by immunohistochemistry (IHC) on DBE and NDBE tissues. Near-infrared fluorescent tracers targeting the proteins were developed and evaluated ex vivo on fresh human specimens. In total, 1976 overexpressed genes were identified in EAC (n = 64) compared to NDBE (n = 66) at RNA level. Prioritization and IHC validation revealed SPARC, SULF1, PKCι, and DDR1 (all p < 0.0001) as the most attractive imaging protein targets for DBE detection. Newly developed tracers SULF1-800CW and SPARC-800CW both showed higher fluorescence intensity in DBE tissue compared to paired non-dysplastic tissue. This study identified SPARC, SULF1, PKCι, and DDR1 as promising targets for FME to differentiate DBE from NDBE tissue, for which SULF1-800CW and SPARC-800CW were successfully ex vivo evaluated. Clinical studies should further validate these findings.

2022 Scientific Article in Numerical Functional Analysis and Optimization Numer. Funct. Anal. Optim. 43, 755-795 (2022)

Recovery of atomic measures on the unit sphere.

We study the problem of recovering an atomic measure on the unit 2-sphere (Formula presented.) given finitely many moments with respect to spherical harmonics. The analysis relies on the formulation of this problem as an optimization problem on the space of bounded Borel measures on (Formula presented.) as it was considered by Y. de Castro & F. Gamboa (J. Math. Anal. Appl. 395(1):336–354, 2012) and E. Candés & C. Fernandez-Granda (J. Fourier Anal. Appl. 19(6):1229–1254, 2013). We construct a dual certificate using a kernel given in an explicit form and make a concrete analysis of the interpolation problem. Numerical examples are provided and analyzed.

2022 Scientific Article in Methods in Molecular Biology Methods Mol. Biol. 2419, 747-763 (2022)

Tissue clearing approaches in atherosclerosis.

Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.

2022 Scientific Article in Cells Cells 11:775 (2022)

Combining HDAC and MEK inhibitors with radiation against glioblastoma-derived spheres.

Glioblastoma stem-like cells (GSLCs) in glioblastoma limit effective treatment and promote therapeutic resistance and tumor recurrence. Using a combined radiation and drugscreening platform, we tested the combination of a histone deacetylase inhibitor (HDACi) and MAPK/ERK kinase inhibitor (MEKi) with radiation to predict the efficacy against GSLCs. To mimic a stem-like phenotype, glioblastoma-derived spheres were used and treated with a combination of HDACi (MS-275) and MEKi (TAK-733 or trametinib) with 4 Gy irradiation. The sphere-forming ability after the combined radiochemotherapy was investigated using a sphere formation assay, while the expression levels of the GSLC markers (CD44, Nestin and SOX2) after treatment were analyzed using Western blotting and flow cytometry. The combined radiochemotherapy treatment inhibited the sphere formation in both glioblastoma-derived spheres, decreased the expression of the GSLC markers in a cell-line dependent manner and increased the dead cell population. Finally, we showed that the combined treatment with radiation was more effective at reducing the GSLC markers compared to the standard treatment of temozolomide and radiation. These results suggest that combining HDAC and MEK inhibition with radiation may offer a new strategy to improve the treatment of glioblastoma.

2022 Scientific Article in Journal of Biomedical Optics J. Biomed. Opt. 27:074704 (2022)

Standardization and implementation of fluorescence molecular endoscopy in the clinic.

SIGNIFICANCE: Near-infrared fluorescence molecular endoscopy (NIR-FME) is an innovative technique allowing for in vivo visualization of molecular processes in hollow organs. Despite its potential for clinical translation, NIR-FME still faces challenges, for example, the lack of consensus in performing quality control and standardization of procedures and systems. This may hamper the clinical approval of the technology by authorities and its acceptance by endoscopists. Until now, several clinical trials using NIR-FME have been performed. However, most of these trials had different study designs, making comparison difficult. AIM: We describe the need for standardization in NIR-FME, provide a pathway for setting up a standardized clinical study, and describe future perspectives for NIR-FME. Body: Standardization is challenging due to many parameters. Invariable parameters refer to the hardware specifications. Variable parameters refer to movement or tissue optical properties. Phantoms can be of aid when defining the influence of these variables or when standardizing a procedure. CONCLUSION: There is a need for standardization in NIR-FME and hurdles still need to be overcome before a widespread clinical implementation of NIR-FME can be realized. When these hurdles are overcome, clinical outcomes can be compared and systems can be benchmarked, enabling clinical implementation.

2022 Scientific Article in Photoacoustics Photoacoustics 25:100333 (2022)

360º optoacoustic capsule endoscopy at 50 Hz for esophageal imaging.

Gastrointestinal (GI) endoscopy is a common medical diagnostic procedure used for esophageal cancer detection. Current emerging capsule optoacoustic endoscopes, however, suffer from low pulse repetition rates and slow scanning units limit attainable imaging frame rates. Consequently, motion artifacts result in inaccurate spatial mapping and misinterpretation of data. To overcome these limitations, we report a 360º, 50 Hz frame rate, distal scanning capsule optoacoustic endoscope. The translational capability of the instrument for human GI tract imaging was characterized with an Archimedean spiral phantom consisting of twelve 100 µm sutures, a stainless steel mesh with a pitch of 3 mm and an ex vivo pig esophagus sample. We estimated an imaging penetration depth of ~0.84 mm in vivo by immersing the mesh phantom in intralipid solution to simulate light scattering in human esophageal tissue and validated our findings ex vivo using pig esophagus. This proof-of-concept study demonstrates the translational potential of the proposed video-rate endoscope for human GI tract imaging.

2022 Review in Pharmaceutics Pharmaceutics 14:362 (2022)

Design principles governing the development of theranostic anticancer agents and their nanoformulations with photoacoustic properties.

The unmet need to develop novel approaches for cancer diagnosis and treatment has led to the evolution of theranostic agents, which usually include, in addition to the anticancer drug, an imaging agent based mostly on fluorescent agents. Over the past few years, a non-invasive pho-toacoustic imaging modality has been effectively integrated into theranostic agents. Herein, we shed light on the design principles governing the development of theranostic agents with photoa-coustic properties, which can be formulated into nanocarriers to enhance their potency. Specifi-cally, we provide an extensive analysis of their individual constituents including the imaging dyes, drugs, linkers, targeting moieties, and their formulation into nanocarriers. Along these lines, we present numerous relevant paradigms. Finally, we discuss the clinical relevance of the specific strategy, as also the limitations and future perspectives, and through this review, we envisage paving the way for the development of theranostic agents endowed with photoacoustic properties as effective anticancer medicines.

2022 Scientific Article in Cancer communications Cancer Comm. 42, 517-535 (2022)

Spatial metabolomics for evaluating response to neoadjuvant therapy in non-small cell lung cancer patients.

BACKGROUND: The response to neoadjuvant chemotherapy (NAC) differs substantially among individual patients with non-small cell lung cancer (NSCLC). Major pathological response (MPR) is a histomorphological read-out used to assess treatment response and prognosis in patients NSCLC after NAC. Although spatial metabolomics is a promising tool for evaluating metabolic phenotypes, it has not yet been utilized to assess therapy responses in patients with NSCLC. We evaluated the potential application of spatial metabolomics in cancer tissues to assess the response to NAC, using a metabolic classifier that utilizes mass spectrometry imaging combined with machine learning. METHODS: Resected NSCLC tissue specimens obtained after NAC (n = 88) were subjected to high-resolution mass spectrometry, and these data were used to develop an approach for assessing the response to NAC in patients with NSCLC. The specificities of the generated tumor cell and stroma classifiers were validated by applying this approach to a cohort of biologically matched chemotherapy-naïve patients with NSCLC (n = 85). RESULTS: The developed tumor cell metabolic classifier stratified patients into different prognostic groups with 81.6% accuracy, whereas the stroma metabolic classifier displayed 78.4% accuracy. By contrast, the accuracies of MPR and TNM staging for stratification were 62.5% and 54.1%, respectively. The combination of metabolic and MPR classifiers showed slightly lower accuracy than either individual metabolic classifier. In multivariate analysis, metabolic classifiers were the only independent prognostic factors identified (tumor: P = 0.001, hazards ratio [HR] = 3.823, 95% confidence interval [CI] = 1.716-8.514; stroma: P = 0.049, HR = 2.180, 95% CI = 1.004-4.737), whereas MPR (P = 0.804; HR = 0.913; 95% CI = 0.445-1.874) and TNM staging (P = 0.078; HR = 1.223; 95% CI = 0.977-1.550) were not independent prognostic factors. Using Kaplan-Meier survival analyses, both tumor and stroma metabolic classifiers were able to further stratify patients as NAC responders (P < 0.001) and non-responders (P < 0.001). CONCLUSIONS: Our findings indicate that the metabolic constitutions of both tumor cells and the stroma are valuable additions to the classical histomorphology-based assessment of tumor response.

2022 Scientific Article in Journal of Biophotonics J. Biophotonics 15:e202100334 (2022)

Weighted model-based optoacoustic reconstruction for partial-view geometries.

Acoustic heterogeneities in biological samples are known to cause artefacts in tomographic optoacoustic (photoacoustic) image reconstruction. A statistical weighted model-based reconstruction approach was previously introduced to mitigate such artefacts. However, this approach does not reliably provide high-quality reconstructions for partial-view imaging systems, which are common in preclinical and clinical optoacoustics. In this paper, the capability of the weighted model-based algorithm is extended to generate optoacoustic reconstructions with less distortions for partial-view geometry data. This is achieved by manipulating the weighting scheme based on the detector geometry. Using partial-view optoacoustic tomography data from a tissue-mimicking phantom containing a strong acoustic reflector, tumors grafted onto mice, and a mouse brain with intact skull, the proposed partial-view-corrected weighted model-based algorithm is shown to mitigate reflection artefacts in reconstructed images without distorting structures or boundaries, compared to both conventional model-based and the weighted model-based algorithms. It is also demonstrated that the partial-view-corrected weighted model-based algorithm has the additional advantage of suppressing streaking artefacts due to the partial-view geometry itself in the presence of a very strong optoacoustic chromophore. Due to its enhanced performance, the partial-view-corrected weighted model-based algorithm may prove useful for improving the quality of partial-view multispectral optoacoustic tomography, leading to enhanced visualization of functional parameters such as tissue oxygenation. This article is protected by copyright. All rights reserved.

2022 Scientific Article in Science Translational Medicine Sci. Transl. Med. 14:eabm8059 (2022)

Enabling precision monitoring of psoriasis treatment by optoacoustic mesoscopy.

Psoriasis is a widespread inflammatory skin disease affecting about 2% of the general population. Recently, treatments that specifically target key proinflammatory cytokines driving the disease have been developed to complement conventional therapies with unspecific antiproliferative or anti-inflammatory effects. Efficient monitoring of treatment efficacy in the context of precision medicine and the assessment of new therapeutics require accurate noninvasive readouts of disease progression. However, characterization of psoriasis treatment remains subjective based on visual and palpatory clinical assessment of features observed on the skin surface. We hypothesized that optoacoustic (photoacoustic) mesoscopy could offer label-free assessment of inflammation biomarkers, extracted from three-dimensional (3D) high-resolution images of the human skin, not attainable by other noninvasive methods. We developed a second-generation ultra-broadband optoacoustic mesoscopy system, featuring sub-10-μm resolution and advanced motion correction technology, and performed 80 longitudinal measurements of 20 psoriatic skin plaques in humans under conventional inpatient treatment or receiving biologics with concomitant topical corticosteroid treatment. Optoacoustic image analysis revealed inflammatory and morphological skin features that indicated treatment efficacy with sensitivity, accuracy, and precision that was not possible using clinical metrics. We identify 3D imaging biomarkers that reveal responses to treatment and offer the potential to facilitate disease and treatment characterization. Our findings suggest that optoacoustic mesoscopy may offer a method of choice for yielding both qualitative and quantitative evaluations of skin treatments that are inaccessible by other methods, potentially enabling optimized therapies and precision medicine in dermatology.

2022 Scientific Article in Nature Communications Nat. Commun. 13:2803 (2022)

Fast raster-scan optoacoustic mesoscopy enables assessment of human melanoma microvasculature in vivo.

Melanoma is associated with angiogenesis and vascular changes that may extend through the entire skin depth. Three-dimensional imaging of vascular characteristics in skin lesions could therefore allow diagnostic insights not available by conventional visual inspection. Raster-scan optoacoustic mesoscopy (RSOM) images microvasculature through the entire skin depth with resolutions of tens of micrometers; however, current RSOM implementations are too slow to overcome the strong breathing motions on the upper torso where melanoma lesions commonly occur. To enable high-resolution imaging of melanoma vasculature in humans, we accelerate RSOM scanning using an illumination scheme that is coaxial with a high-sensitivity ultrasound detector path, yielding 15 s single-breath-hold scans that minimize motion artifacts. We apply this Fast RSOM to image 10 melanomas and 10 benign nevi in vivo, showing marked differences between malignant and benign lesions, supporting the possibility to use biomarkers extracted from RSOM imaging of vasculature for lesion characterization to improve diagnostics.

