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Based on analysis of 819 human biopsies, essential derangement in diabetic nephropathy consists of accumulation of worn-out glomerular basement membrane in the mesangium that may advance to global sclerosis. The most frequent pathway to nephron dropout starts with the penetration of glomerular capillaries into Bowman’s capsule (BC), delivering an exudate into BC that spreads around the entire glomerular circumference and via the glomerulotubular junction onto the tubule, resulting in glomerular sclerosis and chronic tubulointerstitial damage.

PurposeEwing sarcoma (EwS) is a highly aggressive bone- or soft tissue-associated malignancy mostly affecting children, adolescents, and young adults. Although multimodal therapies have strongly improved patients’ overall survival over the past decades, the development of prognostic biomarkers for risk-based patient stratification and more effective therapies with less adverse effects is stagnating. Thus, new personalized medicine approaches are urgently required.Experimental designGene expression data of EwS and normal tissues were crossed with survival data to identify highly overexpressed, prognostically relevant, and actionable potential targets. RNA-interference and dose-response assays as well as tissue-microarray analyses were carried out to explore the functional role and druggability of a prominent candidate gene in vitro and in vivo, and to validate its suitability as a prognostic biomarker.ResultsEmploying a multilayered screening approach, we discover ribonucleotide reductase regulatory subunit M2 (RRM2) as a promising therapeutic target and prognostic biomarker in EwS. Through analysis of two independent EwS patient cohorts, we show that RRM2 mRNA and protein overexpression is associated with an aggressive clinical phenotype and poor patients’ overall survival. In agreement, RRM2 silencing as well as pharmacological inhibition by the specific inhibitor triapine (3-AP) significantly reduces EwS growth in vitro and in vivo. Furthermore, we present evidence that pharmacological RRM2 inhibition by triapine can overcome chemoresistance against doxorubicin or gemcitabine, and synergize with cell cycle checkpoint inhibitors (CHEK1 or WEE1).ConclusionsBased on the aggressive phenotype mediated by and the druggability of RRM2 our results provide a translational rationale for exploiting RRM2 as a novel therapeutic target in EwS and prompt further clinical investigations.

Background and Aims Tight junction (TJ) proteins have been suggested as molecular correlates for peritoneal semi-permeability and dialytic transport function in patients on peritoneal dialysis. Junction abundance in healthy individuals, in those with CKD5 and in patients on PD has not been described yet, the relation with peritoneal solute transport is unknown. Method Junction and transporter expression was analysed in multi-omics data sets from microdissected omental arterioles in children with normal renal function, CKD5 and on PD with low and high glucose degradation product (GDP) content (n=6/group). Parietal peritoneal tight junction proteins CLDN-1,-2,-3,-4,-5,-15, the adapter protein of claudins to actin cytoskeleton protein, zonula occludens-1 (ZO-1), the tricellular junction protein tricellulin (TriC), and transcellular transporters for sodium (ENaC), glucose (SGLT-1) and phosphate (PIT-1) were quantified in 40 non-CKD individuals, 20 children with CKD5 and 20 and 15 children on low- and high-GDP PD by quantitative, digital immunohistochemistry. Findings were correlated to 2-hour peritoneal equilibration test data obtained within 6 months of biopsy sampling (n=23). Primary human umbilical vein endothelial cells (HUVEC) were used to study the effects of single PD compounds on transepithelial electrical resistance (TER) and molecular size-dependent paracellular transport capacity. Co-stained monolayers were visualized by confocal microscopy. Single junction molecule localization and clustering were analysed by super resolution microscopy. Results Transcriptome and proteome pathway enrichment analysis of arteriolar junction and membrane protein demonstrated regulation in CKD5 versus health, and differential regulation by low- and high-GDP PD versus CKD5. In the parietal peritoneum all junctions and cellular transporters were expressed in endothelial and mesothelial cells. Pore forming CLDN-2, -4 and -15 were localized also in submesothelial immune cells. Parietal peritoneal junction abundance was age-dependent and also modified by CKD5 and PD. Mesothelial and endothelial abundance of the selective cation/water channel CLDN-2 increased in patients on low- and high-GDP PD fluids. Adaptor protein ZO-1 was upregulated in low GDP-PD versus CKD5, while sealing proteins CLDN -3 and -5 were downregulated. D/P creatinine, D/P phosphate, D/D0 glucose were similar in CKD5 and PD groups. D/P creatinine correlated with mesothelial CLDN-15, with arteriolar CLDN-2 and TriC and with endothelial ENaC. D/P phosphate correlated with endothelial CLDN-15, D/D0 glucose with mesothelial CLDN-4 and arteriolar CLDN-2. Capillary ZO-1 correlated with 24-h ultrafiltration standardized to body surface area and dialytic glucose exposure. In vitro, TER was decreased by low pH, glucose and 0.5µM methylglyoxal after 5h. Alanyl-glutamine (AlaGln) dose-dependently increased TER, and reduced 10kDa and 70kDa solute at 24mM, increased the abundance of ZO-1 and CLDN5 at cell-cell contacts, and on nanoscale clustering of the pore-forming CLDN2 and CLDN5. Conclusion Abundance of parietal peritoneal sealing and pore forming junctions and transcellular solute transporters varies with cell type and age and is differentially regulated by PD and associated with dialytic transport function. Our preliminary analyses illustrate the role of junctions and cellular transporters for solute transport across the peritoneal mesothelial and endothelial cell barrier. In-depth understanding of specific molecular functions should provide targets for modulation to improve efficacy of PD.

