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Abstract Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Glomerular hyperfiltration and albuminuria subject the proximal tubule (PT) to a subsequent elevation of workload, growth, and hypoxia. Hypoxia plays an ambiguous role in the development and progression of DKD and shall be clarified in our study. PT-von-Hippel-Lindau (Vhl)-deleted mouse model in combination with streptozotocin (STZ)-induced type I diabetes mellitus (DM) was phenotyped. In contrary to PT-Vhl-deleted STZ-induced type 1 DM mice, proteinuria and glomerular hyperfiltration occurred in diabetic control mice the latter due to higher nitric oxide synthase 1 and sodium and glucose transporter expression. PT Vhl deletion and DKD share common alterations in gene expression profiles, including glomerular and tubular morphology, and tubular transport and metabolism. Compared to diabetic control mice, the most significantly altered in PT Vhl-deleted STZ-induced type 1 DM mice were Ldc-1, regulating cellular oxygen consumption rate, and Zbtb16, inhibiting autophagy. Alignment of altered genes in heat maps uncovered that Vhl deletion prior to STZ-induced DM preconditioned the kidney against DKD. HIF-1α stabilization leading to histone modification and chromatin remodeling resets most genes altered upon DKD towards the control level. These data demonstrate that PT HIF-1α stabilization is a hallmark of early DKD and that targeting hypoxia prior to the onset of type 1 DM normalizes renal cell homeostasis and prevents DKD development.

IntroductionMediastinal tumors, particularly non-neuroendocrine thymic epithelial tumors (TET) are relatively uncommon, posing challenges for extensive epidemiological studies. This study presents a comprehensive analysis of these tumors in the United States (US) and Germany (GER) from 1999 to 2019.MethodsPatients aged 0-19 (n=478) and ≥20 years (n=17,459) diagnosed with malignant tumors of the anterior mediastinum were identified from the Surveillance, Epidemiology, and End Results registry (SEER) and the Zentrum für Krebsregisterdaten (ZfKD) databases.ResultsAmong patients aged ≥20 years, TETs accounted for the most prevalent anterior mediastinal tumors (US/GER: 63%/64%), followed by lymphomas (14%/8%). For patients

Background: Economic restrictions and workforce cuts have continually challenged conventional autopsies. Recently, the COVID-19 pandemic has added tissue quality and safety requirements to the investigation of this disease, thereby launching efforts to upgrade autopsy strategies. Methods: In this proof-of-concept study, we performed bedside ultrasound-guided minimally invasive autopsy (US-MIA) in the ICU of critically ill COVID-19 patients using a structured protocol to obtain non-autolyzed tissue. Biopsies were assessed for their quality (vitality) and length of biopsy (mm) and for diagnosis. The efficiency of the procedure was monitored in five cases by recording the time of each step and safety issues by swabbing personal protective equipment and devices for viral contamination. Findings: Ultrasound examination and tissue procurement required a mean time period of 13 min and 54 min, respectively. A total of 318 multiorgan biopsies were obtained from five patients. Quality and vitality standards were fulfilled, which not only allowed for specific histopathological diagnosis but also the reliable detection of SARS-CoV-2 virions in unexpected organs using electronic microscopy and RNA-expressing techniques. Interpretation: Bedside multidisciplinary US-MIA allows for the fast and efficient acquisition of autolytic-free tissue and offers unappreciated potential to overcome the limitations of research in postmortem studies.

Abstract Bladder cancer is one of the top 10 frequently occurring cancers and leads to most cancer deaths worldwide. Recently, blue light (BL) cystoscopy-based photodynamic diagnosis was introduced as a unique technology to enhance the detection of bladder cancer, particularly for the detection of flat and small lesions. Here, we aim to demonstrate a BL image-based artificial intelligence (AI) diagnostic platform using 216 BL images, that were acquired in four different urological departments and pathologically identified with respect to cancer malignancy, invasiveness, and grading. Thereafter, four pre-trained convolution neural networks were utilized to predict image malignancy, invasiveness, and grading. The results indicated that the classification sensitivity and specificity of malignant lesions are 95.77% and 87.84%, while the mean sensitivity and mean specificity of tumor invasiveness are 88% and 96.56%, respectively. This small multicenter clinical study clearly shows the potential of AI based classification of BL images allowing for better treatment decisions and potentially higher detection rates.