2022 Scientific Article in Nature Communications Nat. Commun. 13:2659 (2022)

Selective plasticity of callosal neurons in the adult contralesional cortex following murine traumatic brain injury.

Traumatic brain injury (TBI) results in deficits that are often followed by recovery. The contralesional cortex can contribute to this process but how distinct contralesional neurons and circuits respond to injury remains to be determined. To unravel adaptations in the contralesional cortex, we used chronic in vivo two-photon imaging. We observed a general decrease in spine density with concomitant changes in spine dynamics over time. With retrograde co-labeling techniques, we showed that callosal neurons are uniquely affected by and responsive to TBI. To elucidate circuit connectivity, we used monosynaptic rabies tracing, clearing techniques and histology. We demonstrate that contralesional callosal neurons adapt their input circuitry by strengthening ipsilateral connections from pre-connected areas. Finally, functional in vivo two-photon imaging demonstrates that the restoration of pre-synaptic circuitry parallels the restoration of callosal activity patterns. Taken together our study thus delineates how callosal neurons structurally and functionally adapt following a contralateral murine TBI.

2022 Scientific Article in Medical Image Analysis Med. Image Anal. 77:102364 (2022)

Clinical validation of saliency maps for understanding deep neural networks in ophthalmology.

Deep neural networks (DNNs) have achieved physician-level accuracy on many imaging-based medical diagnostic tasks, for example classification of retinal images in ophthalmology. However, their decision mechanisms are often considered impenetrable leading to a lack of trust by clinicians and patients. To alleviate this issue, a range of explanation methods have been proposed to expose the inner workings of DNNs leading to their decisions. For imaging-based tasks, this is often achieved via saliency maps. The quality of these maps are typically evaluated via perturbation analysis without experts involved. To facilitate the adoption and success of such automated systems, however, it is crucial to validate saliency maps against clinicians. In this study, we used three different network architectures and developed ensembles of DNNs to detect diabetic retinopathy and neovascular age-related macular degeneration from retinal fundus images and optical coherence tomography scans, respectively. We used a variety of explanation methods and obtained a comprehensive set of saliency maps for explaining the ensemble-based diagnostic decisions. Then, we systematically validated saliency maps against clinicians through two main analyses — a direct comparison of saliency maps with the expert annotations of disease-specific pathologies and perturbation analyses using also expert annotations as saliency maps. We found the choice of DNN architecture and explanation method to significantly influence the quality of saliency maps. Guided Backprop showed consistently good performance across disease scenarios and DNN architectures, suggesting that it provides a suitable starting point for explaining the decisions of DNNs on retinal images.

2022 Scientific Article in Engineering in Life Sciences Eng. Life Sci. 22, 100-114 (2022)

Exploitation of the ribosomal protein L10 R98S mutation to enhance recombinant protein production in mammalian cells.

Mammalian cells are commonly used to produce recombinant protein therapeutics, but suffer from a high cost per mg of protein produced. There is therefore great interest in improving protein yields to reduce production cost. We present an entirely novel approach to reach this goal through direct engineering of the cellular translation machinery by introducing the R98S point mutation in the catalytically essential ribosomal protein L10 (RPL10-R98S). Our data support that RPL10-R98S enhances translation levels and fidelity and reduces proteasomal activity in lymphoid Ba/F3 and Jurkat cell models. In HEK293T cells cultured in chemically defined medium, knock-in of RPL10-R98S was associated with a 1.7- to 2.5-fold increased production of four transiently expressed recombinant proteins and 1.7-fold for one out of two stably expressed proteins. In CHO-S cells, eGFP reached a 2-fold increased expression under stable but not transient conditions, but there was no production benefit for monoclonal antibodies. The RPL10-R98S associated production gain thus depends on culture conditions, cell type, and the nature of the expressed protein. Our study demonstrates the potential for using a ribosomal protein mutation for pharmaceutical protein production gains, and further research on how various factors influence RPL10-R98S phenotypes can maximize its exploitability for the mammalian protein production industry.

2022 Scientific Article in EJNMMI Research EJNMMI Res. 12:2 (2022)

CXCR4 peptide-based fluorescence endoscopy in a mouse model of Barrett's esophagus.

BACKGROUND: Near-infrared (NIR) fluorescence imaging has been emerging as a promising strategy to overcome the high number of early esophageal adenocarcinomas missed by white light endoscopy and random biopsy collection. We performed a preclinical assessment of fluorescence imaging and endoscopy using a novel CXCR4-targeted fluorescent peptide ligand in the L2-IL1B mouse model of Barrett's esophagus. METHODS: Six L2-IL1B mice with advanced stage of disease (12-16 months old) were injected with the CXCR4-targeted, Sulfo-Cy5-labeled peptide (MK007), and ex vivo wide-field imaging of the whole stomach was performed 4 h after injection. Before ex vivo imaging, fluorescence endoscopy was performed in three L2-IL1B mice (12-14 months old)  by a novel imaging system with two L2-IL1B mice used as negative controls. RESULTS: Ex vivo imaging and endoscopy in L2-IL1B mice showed that the CXCR4-targeted MK007 accumulated mostly in the dysplastic lesions with a mean target-to-background ratio > 2. The detection of the Sulfo-Cy5 signal in dysplastic lesions and its co-localization with CXCR4 stained cells  by confocal microscopy further confirmed the imaging results. CONCLUSIONS: This preliminary preclinical study shows that CXCR4-targeted fluorescence endoscopy using MK007 can detect dysplastic lesions in a mouse model of Barrett's esophagus. Further investigations are needed to assess its use in the clinical setting.

2022 Review in Journal of Nuclear Medicine J. Nucl. Med. 63, 640-645 (2022)

A guideline for clinicians performing clinical studies with fluorescence imaging.

Fluorescence imaging is an emerging imaging technique that has shown many benefits for clinical care. Currently, the field is in rapid clinical translation, and an unprecedented number of clinical trials are performed. Clinicians are inundated with numerous opportunities and combinations of different imaging modalities. To streamline this process, a multidisciplinary approach is needed with drug discovery, software and systems engineering, and translational medicine. Here, we discuss the main constituents of a uniform fluorescence imaging protocol to match the clinical need and ensure consistent study designs and reliable data collection in clinical trials. In an era in which the potential of fluorescence imaging has become evident, consistent conduct of studies, data analysis, and data interpretation is essential for implementation into the standard of care.

2022 Scientific Article in Nanomedicine Nanomed. 40:102511 (2022)

Dynamic tracing using ultra-bright labelling and multi-photon microscopy identifies endothelial uptake of poloxamer 188 coated poly(lactic-co-glycolic acid) nano-carriers in vivo.

The potential of poly(lactic-co-glycolic acid) (PLGA) to design nanoparticles (NPs) and target the central nervous system remains to be exploited. In the current study we designed fluorescent 70-nm PLGA NPs, loaded with bulky fluorophores, thereby making them significantly brighter than quantum dots in single-particle fluorescence measurements. The high brightness of NPs enabled their visualization by intravital real-time 2-photon microscopy. Subsequently, we found that PLGA NPs coated with pluronic F-68 circulated in the blood substantially longer than uncoated NPs and were taken up by cerebro-vascular endothelial cells. Additionally, confocal microscopy revealed that coated PLGA NPs were present in late endothelial endosomes of cerebral vessels within 1hour after systemic injection and were more readily taken up by endothelial cells in peripheral organs. The combination of ultra-bright NPs and in vivo imaging may thus represent a promising approach to reduce the gap between development and clinical application of nanoparticle-based drug carriers.

2022 Scientific Article in Clinical and translational medicine Clin. Transl. Med. 12:e666 (2022)

The synergism of spatial metabolomics and morphometry improves machine learning-based renal tumour subtype classification.

2022 Scientific Article in Nature Communications Nat. Commun. 13:1589 (2022)

Adrenal tropism of SARS-CoV-2 and adrenal findings in a post-mortem case series of patients with severe fatal COVID-19.

Progressive respiratory failure and hyperinflammatory response is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. Despite mounting evidence of disruption of the hypothalamus-pituitary-adrenal axis in COVID-19, relatively little is known about the tropism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to adrenal glands and associated changes. Here we demonstrate adrenal viral tropism and replication in COVID-19 patients. Adrenal glands showed inflammation accompanied by inflammatory cell death. Histopathologic analysis revealed widespread microthrombosis and severe adrenal injury. In addition, activation of the glycerophospholipid metabolism and reduction of cortisone intensities were characteristic for COVID-19 specimens. In conclusion, our autopsy series suggests that SARS-CoV-2 facilitates the induction of adrenalitis. Given the central role of adrenal glands in immunoregulation and taking into account the significant adrenal injury observed, monitoring of developing adrenal insufficiency might be essential in acute SARS-CoV-2 infection and during recovery.

2022 Scientific Article in Cell Metabolism Cell Metab. 34, 473-486.e9 (2022)

A macrophage-hepatocyte glucocorticoid receptor axis coordinates fasting ketogenesis.

Fasting metabolism and immunity are tightly linked; however, it is largely unknown how immune cells contribute to metabolic homeostasis during fasting in healthy subjects. Here, we combined cell-type-resolved genomics and computational approaches to map crosstalk between hepatocytes and liver macrophages during fasting. We identified the glucocorticoid receptor (GR) as a key driver of fasting-induced reprogramming of the macrophage secretome including fasting-suppressed cytokines and showed that lack of macrophage GR impaired induction of ketogenesis during fasting as well as endotoxemia. Mechanistically, macrophage GR suppressed the expression of tumor necrosis factor (TNF) and promoted nuclear translocation of hepatocyte GR to activate a fat oxidation/ketogenesis-related gene program, cooperatively induced by GR and peroxisome proliferator-activated receptor alpha (PPARα) in hepatocytes. Together, our results demonstrate how resident liver macrophages directly influence ketogenesis in hepatocytes, thereby also outlining a strategy by which the immune system can set the metabolic tone during inflammatory disease and infection.

2022 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 28, 2865-2877 (2022)

Spatial metabolomics identifies distinct tumor-specific subtypes in gastric cancer patients.

PURPOSE: Current systems of gastric cancer (GC) molecular classification include genomic, molecular, and morphological features. GC classification based on tissue metabolomics remains lacking. This study aimed to define metabolically distinct GC subtypes and identify their clinicopathological and molecular characteristics. EXPERIMENTAL DESIGN: Spatial metabolomics by high mass resolution imaging mass spectrometry was performed in 362 GC patients. K-means clustering was used to define tumor and stroma-related subtypes based on tissue metabolites. The identified subtypes were linked with clinicopathological characteristics, molecular features, and metabolic signatures. Responses to trastuzumab treatment were investigated across the subtypes by introducing an independent patient cohort with HER2-positive GC from a multicenter observational study. RESULTS: Three tumor- and three stroma-specific subtypes with distinct tissue metabolite patterns were identified. Tumor-specific subtype T1(HER2+MIB+CD3+) positively correlated with HER2, MIB1, DEFA-1, CD3, CD8, FOXP3, but negatively correlated with MMR. Tumor-specific subtype T2(HER2-MIB-CD3-) negatively correlated with HER2, MIB1, CD3, FOXP3, but positively correlated with MMR. Tumor-specific subtype T3(pEGFR+) positively correlated with pEGFR. Patients with tumor subtype T1(HER2+MIB+CD3+) had elevated nucleotide levels, enhanced DNA metabolism, and a better prognosis than T2(HER2-MIB-CD3-) and T3(pEGFR+). An independent validation cohort confirmed that the T1 subtype benefited from trastuzumab therapy. Stroma-specific subtypes had no association with clinicopathological characteristics, however linked to distinct metabolic pathways and molecular features. CONCLUSIONS: Patient subtypes derived by tissue-based spatial metabolomics are a valuable addition to existing GC molecular classification systems. Metabolic differences between the subtypes and their associations with molecular features could provide a valuable tool to aid in selecting specific treatment approaches.

2022 Scientific Article in Nature Methods Nat. Methods 19, 171–178 (2022)

Squidpy: A scalable framework for spatial omics analysis.