The CD73 pathway is an important anti-inflammatory mechanism in various disease settings. Observations in mouse models suggested that CD73 might have a protective role in kidney damage, however, no direct evidence of its role in human kidney disease has been described to date. Here, we hypothesized that podocyte injury in human kidney diseases alters CD73 expression that may facilitate the diagnosis of podocytopathies. We assessed the expression of CD73 and one of its functionally important targets, the C-C chemokine receptor type 2 (CCR2), in podocytes from kidney biopsies of 39 patients with podocytopathy (including focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulonephritis (MGN) and amyloidosis) and a control group. Podocyte CD73 expression in each of the disease groups was significantly increased in comparison to controls (p &lt, 0.001–p &lt, 0.0001). Moreover, there was a marked negative correlation between CD73 and CCR2 expression, as confirmed by immunohistochemistry and immunofluorescence (Pearson r = −0.5068, p = 0.0031, Pearson r = −0.4705, p = 0.0313, respectively), thus suggesting a protective role of CD73 in kidney injury. Finally, we identify CD73 as a novel potential diagnostic marker of human podocytopathies, particularly of MCD that has been notorious for the lack of pathological features recognizable by light microscopy and immunohistochemistry.

Background The diagnosis of most cancers is made by a board-certified pathologist based on a tissue biopsy under the microscope. Recent research reveals a high discordance between individual pathologists. For melanoma, the literature reports on 25–26% of discordance for classifying a benign nevus versus malignant melanoma. A recent study indicated the potential of deep learning to lower these discordances. However, the performance of deep learning in classifying histopathologic melanoma images was never compared directly to human experts. The aim of this study is to perform such a first direct comparison. Methods A total of 695 lesions were classified by an expert histopathologist in accordance with current guidelines (350 nevi/345 melanoma). Only the haematoxylin & eosin (H&E) slides of these lesions were digitalised via a slide scanner and then randomly cropped. A total of 595 of the resulting images were used to train a convolutional neural network (CNN). The additional 100 H&E image sections were used to test the results of the CNN in comparison to 11 histopathologists. Three combined McNemar tests comparing the results of the CNNs test runs in terms of sensitivity, specificity and accuracy were predefined to test for significance (p Findings The CNN achieved a mean sensitivity/specificity/accuracy of 76%/60%/68% over 11 test runs. In comparison, the 11 pathologists achieved a mean sensitivity/specificity/accuracy of 51.8%/66.5%/59.2%. Thus, the CNN was significantly (p = 0.016) superior in classifying the cropped images. Interpretation With limited image information available, a CNN was able to outperform 11 histopathologists in the classification of histopathological melanoma images and thus shows promise to assist human melanoma diagnoses.

Localization microscopy (LM) allows to acquire pointillistic superresolution images of biological structures on the nanoscale. However, current structure reconstruction and segmentation approaches suffer from either exclusion of small structures or strong dependence on a-priori knowledge. We propose reconstruction methods based on compressed sensing (CS) denoising in combination with the isodata threshold for segmentation. The methods are verified on artificial test data. For the denoising, a Haar dictionary and a KSVD dictionary learning on artificial data are used. Both methods perform significantly better than the reference algorithm, a linear density filter, in terms of root-mean-square deviation from the ground truth. Furthermore, exemplary results on real LM data of irradiated cell nuclei with Heterochromatin labeling make small structures visible that are suppressed by the reference method. CS denoising demonstrates promising results for reconstruction of LM data.

Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.

Keratinocytes have a pivotal role in the regulation of immune responses, but the impact of antigen presentation by these cells is still poorly understood, particularly in a situation where the antigen will be presented only in adult life. Here, we generated a transgenic mouse model in which keratinocytes exclusively present a myelin basic protein (MBP) peptide covalently linked to the major histocompatibility complex class II β-chain, solely under inflammatory conditions. In these mice, inflammation caused by epicutaneous contact sensitizer treatment resulted in keratinocyte-mediated expansion of MBP-specific CD4(+) T cells in the skin. Moreover, repeated contact sensitizer application preceding a systemic MBP immunization reduced the reactivity of the respective CD4(+) T cells and lowered the symptoms of the resulting experimental autoimmune encephalomyelitis. This downregulation was CD4(+) T-cell-mediated and dependent on the presence of the immune modulator Dickkopf-3. Thus, presentation of a neo self-antigen by keratinocytes in the inflamed, adult skin can modulate CD4(+) T-cell auto-aggression at a distal organ.

Originally designed for optical storage, fluorescent nuclear track detectors (FNTD) based on single aluminum oxide crystals contain aggregate color centers that show permanent radiochromic transformation when bombarded with ionizing radiation. Transformed centers produce high-yield fluorescence at 750 nm when stimulated at 620 nm. This enables non-destructive readout using confocal laser scanning microscopes (CLSMs). Since the intensity signal depends on the local energy deposition, 3D particle trajectories through the crystal can be assessed. Together with the excellent sensitivity of FNTDs, this enables derivation of information on track location, direction, energy loss, etc. over the entire particle and energy range found in ion beam therapy. Effects such as projectile fragmentation and secondary electron trajectories can be studied in detail with diffraction-limited resolution. Due to their biocompatibility, autoclavability and since post-irradiation chemical processing is not needed, FNTDs can show significant superiority to existing technologies such as plastic nuclear track detectors (e.g. CR-39). The Heavy Ion Therapy Research Group at the German Cancer Research Center studies FNTD technology for application on three main fields: (a) Fundamental dosimetry quantities (w-value, I-value) in ion beams: FNTDs allow for determination of particle fluence and range with very high accuracy. (b) In vivo track-based assessment of dose to organs at risk during therapy: FNTDs represent one of a few systems that enable biological dose estimation which is the essential predictor for clinical outcome in ion beam therapy. In addition, FNTDs are small, resilient, wireless and biocompatible and can, therefore, be used within phantoms, animal models or even patients. (c) Radiobiology: Our group was the first to use FNTDs as substrate for cell ("Cell-Fit-HD"). This enables to correlate microscopic physical parameters and subcellular/cell response both in fixed and living cell and study cellular processes fundamental to ion beam radiotherapy that are hitherto little understood. The talk will present the basic principle of FNTD technology, our group’s technical implementation as well as the latest methodological developments and application results.

We introduce a fast spectral imaging system using an electron-multiplying charge-coupled device (EM-CCD) as a detector. Our system is combined with a custom-built two-photon excitation laser scanning microscope and has 80 detection channels, which allow for high spectral resolution and fast frame acquisition without any loss of spectral information. To demonstrate the efficiency of our approach, we applied this technology to monitor fluorescent proteins and quantum dot-labeled G protein-coupled receptors in living cells as well as autofluorescence in tissue samples.

We present an implementation of fluorescence correlation spectroscopy with spectrally resolved detection based on a combined commercial confocal laser scanning/fluorescence correlation spectroscopy microscope. We have replaced the conventional detection scheme by a prism-based spectrometer and an electron-multiplying charge-coupled device camera used to record the photons. This allows us to read out more than 80,000 full spectra per second with a signal-to-noise ratio and a quantum efficiency high enough to allow single photon counting. We can identify up to four spectrally different quantum dots in vitro and demonstrate that spectrally resolved detection can be used to characterize photophysical properties of fluorophores by measuring the spectral dependence of quantum dot fluorescence emission intermittence. Moreover, we can confirm intracellular cross-correlation results as acquired with a conventional setup and show that spectral flexibility can help to optimize the choice of the detection windows.