Abstract Renal cell carcinoma (RCC) is the deadliest primary genitourinary malignancy typically associated with asymptomatic initial presentation and poorly predictable survival. Next to established risk factors, tumor microenvironment may alter metastatic capacity and immune landscape. Due to their high concentrations, sulfoglycolipids (sulfatides) were among the first well-described antigens in RCC that are associated with worse prognosis. As sulfatide detection in routine diagnostics is not possible, we aimed to test the prognostic value of its protein counterpart, sulfatide-producing enzyme Gal3ST1. We performed retrospective long-term follow up analysis of Gal3ST1 expression as prognostic risk factor in a representative RCC patient cohort. We observed differentially regulated Gal3ST1 expression in all RCC types, being significantly more associated with clear cell RCC than to chromophobe RCC (p = 0.001). Surprisingly, in contrast to published observations from in vitro models, we could not confirm an association between Gal3ST1 expression and a malignant clinical behaviour of the RCC. In our cohort, Gal3ST1 did not significantly influence progression-free survival (Hazard Ratio (HR): 1.7 95% CI (0.6–4.9), p = 0.327). Particularly after adjusting for histology, T-stage, N-status and M-status at baseline, we observed no independent prognostic effect (HR = 1.0 95% CI (0.3–3.3), p = 0.96). The analysis of Gal3ST1 mRNA expression in a TCGA dataset supported the results of our cohort. Thus, Gal3ST1 might help to differentiate between chromophobe RCC and other frequent RCC entities but—despite previously published data from cell culture models—does not qualify as a prognostic marker for RCC. Further investigation of regulatory mechanisms of sulfatide metabolism in human RCC microenvironment is necessary to understand the role of this quantitatively prominent glycosphingolipid in RCC progression.

Abstract Background Growing evidence supports the pivotal role of long non-coding RNAs (lncRNAs) in the regulation of cancer development and progression. Their expression patterns and biological function in muscle invasive bladder cancer (MIBC) remain elusive. Methods Transcript levels of lncRNA miR-31 host gene (MIR31HG) and its splice variants were measured in our MIBC cohort (n = 102) by qRT-PCR, and validated in silico by the TCGA cohort (n = 370). Kaplan-Meier and multiple Cox regression analysis were conducted to evaluate the survival significance of MIR31HG and its splice variants. Functional experiments were performed to examine the proliferation and migration abilities of MIR31HG and its splice variants by knockdown approaches. Results In this study, a decreased expression of MIR31HG was found in bladder cancer cells and tissues, except in the basal subtype. Survival analysis showed that high expression of MIR31HG was associated with poor overall survival (OS) and disease-free survival (DFS) in patients with MIBC of basal subtype. Two splice variants of MIR31HG lacking exon 1 (MIR31HGΔE1) and exon 3 (MIR31HGΔE3) were identified to have specific expression patterns in different molecular subtypes of our MIBC cohort. MIR31HGΔE3 was highly expressed in basal subtype tumors. A high expression of MIR31HGΔE1 and MIR31HGΔE3 was associated with worse OS and DFS in our cohort. In vitro experiments revealed that knockdown of MIR31HG inhibits cell proliferation, colony formation, and migration in bladder cancer. Cell proliferation and migration assays after knockdown of splice variants of MIR31HG showed corresponding roles for the full-length transcript. Conclusions Our study demonstrates that MIR31HG and its splice variants could serve as biomarkers for the classification and prognosis prediction of patients with MIBC.

Abstract Comprehensive transcriptome expression analyses of bladder cancer revealed distinct lncRNA clusters with differential molecular and clinical characteristics. In this study, pivotal lncRNAs were assessed for their impact on survival and their differential expression between the molecular bladder cancer subtypes. FFPE samples from chemotherapy-naïve patients with muscle invasive bladder cancer (MIBC) were analyzed on the Nanostring nCounter platform for absolute quantification. An established 36-gene panel was used for molecular subtype classification into basal, luminal and infiltrated MIBC. In a second step, 14 pivotal lncRNAs were assessed for their molecular subtype attribution, and their predictive value in disease-specific survival. In silico validation was performed on a total of 487 MIBC patients (MDA, TGCA and Chungbuk cohort). Several pivotal lncRNAs showed a distinct molecular subtype attribution: e.g. MALAT1 showed a downregulation in the basal subtype (p = 0.009), TUG1 and CBR3AS1 showed an upregulation in the luminal subtype (p ≤ 0.001). High transcript levels of SNHG16, CBR3AS1 and H19 appeared to be predictive for a shorter disease-specific survival. Patients overexpressing putative oncogenes MALAT1 and TUG1 in MIBC tissue presented prolonged survival, suggesting tumor suppressive effects of both lncRNAs. The Nanostring nCounter proved to be a valid platform for the quantification of low-abundance transcripts including lncRNAs.