Spatial omics data are advancing the study of tissue organization and cellular communication at an unprecedented scale. Flexible tools are required to store, integrate and visualize the large diversity of spatial omics data. Here, we present Squidpy, a Python framework that brings together tools from omics and image analysis to enable scalable description of spatial molecular data, such as transcriptome or multivariate proteins. Squidpy provides efficient infrastructure and numerous analysis methods that allow to efficiently store, manipulate and interactively visualize spatial omics data. Squidpy is extensible and can be interfaced with a variety of already existing libraries for the scalable analysis of spatial omics data.

2022 Scientific Article in Cell Metabolism Cell Metab. 34, 329-345.e8 (2022)

Atlas of exercise metabolism reveals time-dependent signatures of metabolic homeostasis.

Tissue sensitivity and response to exercise vary according to the time of day and alignment of circadian clocks, but the optimal exercise time to elicit a desired metabolic outcome is not fully defined. To understand how tissues independently and collectively respond to timed exercise, we applied a systems biology approach. We mapped and compared global metabolite responses of seven different mouse tissues and serum after an acute exercise bout performed at different times of the day. Comparative analyses of intra- and inter-tissue metabolite dynamics, including temporal profiling and blood sampling across liver and hindlimb muscles, uncovered an unbiased view of local and systemic metabolic responses to exercise unique to time of day. This comprehensive atlas of exercise metabolism provides clarity and physiological context regarding the production and distribution of canonical and novel time-dependent exerkine metabolites, such as 2-hydroxybutyrate (2-HB), and reveals insight into the health-promoting benefits of exercise on metabolism.

2022 Scientific Article in Optica Optica 9, 32-41 (2022)

Deep learning-based quantitative optoacoustic tomography of deep tissues in the absence of labeled experimental data.

Deep learning (DL) shows promise for quantitating anatomical features and functional parameters of tissues in quantitative optoacoustic tomography (QOAT), but its application to deep tissue is hindered by a lack of ground truth data. We propose DL-based "QOAT-Net,"which functions without labeled experimental data: A dual-path convolutional network estimates absorption coefficients after training with data-label pairs generated via unsupervised "simulation-to-experiment"data translation. In simulations, phantoms, and ex vivo and in vivo tissues, QOAT-Net affords quantitative absorption images with high spatial resolution. This approach makes DL-based QOAT and other imaging applications feasible in the absence of ground truth data.

2022 Scientific Article in Photoacoustics Photoacoustics 25:100301 (2022)

Alginate beads as a highly versatile test-sample for optoacoustic imaging.

Test-samples are necessary for the development of emerging imaging approaches such as optoacoustics (OA); these can be used to benchmark new labeling agents and instrumentation, or to characterize image analysis algorithms or the inversion required to form the three-dimensional reconstructions. Alginate beads (AlBes) loaded with labeled mammalian or bacterial cells provide a method of creating defined structures of controllable size and photophysical characteristics and are well-suited for both in vitro and in vivo use. Here we describe a simple and rapid method for efficient and reproducible production of AlBes with specific characteristics and show three example applications with multispectral OA tomography imaging. We show the advantage of AlBes for studying and eventually improving photo-switching OA imaging approaches. As highly defined, homogeneous, quasi point-like signal sources, AlBes might hold similar advantages for studying other agents, light-fluence models, or the impact of detection geometries on correct image formation in the near future.

2022 Scientific Article in Clinical Cancer Research Clin. Cancer Res. 28, 1038-1052 (2022)

Therapy-related transcriptional subtypes in matched primary and recurrent head and neck cancer.

PURPOSE: The genetic relatedness between primary and recurrent head and neck squamous cell carcinomas (HNSCC) reflects the extent of heterogeneity and therapy-driven selection of tumor subpopulations. Yet, current treatment of recurrent HNSCC ignores the molecular characteristics of therapy-resistant tumor populations. EXPERIMENTAL DESIGN: From 150 tumors, 74 primary HNSCCs were RNA-sequenced and 38 matched primary/recurrent tumor pairs were both, whole-exome and RNA-sequenced. Transcriptome analysis determined the predominant classical (CL), basal (BA) and inflamed-mesenchymal (IMS) transcriptional subtypes according to an established classification. Genomic alterations and clonal compositions of tumors were evaluated from whole-exome data. RESULTS: While CL and IMS subtypes were more common in primary HNSCC with low recurrence rates, the BA subtype was more prevalent and stable in recurrent tumors. The BA subtype was associated with a transcriptional signature of partial epithelial-to-mesenchymal transition (p-emt) and early recurrence. In 44% of matched cases, the dominant subtype changed from primary to recurrent tumors, preferably from IMS to BA or CL. Gene set enrichment analysis identified upregulation of Hypoxia, p-emt and radiation resistance signatures and downregulation of tumor inflammation in recurrences compared to index tumors. A relevant subset of primary/recurrent tumor pairs presented no evidence for a common clonal origin. CONCLUSIONS: Our study showed a high degree of genetic and transcriptional heterogeneity between primary/recurrent tumors, suggesting therapy-related selection of a transcriptional subtype with characteristics unfavorable for therapy. We conclude that therapy decisions should be based on genetic and transcriptional characteristics of recurrences rather than primary tumors to enable optimally tailored treatment strategies.

2022 Scientific Article in Journal of Pathology, The J. Pathol. 256, 202-213 (2022)

Metabolic tumor constitution is superior to tumor regression grading for evaluating response to neoadjuvant therapy of esophageal adenocarcinoma patients.

The response to neoadjuvant therapy can vary widely between individual patients. Histopathological tumor regression grading (TRG) is a strong factor for treatment response and survival prognosis of esophageal adenocarcinoma (EAC) patients following neoadjuvant treatment and surgery. However, TRG systems are usually based on the estimation of residual tumor but do not consider stromal or metabolic changes after treatment. Spatial metabolomics analysis is a powerful tool for molecular tissue phenotyping but has not been used so far in the context of neoadjuvant treatment of esophageal cancer. We used imaging mass spectrometry to assess the potential of spatial metabolomics on tumor and stroma tissue for evaluating therapy response of neoadjuvant-treated EAC patients. With an accuracy of 89.7%, the binary classifier trained on spatial tumor metabolite data proved to be superior for stratifying patients when compared to histopathological response assessment which had an accuracy of 70.5%. Sensitivities and specificities for the poor and favorable survival patient groups ranged from 84.9 to 93.3% using the metabolic classifier and from 62.2 to 78.1% using TRG. The tumor classifier was the only significant prognostic factor (HR 3.38, 95% CI = 1.40-8.12, P = 0.007) when adjusted for clinicopathological parameters such as TRG (HR 1.01, 95% CI = 0.67-1.53, P = 0.968) or stromal classifier (HR 1.856, 95% CI = 0.81-4.25, P = 0.143). The classifier even allowed to further stratify patients within the TRG1-3 categories. The underlying mechanisms of response to treatment has been figured out through network analysis. In summary, metabolic response evaluation outperformed histopathological response evaluation in our study with regard to prognostic stratification. This finding indicates that the metabolic constitution of tumor may have a greater impact on patient survival than the quantity of residual tumor cells or the stroma. This article is protected by copyright. All rights reserved.

2021 Scientific Article in Frontiers in Cardiovascular Medicine Front. Cardiovasc. Med. 8:755968 (2021)

Deep learning predicts heart failure with preserved, mid-range, and reduced left ventricular ejection fraction from patient clinical profiles.

Background: Left ventricular ejection fraction (LVEF) is the gold standard for evaluating heart failure (HF) in coronary artery disease (CAD) patients. It is an essential metric in categorizing HF patients as preserved (HFpEF), mid-range (HFmEF), and reduced (HFrEF) ejection fraction but differs, depending on whether the ASE/EACVI or ESC guidelines are used to classify HF. Objectives: We sought to investigate the effectiveness of using deep learning as an automated tool to predict LVEF from patient clinical profiles using regression and classification trained models. We further investigate the effect of utilizing other LVEF-based thresholds to examine the discrimination ability of deep learning between HF categories grouped with narrower ranges. Methods: Data from 303 CAD patients were obtained from American and Greek patient databases and categorized based on the American Society of Echocardiography and the European Association of Cardiovascular Imaging (ASE/EACVI) guidelines into HFpEF (EF > 55%), HFmEF (50% ≤ EF ≤ 55%), and HFrEF (EF < 50%). Clinical profiles included 13 demographical and clinical markers grouped as cardiovascular risk factors, medication, and history. The most significant and important markers were determined using linear regression fitting and Chi-squared test combined with a novel dimensionality reduction algorithm based on arc radial visualization (ArcViz). Two deep learning-based models were then developed and trained using convolutional neural networks (CNN) to estimate LVEF levels from the clinical information and for classification into one of three LVEF-based HF categories. Results: A total of seven clinical markers were found important for discriminating between the three HF categories. Using statistical analysis, diabetes, diuretics medication, and prior myocardial infarction were found statistically significant (p < 0.001). Furthermore, age, body mass index (BMI), anti-arrhythmics medication, and previous ventricular tachycardia were found important after projections on the ArcViz convex hull with an average nearest centroid (NC) accuracy of 94%. The regression model estimated LVEF levels successfully with an overall accuracy of 90%, average root mean square error (RMSE) of 4.13, and correlation coefficient of 0.85. A significant improvement was then obtained with the classification model, which predicted HF categories with an accuracy ≥93%, sensitivity ≥89%, 1-specificity <5%, and average area under the receiver operating characteristics curve (AUROC) of 0.98. Conclusions: Our study suggests the potential of implementing deep learning-based models clinically to ensure faster, yet accurate, automatic prediction of HF based on the ASE/EACVI LVEF guidelines with only clinical profiles and corresponding information as input to the models. Invasive, expensive, and time-consuming clinical testing could thus be avoided, enabling reduced stress in patients and simpler triage for further intervention.

2021 Review in Frontiers in Oncology Front. Oncol. 11:771335 (2021)

Various uses of PD1/PD-L1 inhibitor in oncology: Opportunities and challenges.

The occurrence and development of cancer are closely related to the immune escape of tumor cells and immune tolerance. Unlike previous surgical, chemotherapy, radiotherapy and targeted therapy, tumor immunotherapy is a therapeutic strategy that uses various means to stimulate and enhance the immune function of the body, and ultimately achieves the goal of controlling tumor cells.With the in-depth understanding of tumor immune escape mechanism and tumor microenvironment, and the in-depth study of tumor immunotherapy, immune checkpoint inhibitors represented by Programmed Death 1/Programmed cell Death-Ligand 1(PD-1/PD-L1) inhibitors are becoming increasingly significant in cancer medication treatment. employ a variety of ways to avoid detection by the immune system, a single strategy is not more effective in overcoming tumor immune evasion and metastasis. Combining different immune agents or other drugs can effectively address situations where immunotherapy is not efficacious, thereby increasing the chances of success and alternative access to alternative immunotherapy. Immune combination therapies for cancer have become a hot topic in cancer treatment today. In this paper, several combination therapeutic modalities of PD1/PD-L1 inhibitors are systematically reviewed. Finally, an analysis and outlook are provided in the context of the recent advances in combination therapy with PD1/PD-L1 inhibitors and the pressing issues in this field.

2021 Scientific Article in Physics in Medicine and Biology Phys. Med. Biol. 66:245020 (2021)

Experimental demonstration of accurate Bragg peak localization with ionoacoustic tandem phase detection (iTPD).

Accurate knowledge of the exact stopping location of ions inside the patient would allow full exploitation of their ballistic properties for patient treatment. The localized energy deposition of a pulsed particle beam induces a rapid temperature increase of the irradiated volume and leads to the emission of ionoacoustic (IA) waves. Detecting the time-of-flight (ToF) of the IA wave allows inferring information on the Bragg peak location and can henceforth be used for in-vivo range verification. A challenge for IA is the poor signal-to-noise ratio at clinically relevant doses and viable machines. We present a frequency-based measurement technique, labeled as ionoacoustic tandem phase detection (iTPD) utilizing lock-in amplifiers. The phase shift of the IA signal to a reference signal is measured to derive the ToF. Experimental IA measurements with a 3.5 MHz lead zirconate titanate (PZT) transducer and lock-in amplifiers were performed in water using 22 MeV proton bursts. A digital iTPD was performed in-silico at clinical dose levels on experimental data obtained from a clinical facility and secondly, on simulations emulating a heterogeneous geometry. For the experimental setup using 22 MeV protons, a localization accuracy and precision obtained through iTPD deviates from a time-based reference analysis by less than 15 mu m. Several methodological aspects were investigated experimentally in systematic manner. Lastly, iTPD was evaluated in-silico for clinical beam energies indicating that iTPD is in reach of sub-mm accuracy for fractionated doses < 5 Gy. iTPD can be used to accurately measure the ToF of IA signals online via its phase shift in frequency domain. An application of iTPD to the clinical scenario using a single pulsed beam is feasible but requires further development to reach <1 Gy detection capabilities.

2021 Scientific Article in Scientific Reports Sci. Rep. 11:24430 (2021)

Reporter gene-based optoacoustic imaging of E. coli targeted colon cancer in vivo.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.

2021 Scientific Article in Cell Reports Cell Rep. 37:110161 (2021)

Optogenetic activation of striatal D1R and D2R cells differentially engages downstream connected areas beyond the basal ganglia.

The basal ganglia (BG) are a group of subcortical nuclei responsible for motor and executive function. Central to BG function are striatal cells expressing D1 (D1R) and D2 (D2R) dopamine receptors. D1R and D2R cells are considered functional antagonists that facilitate voluntary movements and inhibit competing motor patterns, respectively. However, whether they maintain a uniform function across the striatum and what influence they exert outside the BG is unclear. Here, we address these questions by combining optogenetic activation of D1R and D2R cells in the mouse ventrolateral caudoputamen with fMRI. Striatal D1R/D2R stimulation evokes distinct activity within the BG-thalamocortical network and differentially engages cerebellar and prefrontal regions. Computational modeling of effective connectivity confirms that changes in D1R/D2R output drive functional relationships between these regions. Our results suggest a complex functional organization of striatal D1R/D2R cells and hint toward an interconnected fronto-BG-cerebellar network modulated by striatal D1R and D2R cells.

2021 Scientific Article in Biomedicines Biomedicines 9:1696 (2021)

Multi-aspect optoacoustic imaging of breast tumors under chemotherapy with exogenous and endogenous contrasts: Focus on apoptosis and hypoxia.

Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.

2021 Scientific Article in EJNMMI Research EJNMMI Res. 11:120 (2021)

Native glycan fragments detected by MALDI mass spectrometry imaging are independent prognostic factors in pancreatic ductal adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies to date. The impressively developed stroma that surrounds and modulates the behavior of cancer cells is one of the main factors regulating the PDAC growth, metastasis and therapy resistance. Here, we postulate that stromal and cancer cell compartments differentiate in protein/lipid glycosylation patterns and analyze differences in glycan fragments in those compartments with clinicopathologic correlates. Results: We analyzed native glycan fragments in 109 human FFPE PDAC samples using high mass resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometric imaging (MALDI-FT-ICR-MSI). Our method allows detection of native glycan fragments without previous digestion with PNGase or any other biochemical reaction. With this method, 8 and 18 native glycans were identified as uniquely expressed in only stromal or only cancer cell compartment, respectively. Kaplan–Meier survival model identified glycan fragments that are expressed in cancer cell or stromal compartment and significantly associated with patient outcome. Among cancer cell region-specific glycans, 10 predicted better and 6 worse patient survival. In the stroma, 1 glycan predicted good and 4 poor patient survival. Using factor analysis as a dimension reduction method, we were able to group the identified glycans in 2 factors. Multivariate analysis revealed that these factors can be used as independent survival prognostic elements with regard to the established Union for International Cancer Control (UICC) classification both in tumor and stroma regions. Conclusion: Our method allows in situ detection of naturally occurring glycans in FFPE samples of human PDAC tissue and highlights the differences among glycans found in stromal and cancer cell compartment offering a basis for further exploration on the role of specific glycans in cancer–stroma communication.

2021 Scientific Article in European Journal of Nuclear Medicine and Molecular Imaging Eur. J. Nucl. Med. Mol. Imaging, DOI: 10.1007/s00259-021-05582-y (2021)

Targeted Hsp70 fluorescence molecular endoscopy detects dysplasia in Barrett's esophagus.

PURPOSE: The incidence of esophageal adenocarcinoma (EAC) has been increasing for decades without significant improvements in treatment. Barrett's esophagus (BE) is best established risk factor for EAC, but current surveillance with random biopsies cannot predict progression to cancer in most BE patients due to the low sensitivity and specificity of high-definition white light endoscopy. METHODS: Here, we evaluated the membrane-bound highly specific Hsp70-specific contrast agent Tumor-Penetrating Peptide (Hsp70-TPP) in guided fluorescence molecular endoscopy biopsy. RESULTS: Hsp70 was significantly overexpressed as determined by IHC in dysplasia and EAC compared with non-dysplastic BE in patient samples (n = 12) and in high-grade dysplastic lesions in a transgenic (L2-IL1b) mouse model of BE. In time-lapse microscopy, Hsp70-TPP was rapidly taken up and internalized  by human BE dysplastic patient-derived organoids. Flexible fluorescence endoscopy of the BE mouse model allowed a specific detection of Hsp70-TPP-Cy5.5 that corresponded closely with the degree of dysplasia but not BE. Ex vivo application of Hsp70-TPP-Cy5.5 to freshly resected whole human EAC specimens revealed a high (> 4) tumor-to-background ratio and a specific detection of previously undetected tumor infiltrations. CONCLUSION: In summary, these findings suggest that Hsp70-targeted imaging using fluorescently labeled TPP peptide may improve tumor surveillance in BE patients.

2021 Scientific Article in Biochemistry Biochemistry 86, 1434-1445 (2021)

Inhibition of miR-21 promotes cellular senescence in NT2-derived astrocytes.

Abstract: Astrocytes are the main homeostatic cells in the central nervous system (CNS) that provide mechanical, metabolic, and trophic support to neurons. Disruption of their physiological role or acquisition of senescence-associated phenotype can contribute to the CNS dysfunction and pathology. However, molecular mechanisms underlying the complex physiology of astrocytes are explored insufficiently. Recent studies have shown that miRNAs are involved in the regulation of astrocyte function through different mechanisms. Although miR-21 has been reported as an astrocytic miRNA with an important role in astrogliosis, no link between this miRNA and cellular senescence of astrocytes has been identified. To address the role of miR-21 in astrocytes, with special focus on cellular senescence, we used NT2/A (astrocytes derived from NT2/D1 cells). Downregulation of miR-21 expression in both immature and mature NT2/A by the antisense technology induced the arrest of cell growth and premature cellular senescence, as indicated by senescence hallmarks such as increased expression of cell cycle inhibitors p21 and p53 and augmented senescence-associated β-galactosidase activity. Additionally, in silico analysis predicted many of the genes, previously shown to be upregulated in astrocytes with the irradiation-induced senescence, as miR-21 targets. Taken together, our results point to miR-21 as a potential regulator of astrocyte senescence. To the best of our knowledge, these are the first data showing the link between miR-21 and cellular senescence of astrocytes. Since senescent astrocytes are associated with different CNS pathologies, development of novel therapeutic strategies based on miRNA manipulation could prevent senescence and may improve the physiological outcome.

2021 Scientific Article in Analytical Chemistry Anal. Chem. 93, 15323-15330 (2021)

Wide-field mid-infrared hyperspectral imaging by snapshot phase contrast measurement of optothermal excitation.

Vibrational microscopy methods based on Raman scattering or infrared absorption provide a label-free approach for chemical-contrast imaging, but employ point-by-point scanning and impose a compromise between the imaging speed and field-of-view (FOV). Optothermal microscopy has been proposed as a promising imaging modality to avoid this compromise, although at restrictively small FOVs capable of imaging only few cells. Here, we present wide-field optothermal mid-infrared microscopy (WOMiM) for wide-field chemical-contrast imaging based on snapshot pump-probe detection of optothermal signal, using a custom-made condenser-free phase contrast microscopy to capture the phase change of samples after mid-infrared irradiation. We achieved chemical contrast for FOVs up to 180 μm in diameter, yielding 10-fold larger imaging areas than the state-of-the-art, at imaging speeds of 1 ms/frame. The maximum possible imaging speed of WOMiM was determined by the relaxation time of optothermal heat, measured to be 32.8 μs in water, corresponding to a frame rate of μ30 kHz. This proof-of-concept demonstrates that vibrational imaging can be achieved at an unprecedented imaging speed and large FOV with the potential to significantly facilitate label-free imaging of cellular dynamics.

2021 Scientific Article in ACS Photonics ACS Photonics 8, 3346–3356 (2021)

Optoacoustic effect in a hybrid multilayered membrane deposited on a hollow-core microstructured optical waveguide.

Modern imaging technologies, including optoacoustic endoscopy, are based on the optoacoustic effect. Much promise is offered by the all-optical fiber-based approach, because fiber has a miniature cross section, is highly sensitive, and can be used in a variety of imaging and therapeutic techniques. We developed a probe based on a hollow-core microstructured optical waveguide (HC-MOW) with a hybrid nanostructured membrane. The membrane consisted of a free-standing single-walled carbon nanotube film and a Bragg reflector, which can be used as a source and a detector of ultrasound. Membrane vibrations were excited with an IR laser pulse and were read out by recording the intensity of the reflected visible CW laser light. We explained the nature of the intensity modulation of the reflected light and supported our explanation with numerical simulations of the membrane's vibration eigenfrequencies and thermal distribution. The membrane vibrations were also observed with raster-scanning optoacoustic mesoscopy. The transmittance of the HC-MOW between 400 nm and 6.5 μm and that of the hybrid nanostructured membrane in the NIR range enable potential optoacoustic sensing in the IR fingerprint region of biomolecules. This permits the optoacoustic probe to be used for medical endoscopic purposes.

2021 Scientific Article in International Journal of Molecular Sciences Int. J. Mol. Sci. 22:12275 (2021)

Encapsulin based self‐assembling iron‐containing protein nanoparticles for stem cells mri visualization.

Over the past decade, cell therapy has found many applications in the treatment of different diseases. Some of the cells already used in clinical practice include stem cells and CAR‐T cells. Compared with traditional drugs, living cells are much more complicated systems that must be strictly controlled to avoid undesirable migration, differentiation, or proliferation. One of the approaches used to prevent such side effects involves monitoring cell distribution in the human body by any noninvasive technique, such as magnetic resonance imaging (MRI). Long‐term tracking of stem cells with artificial magnetic labels, such as magnetic nanoparticles, is quite problematic because such labels can affect the metabolic process and cell viability. Additionally, the concentration of exogenous labels will decrease during cell division, leading to a corresponding decrease in signal intensity. In the current work, we present a new type of genetically encoded label based on encapsulin from Myxococcus xanthus bacteria, stably expressed in human mesenchymal stem cells (MSCs) and coexpressed with ferroxidase as a cargo protein for nanoparticles’ synthesis inside encapsulin shells. mZip14 protein was expressed for the enhancement of iron transport into the cell. Together, these three proteins led to the synthesis of iron‐containing nanoparticles in mesenchymal stem cells—without affecting cell viability—and increased contrast properties of MSCs in MRI.

2021 Scientific Article in Cell Discovery Cell Discov. 7:105 (2021)

Multiomics interrogation into HBV (Hepatitis B virus)-host interaction reveals novel coding potential in human genome, and identifies canonical and non-canonical proteins as host restriction factors against HBV.

Hepatitis B Virus (HBV) constitutes a major threat to global public health. Current understanding of HBV-host interaction is yet limited. Here, ribosome profiling, quantitative mass spectrometry and RNA-sequencing were conducted on a recently established HBV replication system, through which we identified multiomic differentially expressed genes (DEGs) that HBV orchestrated to remodel host proteostasis networks. Our multiomics interrogation revealed that HBV induced significant changes in both transcription and translation of 35 canonical genes including PPP1R15A, PGAM5 and SIRT6, as well as the expression of at least 15 non-canonical open reading frames (ncORFs) including ncPON2 and ncGRWD1, thus revealing an extra coding potential of human genome. Overexpression of these five genes but not the enzymatically deficient SIRT6 mutants suppressed HBV replication while knockdown of SIRT6 had opposite effect. Furthermore, the expression of SIRT6 was down-regulated in patients, cells or animal models of HBV infection. Mechanistic study further indicated that SIRT6 directly binds to mini-chromosome and deacetylates histone H3 lysine 9 (H3K9ac) and histone H3 lysine 56 (H3K56ac), and chemical activation of endogenous SIRT6 with MDL800 suppressed HBV infection in vitro and in vivo. By generating the first multiomics landscape of host-HBV interaction, our work is thus opening a new avenue to facilitate therapeutic development against HBV infection.

2021 Scientific Article in Cellular Microbiology Cell. Microbiol. 23:e13399 (2021)

Hepatitis B virus envelope proteins can serve as therapeutic targets embedded in the host cell plasma membrane.