Background: The simultaneous occurrence of impaired kidney function and paraproteinemia is common in our constantly aging society. Both can be independent entities; however, renal insufficiency can also be caused by the paraprotein. We assessed all kidney biopsies in patients with monoclonal gammopathy in our clinic over the past 20 years and evaluated the histological results. Methods: Biopsies were systematically performed in nearly all patients with paraproteinemia and impaired kidney function (n = 178). The histological findings were systematically evaluated and correlated with the initial clinical diagnosis. Results: We found cast nephropathy (CN) in n = 66 (37.1%) biopsies, AL amyloidosis in n = 31 (17.4%) biopsies, monoclonal immunoglobulin deposition disease (MIDD) in n = 7 (3.9%) biopsies and other renal diseases (ORDs) in n = 74 (41.6%) biopsies. In the latter group, paraprotein-associated changes were found in 37 of 74 (50%) patients, whereas paraprotein-independent changes were found in the other half. Whereas, in the group of patients with MGUS, the findings were heterogenous, most of the patients with known multiple myeloma (MM) or B-NHL showed malignancy-associated changes in the kidney. The biopsy changed the diagnoses in a significant proportion of the patients: The group of patients with MM grew from 71 to 112 patients, whereas, in the MGUS group, only 31 of 44 patients remained. Conclusion: Kidney biopsies in patients with paraproteinemia and renal impairment show a wide range of findings that can lead to a change in diagnosis.

Introduction: This study investigates whether it is possible to predict a final diagnosis based on a written nephropathological description—as a surrogate for image analysis—using various NLP methods. Methods: For this work, 1107 unlabelled nephropathological reports were included. (i) First, after separating each report into its microscopic description and diagnosis section, the diagnosis sections were clustered unsupervised to less than 20 diagnostic groups using different clustering techniques. (ii) Second, different text classification methods were used to predict the diagnostic group based on the microscopic description section. Results: The best clustering results (i) could be achieved with HDBSCAN, using BoW-based feature extraction methods. Based on keywords, these clusters can be mapped to certain diagnostic groups. A transformer encoder-based approach as well as an SVM worked best regarding diagnosis prediction based on the histomorphological description (ii). Certain diagnosis groups reached F1-scores of up to 0.892 while others achieved weak classification metrics. Conclusion: While textual morphological description alone enables retrieving the correct diagnosis for some entities, it does not work sufficiently for other entities. This is in accordance with a previous image analysis study on glomerular change patterns, where some diagnoses are associated with one pattern, but for others, there exists a complex pattern combination.

Muscle-invasive bladder cancer (MIBC) is characterized by high recurrence and rapid progression. Progression is linked to changes in glycan structures and altered levels of glycosyltransferases. The relationship of mRNA expression by glycosyltransferase genes B4GALT1, EXT1, MGAT5B, and POFUT1 to the probability of surviving MIBC after radical cystectomy has not yet been investigated.mRNA expression was analyzed using qRT-PCR in formalin-fixed and paraffin-embedded tumor samples (n = 105; 74% male patients and 26% female patients; median age = 72 years), correlated with histopathological variables, and evaluated by means of multivariable Cox regression analysis regarding to overall survival (OS), cancer-specific survival (CSS), and disease-free survival (DFS).Multivariable Cox regression analysis identified POFUT1 mRNA expression as superior prognostic marker, compared with currently used histological tumor stage methods, for CSS by MIBC patients following radical cystectomy. Thus, the patients with low POFUT1 mRNA were at a 4.9-fold greater risk for cancer-specific death according to the multivariable analysis (p = 0.0001). Low mRNA levels predicted poor survival according to the Kaplan-Meier analysis ((POFUT1:OS p = 0.0014; CSS p = 0.0007; DFS p = 0.0088); (EXT1:OS p = 0.0150; CSS p = 0.0130; DFS p = 0.0286); (B4GALT1:CSS p = 0.0134; DFS p = 0.0493)). A subgroup analysis of patients without lymph node metastasis (pN−; n = 73) indicated that low expression of POFUT1 predicted reduced OS (p = 0.0073), CSS (p = 0.0058,) and DSS (p = 0.0079).Low levels of POFUT1 mRNA are an independent prognostic indicator for OS and CSS in MIBC patients following radical cystectomy. This finding demonstrates the importance of altered glycosylation for the progress of MIBC.