Hepatitis B virus (HBV) infection is a major health threat causing 880,000 deaths each year. Available therapies control viral replication, but do not cure HBV leaving patients at risk to develop hepatocellular carcinoma. Here we show that HBV envelope proteins (HBs) - besides their integration into endosomal membranes - become embedded in the plasma membrane where they can be targeted by redirected T-cells. HBs was detected on the surface of HBV-infected cells, in livers of mice replicating HBV and in HBV-induced hepatocellular carcinoma. Staining with HBs-specific recombinant antibody MoMab recognizing a conformational epitope indicated that membrane-associated HBs remains correctly folded in HBV-replicating cells in cell culture and in livers of HBV-transgenic mice in vivo. MoMab coated onto superparamagnetic iron oxide nanoparticles allowed to detect membrane-associated HBs after HBV infection by electron microscopy in distinct stretches of the hepatocyte plasma membrane. Last not least we demonstrate that HBs located to the cell surface allows therapeutic targeting of HBV-positive cells by T-cells either engrafted with a chimeric antigen receptor or redirected by bispecific, T-cell engager antibodies. This article is protected by copyright. All rights reserved.

2021 Scientific Article in Nature Biotechnology Nat. Biotechnol., DOI: 10.1038/s41587-021-01100-5 (2021)

Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging.

Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.Calcium and other analytes can be imaged at super-resolution and in vivo with photo-switchable sensors.

2021 Scientific Article in Physics & Imaging in Radiation Oncology Phys. Imag. Radiat. Oncology 20, 11-16 (2021)

In-vivo X-ray dark-field computed tomography for the detection of radiation-induced lung damage in mice.

Background and Purpose: Radiotherapy of thoracic tumours can lead to side effects in the lung, which may benefit from early diagnosis. We investigated the potential of X-ray dark-field computed tomography by a proof-of-principle murine study in a clinically relevant radiotherapeutic setting aiming at the detection of radiation-induced lung damage. Material and Methods: Six mice were irradiated with 20 Gy to the entire right lung. Together with five unirradiated control mice, they were imaged using computed tomography with absorption and dark-field contrast before and 16 weeks post irradiation. Mean pixel values for the right and left lung were calculated for both contrasts, and the right-to-left-ratio R of these means was compared. Radiologists also assessed the tomograms acquired 16 weeks post irradiation. Sensitivity, specificity, inter- and intra-reader accuracy were evaluated. Results: In absorption contrast the group-average of R showed no increase in the control group and increased by 7% (p = 0.005) in the irradiated group. In dark-field contrast, it increased by 2% in the control group and by 14% (p = 0.005) in the irradiated group. Specificity was 100% for both contrasts but sensitivity was almost four times higher using dark-field tomography. Two cases were missed by absorption tomography but were detected by dark-field tomography. Conclusions: The applicability of X-ray dark-field computed tomography for the detection of radiation-induced lung damage was demonstrated in a pre-clinical mouse model. The presented results illustrate the differences between dark-field and absorption contrast and show that dark-field tomography could be advantageous in future clinical settings.

2021 Review in Hautarzt, Der Hautarzt 72, 1025-1038 (2021)

Optoakustische Bildgebung – innovative Bildgebungsverfahren auf dem Vormarsch.

Die optoakustische Bildgebung (OAB) hat sich in den letzten Jahren stetigweiterentwickelt. Mittels teils gepulsten Lichts in verschiedenen Wellenlängen werdenunterschiedliche Farbträger (Chromophore) zur Bildung von Schallwellen angeregt.Diese werden von den neu entwickelten Systemen detektiert und mittels verschiedenerAlgorithmen in dreidimensionale Bilder umgewandelt. Die Technik zeichnet sich durchein gutes Verhältnis von Kontrast und Eindringtiefe aus und kann aufgrund ihrerSkalierbarkeit makro-, meso- und mikroskopische Bilder erzeugen. Die optoakustischeMakroskopie bestrahlt das zu untersuchende Areal breit mit Laserlicht. Hierdurchkönnen Abbildungen mit hoher Eindringtiefe erzeugt werden, jedoch lediglich miteiner mittleren Auflösung. Klinisch interessante Anwendungsfelder sind z.B. dieErgebnisse von ex-vivo untersuchten Sentinellymphknoten mittels makroskopischerOptoakustik. Aufgrund der Fähigkeit der OAB Melanin darzustellen zeigte sicheine bisherigen bildgebenden Methoden, jedoch nicht der Histologie überlegeneDetektionsrate für Metastasen. Die Fähigkeit, mit einer guten Auflösung dermale undepidermale Strukturen, besonders Gefäße, darzustellen, macht sich die optoakustischeMesoskopie bei der Untersuchung entzündlicher Hauterkrankungen zunutze undkönnte künftig zur Überprüfung des Therapieerfolges, z.B. durch Biologika bei Psoriasisvulgaris oder dem atopischen Ekzem, beitragen. Die bisher v. a. auf präklinische In-vivo-Forschung beschränkte optoakustische Mikroskopie könnte künftig zur Detektion nochfeinerer Gefäßstrukturen und deren Veränderungen verwendet werden. Die klinischenMöglichkeiten der OAB scheinen bisher sehr großen Nutzen bieten zu können und sindein aktuell stark untersuchtes Forschungsfeld.

2021 Review in Nature biomedical engineering Nat. Bio. Eng. 6, 503-514 (2021)

Intraoperative imaging in pathology-assisted surgery.

The pathological assessment of surgical specimens during surgery can reduce the incidence of positive resection margins, which otherwise can result in additional surgeries or aggressive therapeutic regimens. To improve patient outcomes, intraoperative spectroscopic, fluorescence-based, structural, optoacoustic and radiological imaging techniques are being tested on freshly excised tissue. The specific clinical setting and tumour type largely determine whether endogenous or exogenous contrast is to be detected and whether the tumour specificity of the detected biomarker, image resolution, image-acquisition times or penetration depth are to be prioritized. In this Perspective, we describe current clinical standards for intraoperative tissue analysis and discuss how intraoperative imaging is being implemented. We also discuss potential implementations of intraoperative pathology-assisted surgery for clinical decision-making.

2021 Scientific Article in Cancer Research Cancer Res. 81, 5862-5875 (2021)

Patterns of carbon-bound exogenous compounds in lung cancer patients and association with disease pathophysiology.

Asymptomatic anthracosis is the accumulation of black carbon particles in adult human lungs. It is a common occurrence, but the pathophysiological significance of anthracosis is debatable. Using in situ high mass resolution matrix-assisted laser desorption/ionization (MALDI) fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry imaging analysis, we discovered noxious carbon-bound exogenous compounds, such as polycyclic aromatic hydrocarbons (PAHs), tobacco-specific nitrosamines, or aromatic amines, in a series of 330 lung cancer patients in highly variable and unique patterns. The characteristic nature of carbon-bound exogenous compound had a strong association with patient outcome, tumor progression, the tumor immune microenvironment, PD-L1 expression, and DNA damage. Spatial correlation network analyses revealed substantial differences in the metabolome of tumor cells compared to tumor stroma depending on carbon-bound exogenous compounds. Overall, the bioactive pool of exogenous compounds is associated with several changes in lung cancer pathophysiology and correlates with patient outcome. Given the high prevalence of anthracosis in the lungs of adult humans, future work should investigate the role of carbon-bound exogenous compounds in lung carcinogenesis and lung cancer therapy.

2021 Scientific Article in Clinical and translational medicine Clin. Transl. Med. 11:e547 (2021)

Metabolomic therapy response prediction in pretherapeutic tissue biopsies for trastuzumab in patients with HER2-positive advanced gastric cancer.

2021 Scientific Article in Scientific Reports Sci. Rep. 11:18370 (2021)

Speckle reduction in ultrasound endoscopy using refraction based elevational angular compounding.

Endoscopic ultrasonography (EUS) is a safe, real-time diagnostic and therapeutic tool. Speckle noise, inherent to ultrasonography, degrades the diagnostic precision of EUS. Elevational angular compounding (EAC) can provide real-time speckle noise reduction; however, EAC has never been applied to EUS because current implementations require costly and bulky arrays and are incompatible with the tight spatial constraints of hollow organs. Here we develop a radial implementation of a refraction-based elevational angular compounding technique (REACT) for EUS and demonstrate for the first time spatial compounding in a radial endoscopy. The proposed implementation was investigated in cylindrical phantoms and demonstrated superior suppression of ultrasound speckle noise and up to a two-fold improvement in signal- and contrast- ratios, compared to standard image processing techniques and averaging. The effect of elevational angular deflection on image fidelity was further investigated in a phantom with lymph node-like structures to determine the optimum elevational angular width for high speckle reduction efficiency while maintaining image fidelity. This study introduces REACT as a potential compact and low-cost solution to impart current radial echo-endoscopes with spatial compounding, which could enable accurate identification and precise sizing of lymph nodes in staging of gastrointestinal tract cancers.

2021 Scientific Article in Molecular Metabolism Mol. Metab. 54:101330 (2021)

Vertical sleeve gastrectomy triggers fast β-cell recovery upon overt diabetes.

While the effectiveness of bariatric surgery in restoring β-cell function has been described in type-2 diabetes (T2D) patients and animal models for years, the mechanistic underpinnings are largely unknown. The possibility of vertical sleeve gastrectomy (VSG) to rescue a clinically-relevant, late-stage T2D condition and to promote β-cell recovery has not been investigated on a single-cell level. Nevertheless, characterization of the heterogeneity and functional states of β-cells after VSG is a fundamental step to understand mechanisms of glycaemic recovery and to ultimately develop alternative, less-invasive therapies. Here, we report that VSG was superior to calorie restriction in late-stage T2D and rapidly restored normoglycaemia in morbidly obese and overt diabetic db/db mice. Single-cell profiling of islets of Langerhans showed that VSG induced distinct, intrinsic changes in the β-cell transcriptome, but not in that of α-, δ-, and PP-cells. VSG triggered fast β-cell redifferentiation and functional improvement within only two weeks of intervention, which is not seen upon calorie restriction. Furthermore, VSG expanded β-cell area by means of redifferentiation and by creating a proliferation competent β-cell state. Collectively, our study reveals the superiority of VSG in the remission of far-progressed T2D and presents paths of β-cell regeneration and molecular pathways underlying the glycaemic benefits of VSG.

2021 Scientific Article in Communications Biology Comm. Biol. 4:1040 (2021)

Label-free concurrent 5-modal microscopy (Co5M) resolves unknown spatio-temporal processes in wound healing.

The non-invasive investigation of multiple biological processes remains a methodological challenge as it requires capturing different contrast mechanisms, usually not available with any single modality. Intravital microscopy has played a key role in dynamically studying biological morphology and function, but it is generally limited to resolving a small number of contrasts, typically generated by the use of transgenic labels, disturbing the biological system. We introduce concurrent 5-modal microscopy (Co5M), illustrating a new concept for label-free in vivo observations by simultaneously capturing optoacoustic, two-photon excitation fluorescence, second and third harmonic generation, and brightfield contrast. We apply Co5M to non-invasively visualize multiple wound healing biomarkers and quantitatively monitor a number of processes and features, including longitudinal changes in wound shape, microvascular and collagen density, vessel size and fractality, and the plasticity of sebaceous glands. Analysis of these parameters offers unique insights into the interplay of wound closure, vasodilation, angiogenesis, skin contracture, and epithelial reformation in space and time, inaccessible by other methods. Co5M challenges the conventional concept of biological observation by yielding multiple simultaneous parameters of pathophysiological processes in a label-free mode.

2021 Scientific Article in Nature metabolism Nat. Metab. 3, 1202-1216 (2021)

Diet-induced alteration of intestinal stem cell function underlies obesity and prediabetes in mice.

Excess nutrient uptake and altered hormone secretion in the gut contribute to a systemic energy imbalance, which causes obesity and an increased risk of type 2 diabetes and colorectal cancer. This functional maladaptation is thought to emerge at the level of the intestinal stem cells (ISCs). However, it is not clear how an obesogenic diet affects ISC identity and fate. Here we show that an obesogenic diet induces ISC and progenitor hyperproliferation, enhances ISC differentiation and cell turnover and changes the regional identities of ISCs and enterocytes in mice. Single-cell resolution of the enteroendocrine lineage reveals an increase in progenitors and peptidergic enteroendocrine cell types and a decrease in serotonergic enteroendocrine cell types. Mechanistically, we link increased fatty acid synthesis, Ppar signaling and the Insr-Igf1r-Akt pathway to mucosal changes. This study describes molecular mechanisms of diet-induced intestinal maladaptation that promote obesity and therefore underlie the pathogenesis of the metabolic syndrome and associated complications.

2021 Scientific Article in EMBO Molecular Medicine EMBO Mol. Med. 13:e13490 (2021)

Functional multispectral optoacoustic tomography imaging of hepatic steatosis development in mice.