Abstract Background The Oxford classification of IgA nephropathy (IgAN) defines histologic criteria (MEST-C) that provide prognostic information based on the kidney biopsy. There are few data on the predictive impact of this classification in randomized clinical trial settings. Methods We performed an exploratory analysis of MEST-C scores in 70 available renal biopsies from 162 randomized STOP-IgAN trial participants and correlated the results with clinical outcomes. Analyses were performed by researchers blinded to the clinical outcome of the patients. Biopsies had been obtained 6.5 to 95 (median 9.4) months prior to randomization. Results Mesangial hypercellularity (M1) associated with higher annual eGFR-loss during the 3-year trial (M1: − 5.06 ± 5.17 ml/min/1.73 m2, M0: − 0.79 ± 4.50 ml/min/1.73 m2, p = 0.002). An M0-score additionally showed a weak association with full clinical remission, whereas the percentage of patients losing ≥15 ml/min/1.73 m2 over the 3-year trial phase was higher among those scored as M1. Among patients with additional immunosuppression, ESRD occurred more frequently in patients when tubulointerstitial fibrosis (T1/2) was present (T1/2 = 33%, T0 = 0%, p = 0.008). In patients receiving supportive care only, ESRD frequencies were similar (T1/2 = 18%, T0 = 7%, p = 0.603). At randomization, eGFR was significantly lower when tubulointerstitial fibrosis was present (T1/2: 45.2 ± 15.7 ml/min/1.73 m2, T0: 74.6 ± 28.2 ml/min/1.73 m2, p

Recent advances in mass spectrometry (MS)‐based technologies are now set to transform translational cancer proteomics from an idea to a practice. Here, we present a robust proteomic workflow for the analysis of clinically relevant human cancer tissues that allows quantitation of thousands of tumor proteins in several hours of measuring time and a total turnaround of a few days. We applied it to a chemorefractory metastatic case of the extremely rare urachal carcinoma. Quantitative comparison of lung metastases and surrounding tissue revealed several significantly upregulated proteins, among them lysine‐specific histone demethylase 1 (LSD1/KDM1A). LSD1 is an epigenetic regulator and the target of active development efforts in oncology. Thus, clinical cancer proteomics can rapidly and efficiently identify actionable therapeutic options. While currently described for a single case study, we envision that it can be applied broadly to other patients in a similar condition.

Invariant natural killer T (iNKT) cells represent a unique population of CD1d-restricted T lymphocytes expressing an invariant T cell receptor encoded by Vα14-Jα18 and Vα24-Jα18 gene segments in mice and humans, respectively. Recognition of CD1d-loaded endogenous lipid antigen(s) on CD4/CD8-double positive (DP) thymocytes is essential for the development of iNKT cells. The lipid repertoire of DP thymocytes and the identity of the decisive endogenous lipid ligands have not yet been fully elucidated. Glycosphingolipids (GSL) were implicated to serve as endogenous ligands. However, further in vivo investigations were hampered by early embryonal lethality of mice deficient for the key GSL-synthesizing enzyme glucosylceramide (GlcCer) synthase [GlcCer synthase (GCS), EC 2.4.1.80]. We have now analyzed the GSL composition of DP thymocytes and shown that GlcCer represented the sole neutral GSL and the acidic fraction was composed of gangliosides. Furthermore, we report on a mouse model that by combination of Vav-promoter-driven iCre and floxed GCS alleles (VavCreGCSf/f) enabled an efficient depletion of GCS-derived GSL very early in the T cell development, reaching a reduction by 99.6% in DP thymocytes. Although the general T cell population remained unaffected by this depletion, iNKT cells were reduced by approximately 50% in thymus, spleen, and liver and showed a reduced proliferation and an increased apoptosis rate. The Vβ-chains repertoire and development of iNKT cells remained unaltered. The GSL-depletion neither interfered with expression of CD1d, SLAM, and Ly108 molecules nor impeded the antigen presentation on DP thymocytes. These results indicate that GlcCer-derived GSL, in particular GlcCer, contribute to the homeostatic development of iNKT cells.