The increasing worldwide prevalence of obesity, fatty liver diseases and the emerging understanding of the important roles lipids play in various other diseases is generating significant interest in lipid research. Lipid visualization in particular can play a critical role in understanding functional relations in lipid metabolism. We investigated the potential of multispectral optoacoustic tomography (MSOT) as a novel modality to non-invasively visualize lipids in laboratory mice around the 930nm spectral range. Using an obesity-induced non-alcoholic fatty liver disease (NAFLD) mouse model, we examined whether MSOT could detect and differentiate different grades of hepatic steatosis and monitor the accumulation of lipids in the liver quantitatively over time, without the use of contrast agents, i.e. in label-free mode. Moreover, we demonstrate the efficacy of using the real-time clearance kinetics of indocyanine green (ICG) in the liver, monitored by MSOT, as a biomarker to evaluate the organ’s function and assess the severity of NAFLD. This study establishes MSOT as an efficient imaging tool for lipid visualization in preclinical studies, particularly for the assessment of NAFLD.

2021 Scientific Article in Carcinogenesis Carcinogenesis 42, 1068-1078 (2021)

Anti-inflammatory chemoprevention attenuates the phenotype in a mouse model of esophageal adenocarcinoma.

Barrett's esophagus (BE) is the main known precursor condition of esophageal adenocarcinoma (EAC). BE is defined by the presence of metaplasia above the normal squamous columnar junction and has mainly been attributed to gastroesophageal reflux disease and chronic reflux esophagitis. Thus, the rising incidence of EAC in the Western world is probably mediated by chronic esophageal inflammation, secondary to gastroesophageal reflux disease in combination with environmental risk factors such as a Western diet and obesity. However, (at present) risk prediction tools and endoscopic surveillance have shown limited effectiveness. Chemoprevention as an adjunctive approach remains an attractive option to reduce the incidence of neoplastic disease. Here, we investigate the feasibility of chemopreventive approaches in BE and EAC via inhibition of inflammatory signaling in a transgenic mouse model of BE and EAC (L2-IL1B mice), with accelerated tumor formation on a high-fat diet (HFD). L2-IL1B mice were treated with the IL-1 receptor antagonist Anakinra and the nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin or Sulindac. Interleukin-1b antagonism reduced tumor progression in L2-IL1B mice with or without a HFD, whereas both NSAIDs were effective chemoprevention agents in the accelerated HFD-fed L2-IL1B mouse model. Sulindac treatment also resulted in a marked change in the immune profile of L2-IL1B mice. In summary, anti-inflammatory treatment of HFD-treated L2-IL1B mice acted protectively on disease progression. These results from a mouse model of BE support results from clinical trials that suggest that anti-inflammatory medication may be effective in the chemoprevention of EAC.

2021 Scientific Article in Theranostics Theranostics 11, 7813-7828 (2021)

Chemotherapeutic effects on breast tumor hemodynamics revealed by eigenspectra multispectral optoacoustic tomography (eMSOT).

Non-invasive monitoring of hemodynamic tumor responses to chemotherapy could provide unique insights into the development of therapeutic resistance and inform therapeutic decision-making in the clinic. Methods: Here, we examined the longitudinal and dynamic effects of the common chemotherapeutic drug Taxotere on breast tumor (KPL-4) blood volume and oxygen saturation using eigenspectra multispectral optoacoustic tomography (eMSOT) imaging over a period of 41 days. Tumor vascular function was assessed by dynamic oxygen-enhanced eMSOT (OE-eMSOT). The obtained in vivo optoacoustic data were thoroughly validated by ex vivo cryoimaging and immunohistochemical staining against markers of vascularity and hypoxia. Results: We provide the first preclinical evidence that prolonged treatment with Taxotere causes a significant drop in mean whole tumor oxygenation. Furthermore, application of OE-eMSOT showed a diminished vascular response in Taxotere-treated tumors and revealed the presence of static blood pools, indicating increased vascular permeability. Conclusion: Our work has important translational implications and supports the feasibility of eMSOT imaging for non-invasive assessment of tumor microenvironmental responses to chemotherapy.

2021 Scientific Article in Medical Image Analysis Med. Image Anal. 73:102166 (2021)

VERSE: A Vertebrae labelling and segmentation benchmark for multi-detector CT images.

Vertebral labelling and segmentation are two fundamental tasks in an automated spine processing pipeline. Reliable and accurate processing of spine images is expected to benefit clinical decision support systems for diagnosis, surgery planning, and population-based analysis of spine and bone health. However, designing automated algorithms for spine processing is challenging predominantly due to considerable variations in anatomy and acquisition protocols and due to a severe shortage of publicly available data. Addressing these limitations, the Large Scale Vertebrae Segmentation Challenge (VERSE) was organised in conjunction with the International Conference on Medical Image Computing and Computer Assisted Intervention (MICCAI) in 2019 and 2020, with a call for algorithms tackling the labelling and segmentation of vertebrae. Two datasets containing a total of 374 multi-detector CT scans from 355 patients were prepared and 4505 vertebrae have individually been annotated at voxel level by a human-machine hybrid algorithm (https://osf.io/nqjyw/, https://osf.io/t98fz/). A total of 25 algorithms were benchmarked on these datasets. In this work, we present the results of this evaluation and further investigate the performance variation at the vertebra level, scan level, and different fields of view. We also evaluate the generalisability of the approaches to an implicit domain shift in data by evaluating the top-performing algorithms of one challenge iteration on data from the other iteration. The principal takeaway from VERSE: the performance of an algorithm in labelling and segmenting a spine scan hinges on its ability to correctly identify vertebrae in cases of rare anatomical variations. The VERSE content and code can be accessed at: https://github.com/anjany/verse.

2021 Scientific Article in Molecular Metabolism Mol. Metab. 54:101334 (2021)

A point mutation in the Pdia6 gene results in loss of pancreatic β-cell identity causing overt diabetes.

OBJECTIVE: Protein disulfide isomerases (PDIs) are oxidoreductases that are involved in catalyzing the formation and rearrangement of disulfide bonds during protein folding. One of the PDI members is the PDI-associated 6 (PDIA6) protein, which has been shown to carry a vital role in β-cell dysfunction and diabetes. However, very little is known about the function of this protein in β-cells in vivo. This study aimed to describe the consequences of a point mutation in Pdia6 on β-cell development and function. METHODS: We generated an ENU mouse model carrying a missense mutation (Phe175Ser) in the second thioredoxin domain of the Pdia6 gene. Using biochemical and molecular tools, we determined the effects of the mutation on the β-cell development at embryonic day (E)18.5 and β-cell identity as well as function at postnatal stages. RESULTS: Mice homozygous for the Phe175Ser (F175S) mutation were mildly hyperglycemic at weaning and subsequently became hypoinsulinemic and overtly diabetic at the adult stage. Although, no developmental phenotype was detected during embryogenesis, mutant mice displayed reduced insulin-expressing β-cells at P14 and P21 without any changes in the rate of cell death and proliferation. Further analysis revealed an increase in BiP as well as PDI family member PDIA4, however without any concomitant apoptosis and cell death. Instead, the expression of prominent markers of β-cell maturation and function, such as Ins2, Mafa and Slc2a2 along with increased expression of α-cell markers, Mafb and glucagon was observed in adult mice, suggesting loss of β-cell identity. CONCLUSIONS: The data demonstrates that a global Pdia6 mutation renders mice hypoinsulinemic and hyperglycemic. This occurs due to the loss of pancreatic β-cell function and identity, suggesting a critical role of PDIA6 specifically for β-cells.

2021 Scientific Article in Cell Death & Disease Cell Death Dis. 12:723 (2021)

Inhibition of metabotropic glutamate receptor III facilitates sensitization to alkylating chemotherapeutics in glioblastoma.

Glioblastoma (GBM), the most malignant tumor of the central nervous system, is marked by its dynamic response to microenvironmental niches. In particular, this cellular plasticity contributes to the development of an immediate resistance during tumor treatment. Novel insights into the developmental trajectory exhibited by GBM show a strong capability to respond to its microenvironment by clonal selection of specific phenotypes. Using the same mechanisms, malignant GBM do develop intrinsic mechanisms to resist chemotherapeutic treatments. This resistance was reported to be sustained by the paracrine and autocrine glutamate signaling via ionotropic and metabotropic receptors. However, the extent to which glutamatergic signaling modulates the chemoresistance and transcriptional profile of the GBM remains unexplored. In this study we aimed to map the manifold effects of glutamate signaling in GBM as the basis to further discover the regulatory role and interactions of specific receptors, within the GBM microenvironment. Our work provides insights into glutamate release dynamics, representing its importance for GBM growth, viability, and migration. Based on newly published multi-omic datasets, we explored the and characterized the functions of different ionotropic and metabotropic glutamate receptors, of which the metabotropic receptor 3 (GRM3) is highlighted through its modulatory role in maintaining the ability of GBM cells to evade standard alkylating chemotherapeutics. We addressed the clinical relevance of GRM3 receptor expression in GBM and provide a proof of concept where we manipulate intrinsic mechanisms of chemoresistance, driving GBM towards chemo-sensitization through GRM3 receptor inhibition. Finally, we validated our findings in our novel human organotypic section-based tumor model, where GBM growth and proliferation was significantly reduced when GRM3 inhibition was combined with temozolomide application. Our findings present a new picture of how glutamate signaling via mGluR3 interacts with the phenotypical GBM transcriptional programs in light of recently published GBM cell-state discoveries.

2021 Scientific Article in Nature Cell Biology Nat. Cell Biol. 23, 652-663 (2021)

Non-invasive and high-throughput interrogation of exon-specific isoform expression.

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

2021 Review in Methods in Enzymology Methods Enzymol. 657, 365-383 (2021)

A practical guide to photoswitching optoacoustics tomography.

Photochromic proteins and photoswitching optoacoustics (OA) are a promising combination, that allows OA imaging of even small numbers of cells in whole live animals and thus can facilitate a more wide-spread use of OA in life-science and preclinical research. The concept relies on exploiting the modulation achieved by the photoswitching to discriminate the agents' signal from the non-modulating background. Here we share our analysis approaches that can be readily used on data generated with commercial OA tomography imaging instrumentation allowing—depending on the used photoswitching agent and sample—routine visualizations of as little as several hundreds of transgene labeled cells per imaging volume in the live animal.

2021 Scientific Article in Photoacoustics Photoacoustics 23:100283 (2021)

Multispectral optoacoustic tomography of lipid and hemoglobin contrast in human carotid atherosclerosis.

Several imaging techniques aim at identifying features of carotid plaque instability but come with limitations, such as the use of contrast agents, long examination times and poor portability. Multispectral optoacoustic tomography (MSOT) employs light and sound to resolve lipid and hemoglobin content, both features associated with plaque instability, in a label-free, fast and highly portable way. Herein, 5 patients with carotid atherosclerosis, 5 healthy volunteers and 2 excised plaques, were scanned with handheld MSOT. Spectral unmixing allowed visualization of lipid and hemoglobin content within three ROIs: whole arterial cross-section, plaque and arterial lumen. Calculation of the fat-blood-ratio (FBR) value within the ROIs enabled the differentiation between patients and healthy volunteers (P = 0.001) and between plaque and lumen in patients (P = 0.04). Our results introduce MSOT as a tool for molecular imaging of human carotid atherosclerosis and open new possibilities for research and clinical assessment of carotid plaques.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11629:116292D (2021)

Optoacoustic visualization of GCaMP6f labeled deep brain activity in a murine intracardiac perfusion model.

The inability to directly visualize large-scale neural dynamics across the entire mammalian brain in the millisecond temporal resolution regime is among the main limitations of existing neuroimaging methods. Recent advances in optoacoustic imaging systems have led to the establishment of this technology as an alternative method for real-time deep-tissue observations. Particularly, functional optoacoustic neurotomography (FONT) has recently been suggested for three-dimensional imaging of both direct calcium activity and cerebral hemodynamic parameters in rodents. However, the lack of suitable calcium indicators featuring optical absorption peaks within the so-called near-infrared window has hampered the applicability of FONT for imaging neuronal activity deep within the mammalian brain. To surmount this challenge, we developed and validated an intracardially perfused murine brain model labelled with genetically encoded calcium indicator GCaMP6f that closely simulates in vivo conditions. Penetration of light through skull and skin is greatly facilitated after blood is substituted by artificial cerebrospinal fluid (ACSF). The new preparation enabled here the observation of stimulus-evoked calcium dynamics within the mouse brain at penetration depths and spatio-temporal resolution scales not attainable with other neuroimaging techniques.

2021 Scientific Article in Frontiers in Public Health Front. Publ. Health 9:678856 (2021)

Data-independent acquisition proteomics reveals long-term biomarkers in the serum of C57BL/6J mice following local high-dose heart irradiation.