Mammalian kidneys are rich in sulfatides. Papillary sulfatides, especially, contribute to renal adaptation to chronic metabolic acidosis. Due to differences in their cer­amide (Cer) anchors, the structural diversity of renal sulfatides is large. However, the underling biological function of this complexity is not understood. As a compound's function and its tissue location are intimately connected, we analyzed individual renal sulfatide distributions of control and Cer synthase 2 (CerS)2-deficient mice by imaging MS (IMS) and by LC-MS2 (in controls for the cortex, medulla, and papillae separately). To explain locally different structures, we compared our lipid data with regional mRNA levels of corresponding anabolic enzymes. The combination of IMS and in source decay-LC-MS2 analyses revealed exclusive expression of C20-sphingosine-containing sulfatides within the renal papillae, whereas conventional C18-sphingosine-containing compounds were predominant in the medulla, and sulfatides with a C18-phytosphingosine were restricted to special cortical structures. CerS2 deletion resulted in bulk loss of sulfatides with C23/C24-acyl chains, but did not lead to decreased urinary pH, as previously observed in sulfatide-depleted kidneys. The reasons may be the almost unchanged C22-sulfatide levels and constant total renal sulfatide levels due to compensation with C16- to C20-acyl chain-containing compounds. Intriguingly, CerS2-deficient kidneys were completely depleted of phytosphingosine-containing cortical sulfatides without any compensation.

FADD, caspase-8, and cFLIP regulate the outcome of cell death signaling. Mice that constitutively lack these molecules die at an early embryonic age, whereas tissue-specific constitutive deletion of FADD or caspase-8 results in inflammatory skin disease caused by increased necroptosis. The function of cFLIP in the skin in vivo is unknown. In contrast to tissue-specific caspase-8 knockout, we show that mice constitutively lacking cFLIP in the epidermis die around embryonic days 10 and 11. When cFLIP expression was abrogated in adult skin of cFLIPfl/fl-K14CreERtam mice, severe inflammation of the skin with concomitant caspase activation and apoptotic, but not necroptotic, cell death developed. Apoptosis was dependent of autocrine tumor necrosis factor production triggered by loss of cFLIP. In addition, epidermal cFLIP protein was lost in patients with severe drug reactions associated with epidermal apoptosis. Our data demonstrate the importance of cFLIP for the integrity of the epidermis and for silencing of spontaneous skin inflammation.

Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.

IgA nephropathy (IgAN) is a clinically and pathologically heterogeneous disease. Endocapillary proliferation is associated with higher risk of progressive disease, and clinical studies suggest that corticosteroids mitigate this risk. However, corticosteroids are associated with protean cellular effects and significant toxicity. Furthermore the precise mechanism by which they modulate kidney injury in IgAN is not well delineated. To better understand molecular pathways involved in the development of endocapillary proliferation and to identify novel specific therapeutic targets, we evaluated the glomerular transcriptome of microdissected kidney biopsies from 22 patients with IgAN. Endocapillary proliferation was defined according to the Oxford scoring system independently by 3 nephropathologists. We analyzed mRNA expression using microarrays and identified transcripts differentially expressed in patients with endocapillary proliferation compared to IgAN without endocapillary lesions. Next, we employed both transcription factor analysis and in silico drug screening and confirmed that the endocapillary proliferation transcriptome is significantly enriched with pathways that can be impacted by corticosteroids. With this approach we also identified novel therapeutic targets and bioactive small molecules that may be considered for therapeutic trials for the treatment of IgAN, including resveratrol and hydroquinine. In summary, we have defined the distinct molecular profile of a pathologic phenotype associated with progressive renal insufficiency in IgAN. Exploration of the pathways associated with endocapillary proliferation confirms a molecular basis for the clinical effectiveness of corticosteroids in this subgroup of IgAN, and elucidates new therapeutic strategies for IgAN.

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.

Editorial

In 2008, a curricular reform was begun in Slovakia. It focused on introducing a two-level model of curriculum at pre-primary, primary and secondary schools. The reform has been met with mixed reactions from teachers and school administration staff. Our paper provides brief information on the nature of this reform, the content of the reform curricular documents and some of the results of a questionnaire survey regarding the attitudes of teachers in basic schools (primary and lower secondary level of education) towards the reform. Their ratings are examined in three areas: satisfaction with the development of the Slovak school system in the last six years, the importance of curricular changes and the effect of these changes.

In the paper, the framework, the process, the impediments and the perspectivesof the transformation of the Slovak educational system after 1989 are discussed.Individual phases of the transformation process and their characteristics andcontradictions are analysed. The example of the primary level of education is used toillustrate the contradictions and lack of planning inherent in the changes on the levelof educational policy in comparison with the microlevel of schools and teachers. Theanalysis of the current state points to serious limitations to profound systematic changesin teacher education courses.