Background and Purpose: Cardiotoxicity is a well-known adverse effect of radiation therapy. Measurable abnormalities in the heart function indicate advanced and often irreversible heart damage. Therefore, early detection of cardiac toxicity is necessary to delay and alleviate the development of the disease. The present study investigated long-term serum proteome alterations following local heart irradiation using a mouse model with the aim to detect biomarkers of radiation-induced cardiac toxicity. Materials and Methods: Serum samples from C57BL/6J mice were collected 20 weeks after local heart irradiation with 8 or 16 Gy X-ray; the controls were sham-irradiated. The samples were analyzed by quantitative proteomics based on data-independent acquisition mass spectrometry. The proteomics data were further investigated using bioinformatics and ELISA. Results: The analysis showed radiation-induced changes in the level of several serum proteins involved in the acute phase response, inflammation, and cholesterol metabolism. We found significantly enhanced expression of proinflammatory cytokines (TNF-α, TGF-β, IL-1, and IL-6) in the serum of the irradiated mice. The level of free fatty acids, total cholesterol, low-density lipoprotein (LDL), and oxidized LDL was increased, whereas that of high-density lipoprotein was decreased by irradiation. Conclusions: This study provides information on systemic effects of heart irradiation. It elucidates a radiation fingerprint in the serum that may be used to elucidate adverse cardiac effects after radiation therapy.

2021 Scientific Article in Advanced Optical Materials Adv. Opt. Mater. 9:2100256 (2021)

Silicon-photonics point sensor for high-resolution optoacoustic imaging.

The recent development of ultrasound sensing using the silicon-photonics platform has enabled super-resolution optoacoustic imaging not possible by piezoelectric technology or polymeric optical microresonators. The silicon waveguide etalon detector (SWED) design exploits the sub-micrometer light confinement in the cross-section of a silicon strip waveguide to achieve a sensor aperture which is 13-fold to 30-fold smaller than the cutoff wavelength of the sensor. While its performance in near-field scanning optoacoustic imaging has been previously studied, the operational characteristics of this sensor as it relates to conventional optoacoustic imaging applications are not known. Here, for the first time, the application of the SWED in optoacoustic mesoscopy is investigated, the interaction of the sensor with ultrasound in the far-field is characterized, the acoustic point spread function up to a depth of 10 mm is measured, and 3D vasculature-mimicking phantoms are imaged. The measured point spread function of the sensor shows that surface acoustic waves can degrade the lateral resolution. Nevertheless, superior resolution is demonstrated over any state-of-the-art ultrasound sensor, over the whole range of imaging depths that are of interest to optoacoustic mesoscopy. Silicon photonics is proposed as a powerful and promising new platform for ultrasonics and optoacoustics.

2021 Scientific Article in Cell Metabolism Cell Metab. 33, 1685-1700.e9 (2021)

Liver-fibrosis-activated transcriptional networks govern hepatocyte reprogramming and intra-hepatic communication.

Liver fibrosis is a strong predictor of long-term mortality in individuals with metabolic-associated fatty liver disease; yet, the mechanisms underlying the progression from the comparatively benign fatty liver state to advanced non-alcoholic steatohepatitis (NASH) and liver fibrosis are incompletely understood. Using cell-type-resolved genomics, we show that comprehensive alterations in hepatocyte genomic and transcriptional settings during NASH progression, led to a loss of hepatocyte identity. The hepatocyte reprogramming was under tight cooperative control of a network of fibrosis-activated transcription factors, as exemplified by the transcription factor Elf-3 (ELF3) and zinc finger protein GLIS2 (GLIS2). Indeed, ELF3- and GLIS2-controlled fibrosis-dependent hepatokine genes targeting disease-associated hepatic stellate cell gene programs. Thus, interconnected transcription factor networks not only promoted hepatocyte dysfunction but also directed the intra-hepatic crosstalk necessary for NASH and fibrosis progression, implying that molecular "hub-centered" targeting strategies are superior to existing mono-target approaches as currently used in NASH therapy.

2021 Review in Methods in Enzymology Methods Enzymol. 657, 349-364 (2021)

Bioengineered bacterial vesicles for optoacoustics-guided phototherapy.

Genetically engineered bacterial outer membrane vesicles (OMVs) offer promising applications for gene therapy, immunotherapy, and vaccine delivery. Importantly, OMVs are biocompatible, biodegradable, and easy to engineer and produce on a large scale. In this chapter, we discuss the development and application of bioengineered OMVs for optoacoustics-guided phototherapy applications (theranostics). We provide detailed protocols for OMVs preparation, characterization, and in vitro and in vivo validation. The engineered OMVs carry the biopolymer melanin, which generates a strong optoacoustic (OA) signal and intense heat upon absorption of near-infrared (NIR) light, enabling optoacoustics-guided cancer diagnosis and photothermal therapy in vivo.

2021 Scientific Article in Journal of Biophotonics J. Biophotonics 14:e202100048 (2021)

Intravascular molecular-structural imaging with a miniaturized integrated near-infrared fluorescence and ultrasound catheter.

Coronary artery disease (CAD) remains a leading cause of mortality and warrants new imaging approaches to better guide clinical care. We report on a miniaturized, hybrid intravascular catheter and imaging system for comprehensive coronary artery imaging in vivo. Our catheter exhibits a total diameter of 1.0 mm (3.0 French), equivalent to standalone clinical intravascular ultrasound (IVUS) catheters but enables simultaneous near-infrared fluorescence (NIRF) and IVUS molecular-structural imaging. We demonstrate NIRF-IVUS imaging in vitro in coronary stents using NIR fluorophores, and compare NIRF signal strengths for prism and ball lens sensor designs in both low and high scattering media. Next, in vivo intravascular imaging in pig coronary arteries demonstrates simultaneous, co-registered molecular-structural imaging of experimental CAD inflammation on IVUS and distance-corrected NIRF images. The obtained results suggest substantial potential for the NIRF-IVUS catheter to advance standalone IVUS, and enable comprehensive phenotyping of vascular disease to better assess and treat patients with CAD.

2021 Review in Frontiers in Neuroscience Front. Neurosci. 15:655247 (2021)

Photoacoustic neuroimaging - Perspectives on a maturing imaging technique and its applications in neuroscience.

A prominent goal of neuroscience is to improve our understanding of how brain structure and activity interact to produce perception, emotion, behavior, and cognition. The brain's network activity is inherently organized in distinct spatiotemporal patterns that span scales from nanometer-sized synapses to meter-long nerve fibers and millisecond intervals between electrical signals to decades of memory storage. There is currently no single imaging method that alone can provide all the relevant information, but intelligent combinations of complementary techniques can be effective. Here, we thus present the latest advances in biomedical and biological engineering on photoacoustic neuroimaging in the context of complementary imaging techniques. A particular focus is placed on recent advances in whole-brain photoacoustic imaging in rodent models and its influential role in bridging the gap between fluorescence microscopy and more non-invasive techniques such as magnetic resonance imaging (MRI). We consider current strategies to address persistent challenges, particularly in developing molecular contrast agents, and conclude with an overview of potential future directions for photoacoustic neuroimaging to provide deeper insights into healthy and pathological brain processes.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11642:116422K (2021)

Concurrent in vivo tumor ablation and real-time optoacoustic monitoring with a pulsed 1064 nm laser source.

Laser ablation (LA) is gaining acceptance for the treatment of tumors as a viable alternative to surgical resection. In parallel, optoacoustic tomography (OAT) has enabled defining new regimes for diagnosis and characterization of malignant neoplastic lesions with high sensitivity and specificity. Even though pulsed nanosecond lasers are commonly used for both imaging and therapeutic purposes, real-time thermal treatment monitoring with a single laser source has not been previously attempted. Herein, we demonstrate the feasibility of combined OAT and LA by percutaneous irradiation of subcutaneous tumors with a 100 mJ short-pulsed (∼5 ns) laser operating at 1064 nm and 100 Hz pulse repetition frequency. The OAT images rendered with a spherical ultrasound transducer array enabled real-time monitoring of the LA lesion progression, which is essential for determining the optimal treatment end-point. Local changes in the optoacoustic signal intensity associated with the induced temperature changes as well as structural alterations in the tumor vasculature could clearly be observed. The optoacoustic volumetric projections further correlated with crosssections extracted from the excised tumors. This newly enabled capability anticipates new theranostic approaches in cancer research and treatment with potential applicability in a clinical setting.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11642:116420A (2021)

Whole-body visualization of nanoagent kinetics in mice with flash scanning volumetric optoacoustic tomography.

Visualizing whole-body dynamics across entire living organisms is crucial for understanding complex biology, disease progression as well as evaluating efficacy of new drugs and therapies. Existing small animal functional and molecular imaging modalities either suffer from low spatial and temporal resolution, limited penetration depth or poor contrast. In this work, we present flash scanning volumetric optoacoustic tomography (fSVOT) imaging system that enables the acquisition speeds required for visualizing fast kinetics and biodistribution of optical contrast agents across whole mice. fSVOT can render images of intricate vascular and organ anatomy with rich contrast by capitalizing on the large angular coverage of a spherical matrix array transducer rapidly scanned around the mouse. Volumetric (three-dimensional) images with 200 μm resolution can be acquired within 45 seconds, which corresponds to an imaging speed gain of an order of magnitude with respect to existing state-of-the-art modalities offering comparable resolution performance. We demonstrate volumetric tracking and quantification of gold nanorod kinetics and their differential uptake across the spleen, liver and kidneys. Overall, fSVOT offers unprecedented capabilities for multi-scale imaging of pharmacokinetics and bio-distribution of agents with high contrast, resolution and image acquisition speed.

2021 Scientific Article in Proceedings of SPIE Proc. SPIE 11642:116422L (2021)

Transmission-reflection optoacoustic ultrasound (TROPUS) imaging of mammary tumors.

Ultrasound (US) and optoacoustic (OA) imaging provide complementary information for quantitative analysis of the tumor microenvironment. Herein, we demonstrate the unique capabilities of transmission-reflection optoacoustic ultrasound (TROPUS) for characterizing breast cancer in tumor-bearing mice. For this, 4 different mice featuring orthotopic tumor of different sizes were scanned with a full-ring ultrasound transducer array to simultaneously render pulse-echo US images, speed of sound (SoS) maps and OA images. The tumor size, vascular density and its elastic parameters were further quantified in the images. Our results pave the way toward clinical translation of the hybrid TROPUS imaging for tumor detection and characterization.

2021 Scientific Article in IEEE Transactions on Medical Imaging IEEE Trans. Med. Imaging 40, 3349-3357 (2021)

In vivo three-dimensional Raster Scan Optoacoustic Mesoscopy using Frequency Domain Inversion.

Optoacoustic signals are typically reconstructed into images using inversion algorithms applied in the time-domain. However, time-domain reconstructions can be computationally intensive and therefore slow when large amounts of raw data are collected from an optoacoustic scan. Here we consider a fast weighted ω-κ (FWOK) algorithm operating in the frequency domain to accelerate the inversion in raster-scan optoacoustic mesoscopy (RSOM), while seamlessly incorporating impulse response correction with minimum computational burden. We investigate the FWOK performance with RSOM measurements from phantoms and mice in vivo and obtained 360-fold speed improvement over inversions based on the back-projection algorithm in the time-domain. This previously unexplored inversion of in vivo optoacoustic data with impulse response correction in frequency domain reconstructions points to a promising strategy of accelerating optoacoustic imaging computations, toward video-rate tomography.

2021 Scientific Article in Biological Trace Element Research Biol. Trace Elem. Res. 200, 1688-1698 (2021)

Silica nanoparticles induce hepatotoxicity by triggering oxidative damage, apoptosis, and bax-Bcl2 signaling pathway.

The increase in the usage of silica nanoparticles (SiNPs) in the industrial and medical fields has raised concerns about their possible adverse effects on human health. The present study aimed to investigate the potential adverse effects of SiNPs at daily doses of 25 and 100 mg/kg body weight intraperitoneally (i.p.) for 28 consecutive days on markers of liver damage in adult male rats. Results revealed that SiNPs induced a marked increase in serum markers of liver damage, including lactate dehydrogenase (LDH), alanine aminotransferase (ALAT), and aspartate aminotransferase (ASAT). SiNPs also induced an elevation of reactive oxygen species (ROS) production in liver, along with an increase in oxidative stress markers (NO, MDA, PCO, and H2O2), and a decrease in antioxidant enzyme activities (CAT, SOD, and GPx). Quantitative real-time PCR showed that SiNPs also induced upregulation of pro-apoptotic gene expression (including Bax, p53, Caspase-9/3) and downregulation of anti-apoptotic factors Bcl-2. Moreover, histopathological analysis revealed that SiNPs induced hepatocyte alterations, which was accompanied by sinusoidal dilatation, Kupffer cell hyperplasia, and the presence of inflammatory cells in the liver. Taken together, these data showed that SiNPs trigger hepatic damage through ROS-activated caspase signaling pathway, which plays a fundamental role in SiNP-induced apoptosis in the liver.

2021 Scientific Article in European Journal of Endocrinology Eur. J. Endocrinol. 185, 179-191 (2021)

Mass spectrometry imaging identifies metabolic patterns associated with malignant potential in pheochromocytoma and paraganglioma.

OBJECTIVE: Within the past decade, important genetic drivers of pheochromocytoma and paraganglioma (PPGLs) development have been identified. The pathophysiological mechanism that translate these alterations into functional autonomy and potentially malignant behavior have not been elucidated in detail. Here we used MALDI-mass spectrometry imaging (MALDI-MSI) of formalin-fixed paraffin-embedded tissue specimens to comprehensively characterize the metabolic profiles of PPGLs. DESIGN AND METHODS: MALDI-MSI was conducted in 344 PPGLs and results correlated with genetic and phenotypic information. We experimentally silenced genetic drivers by siRNA in PC12 cells to confirm their metabolic impact in vitro. RESULTS: Tissue abundance of kynurenine pathway metabolites such as xanthurenic acid was significantly lower (P = 5.06E-11) in the pseudohypoxia pathway cluster 1 compared to PPGLs of the kinase-driven PPGLs cluster 2. Lower abundance of xanthurenic acid was associated with shorter metastasis-free survival (log-rank tests P = 7.96E-06) and identified as a risk factor for metastasis independent of the genetic status (hazard ratio, 32.6, P = 0.002). Knock-down of Sdhb and Vhl in an in vitro model demonstrated that inositol metabolism and sialic acids were similarly modulated as in tumors of the respective cluster. CONCLUSIONS: The present study has identified distinct tissue metabolomic profiles of PPGLs in relation to tumor genotypes. In addition, we revealed significantly altered metabolites in the kynurenine pathway in metastatic PPGLs, which can aid in the prediction of its malignant potential. However, further validation studies will be required to confirm our findings.

2021 Scientific Article in Science Advances Sci. Adv. 7:eabd1505 (2021)

Noninvasive visualization of electrical conductivity in tissues at the micrometer scale.

Despite its importance in regulating cellular or tissue function, electrical conductivity can only be visualized in tissue indirectly as voltage potentials using fluorescent techniques, or directly with radio waves. These either requires invasive procedures like genetic modification or suffers from limited resolution. Here, we introduce radio-frequency thermoacoustic mesoscopy (RThAM) for the noninvasive imaging of conductivity by exploiting the direct absorption of near-field ultrashort radio-frequency pulses to stimulate the emission of broadband ultrasound waves. Detection of ultrasound rather than radio waves enables micrometer-scale resolutions, over several millimeters of tissue depth. We confirm an imaging resolution of <30 μm in phantoms and demonstrate microscopic imaging of conductivity correlating to physical structures in 1- and 512-cell zebrafish embryos, as well as larvae. These results support RThAM as a promising method for high-resolution, label-free assessment of conductivity in tissues.

2021 Scientific Article in Diabetologia Diabetologia 64, 1850-1865 (2021)

HAND2 is a novel obesity-linked adipogenic transcription factor regulated by glucocorticoid signalling.

Aims/hypothesis: Adipocytes are critical cornerstones of energy metabolism. While obesity-induced adipocyte dysfunction is associated with insulin resistance and systemic metabolic disturbances, adipogenesis, the formation of new adipocytes and healthy adipose tissue expansion are associated with metabolic benefits. Understanding the molecular mechanisms governing adipogenesis is of great clinical potential to efficiently restore metabolic health in obesity. Here we investigate the role of heart and neural crest derivatives-expressed 2 (HAND2) in adipogenesis. Methods: Human white adipose tissue (WAT) was collected from two cross-sectional studies of 318 and 96 individuals. In vitro, for mechanistic experiments we used primary adipocytes from humans and mice as well as human multipotent adipose-derived stem (hMADS) cells. Gene silencing was performed using siRNA or genetic inactivation in primary adipocytes from loxP and or tamoxifen-inducible Cre-ERT2 mouse models with Cre-encoding mRNA or tamoxifen, respectively. Adipogenesis and adipocyte metabolism were measured by Oil Red O staining, quantitative PCR (qPCR), microarray, glucose uptake assay, western blot and lipolysis assay. A combinatorial RNA sequencing (RNAseq) and ChIP qPCR approach was used to identify target genes regulated by HAND2. In vivo, we created a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter (Hand2 ) and performed a large panel of metabolic tests. Results: We found that HAND2 is an obesity-linked white adipocyte transcription factor regulated by glucocorticoids that was necessary but insufficient for adipocyte differentiation in vitro. In a large cohort of humans, WAT HAND2 expression was correlated to BMI. The HAND2 gene was enriched in white adipocytes compared with brown, induced early in differentiation and responded to dexamethasone (DEX), a typical glucocorticoid receptor (GR, encoded by NR3C1) agonist. Silencing of NR3C1 in hMADS cells or deletion of GR in a transgenic conditional mouse model results in diminished HAND2 expression, establishing that adipocyte HAND2 is regulated by glucocorticoids via GR in vitro and in vivo. Furthermore, we identified gene clusters indirectly regulated by the GR–HAND2 pathway. Interestingly, silencing of HAND2 impaired adipocyte differentiation in hMADS and primary mouse adipocytes. However, a conditional adipocyte Hand2 deletion mouse model using Cre under control of the Adipoq promoter did not mirror these effects on adipose tissue differentiation, indicating that HAND2 was required at stages prior to Adipoq expression. Conclusions/interpretation: In summary, our study identifies HAND2 as a novel obesity-linked adipocyte transcription factor, highlighting new mechanisms of GR-dependent adipogenesis in humans and mice. Data availability: Array data have been submitted to the GEO database at NCBI (GSE148699). Graphical abstract: [Figure not available: see fulltext.] AdipoqCre

2021 Scientific Article in Nature Communications Nat. Commun. 12:2999 (2021)

Orphan GPR116 mediates the insulin sensitizing effects of the hepatokine FNDC4 in adipose tissue.

The proper functional interaction between different tissues represents a key component in systemic metabolic control. Indeed, disruption of endocrine inter-tissue communication is a hallmark of severe metabolic dysfunction in obesity and diabetes. Here, we show that the FNDC4-GPR116, liver-white adipose tissue endocrine axis controls glucose homeostasis. We found that the liver primarily controlled the circulating levels of soluble FNDC4 (sFNDC4) and lowering of the hepatokine FNDC4 led to prediabetes in mice. Further, we identified the orphan adhesion GPCR GPR116 as a receptor of sFNDC4 in the white adipose tissue. Upon direct and high affinity binding of sFNDC4 to GPR116, sFNDC4 promoted insulin signaling and insulin-mediated glucose uptake in white adipocytes. Indeed, supplementation with FcsFNDC4 in prediabetic mice improved glucose tolerance and inflammatory markers in a white-adipocyte selective and GPR116-dependent manner. Of note, the sFNDC4-GPR116, liver-adipose tissue axis was dampened in (pre) diabetic human patients. Thus our findings will now allow for harnessing this endocrine circuit for alternative therapeutic strategies in obesity-related pre-diabetes.

2021 Nature Reviews - Endocrinology Nat. Rev. Endocrinol., DOI: 10.1038/s41574-021-00515-z (2021)

Author Correction: Optoacoustic imaging in endocrinology and metabolism (Nature Reviews Endocrinology, (2021), 17, 6, (323-335), 10.1038/s41574-021-00482-5).

In the original version of this article, in Fig. 1c, the icons for oxygenated haemoglobin and deoxygenated haemoglobin were incorrectly shown outside of the artery and vein. This error has now been corrected in the HTML and PDF versions of the article.

2021 Scientific Article in Cell Metabolism Cell Metab. 33, 1155-1170.e10 (2021)

Obesity-associated hyperleptinemia alters the gliovascular interface of the hypothalamus to promote hypertension.

Pathologies of the micro- and macrovascular systems are a hallmark of the metabolic syndrome, which can lead to chronically elevated blood pressure. However, the underlying pathomechanisms involved still need to be clarified. Here, we report that an obesity-associated increase in serum leptin triggers the select expansion of the micro-angioarchitecture in pre-autonomic brain centers that regulate hemodynamic homeostasis. By using a series of cell- and region-specific loss- and gain-of-function models, we show that this pathophysiological process depends on hypothalamic astroglial hypoxia-inducible factor 1α-vascular endothelial growth factor (HIF1α-VEGF) signaling downstream of leptin signaling. Importantly, several distinct models of HIF1α-VEGF pathway disruption in astrocytes are protected not only from obesity-induced hypothalamic angiopathy but also from sympathetic hyperactivity or arterial hypertension. These results suggest that hyperleptinemia promotes obesity-induced hypertension via a HIF1α-VEGF signaling cascade in hypothalamic astrocytes while establishing a novel mechanistic link that connects hypothalamic micro-angioarchitecture with control over systemic blood pressure.

2021 Scientific Article in Photoacoustics Photoacoustics 22:100263 (2021)

Croconaine-based nanoparticles enable efficient optoacoustic imaging of murine brain tumors.

Contrast enhancement in optoacoustic (photoacoustic) imaging can be achieved with agents that exhibit high absorption cross-sections, high photostability, low quantum yield, low toxicity, and preferential bio-distribution and clearance profiles. Based on advantageous photophysical properties of croconaine dyes, we explored croconaine-based nanoparticles (CR780RGD-NPs) as highly efficient contrast agents for targeted optoacoustic imaging of challenging preclinical tumor targets. Initial characterization of the CR780 dye was followed by modifications using polyethylene glycol and the cancer-targeting c(RGDyC) peptide, resulting in self-assembled ultrasmall particles with long circulation time and active tumor targeting. Preferential bio-distribution was demonstrated in orthotopic mouse brain tumor models by multispectral optoacoustic tomography (MSOT) imaging and histological analysis. Our findings showcase particle accumulation in brain tumors with sustainable strong optoacoustic signals and minimal toxic side effects. This work points to CR780RGD-NPs as a promising optoacoustic contrast agent for potential use in the diagnosis and image-guided resection of brain tumors.

2021 Review in Nature Reviews - Endocrinology Nat. Rev. Endocrinol. 17, 323-335 (2021)

Optoacoustic imaging in endocrinology and metabolism.

Imaging is an essential tool in research, diagnostics and the management of endocrine disorders. Ultrasonography, nuclear medicine techniques, MRI, CT and optical methods are already used for applications in endocrinology. Optoacoustic imaging, also termed photoacoustic imaging, is emerging as a method for visualizing endocrine physiology and disease at different scales of detail: microscopic, mesoscopic and macroscopic. Optoacoustic contrast arises from endogenous light absorbers, such as oxygenated and deoxygenated haemoglobin, lipids and water, or exogenous contrast agents, and reveals tissue vasculature, perfusion, oxygenation, metabolic activity and inflammation. The development of high-performance optoacoustic scanners for use in humans has given rise to a variety of clinical investigations, which complement the use of the technology in preclinical research. Here, we review key progress with optoacoustic imaging technology as it relates to applications in endocrinology; for example, to visualize thyroid morphology and function, and the microvasculature in diabetes mellitus or adipose tissue metabolism, with particular focus on multispectral optoacoustic tomography and raster-scan optoacoustic mesoscopy. We explain the merits of optoacoustic microscopy and focus on mid-infrared optoacoustic microscopy, which enables label-free imaging of metabolites in cells and tissues. We showcase current optoacoustic applications within endocrinology and discuss the potential of these technologies to advance research and clinical practice.

2021 Scientific Article in Frontiers in Oncology Front. Oncol. 11:612354 (2021)

Inhibition of HSP90 as a strategy to radiosensitize glioblastoma: Targeting the DNA damage response and beyond.

Radiotherapy is an essential component of multi-modality treatment of glioblastoma (GBM). However, treatment failure and recurrence are frequent and give rise to the dismal prognosis of this aggressive type of primary brain tumor. A high level of inherent treatment resistance is considered to be the major underlying reason, stemming from constantly activated DNA damage response (DDR) mechanisms as a consequence of oncogene overexpression, persistent replicative stress, and other so far unknown reasons. The molecular chaperone heat shock protein 90 (HSP90) plays an important role in the establishment and maintenance of treatment resistance, since it crucially assists the folding and stabilization of various DDR regulators. Accordingly, inhibition of HSP90 represents a multi-target strategy to interfere with DDR function and to sensitize cancer cells to radiotherapy. Using NW457, a pochoxime-based HSP90 inhibitor with favorable brain pharmacokinetic profile, we show here that HSP90 inhibition at low concentrations with per se limited cytotoxicity leads to downregulation of various DNA damag