Publications by authors named "Klaus Jung"

417 Publications

Correcting the Estimation of Viral Taxa Distributions in Next-Generation Sequencing Data after Applying Artificial Neural Networks.

Genes (Basel) 2021 Oct 31;12(11). Epub 2021 Oct 31.

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, D-30559 Hannover, Germany.

Estimating the taxonomic composition of viral sequences in a biological samples processed by next-generation sequencing is an important step in comparative metagenomics. Mapping sequencing reads against a database of known viral reference genomes, however, fails to classify reads from novel viruses whose reference sequences are not yet available in public databases. Instead of a mapping approach, and in order to classify sequencing reads at least to a taxonomic level, the performance of artificial neural networks and other machine learning models was studied. Taxonomic and genomic data from the NCBI database were used to sample labelled sequencing reads as training data. The fitted neural network was applied to classify unlabelled reads of simulated and real-world test sets. Additional auxiliary test sets of labelled reads were used to estimate the conditional class probabilities, and to correct the prior estimation of the taxonomic distribution in the actual test set. Among the taxonomic levels, the biological order of viruses provided the most comprehensive data base to generate training data. The prediction accuracy of the artificial neural network to classify test reads to their viral order was considerably higher than that of a random classification. Posterior estimation of taxa frequencies could correct the primary classification results.
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http://dx.doi.org/10.3390/genes12111755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8624964PMC
October 2021

Mammals Preferred: Reassortment of and Occurs in Mammalian but Not Insect Cells.

Viruses 2021 Aug 27;13(9). Epub 2021 Aug 27.

Institute for Parasitology, Center for Infection Medicine, University of Veterinary Medicine Hannover, 30559 Hanover, Germany.

Reassortment is a viral genome-segment recomposition known for many viruses, including the orthobunyaviruses. The co-infection of a host cell with two viruses of the same serogroup, such as the and the can give rise to novel viruses. One example is the Ngari virus, which has caused major outbreaks of human infections in Central Africa. This study aimed to investigate the potential for reassortment of and the during co-infection studies and the replication properties of the reassortants in different mammalian and insect cell lines. In the co-infection studies, a Ngari-like virus reassortant and a novel reassortant virus, the Batunya virus, arose in BHK-21 cells (). In contrast, no reassortment was observed in the examined insect cells from (Aag2) and (U4.4 and C6/36). The growth kinetic experiments show that both reassortants are replicated to higher titers in some mammalian cell lines than the parental viruses but show impaired growth in insect cell lines.
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http://dx.doi.org/10.3390/v13091702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8473249PMC
August 2021

Host Age and Denture Wearing Jointly Contribute to Oral Colonization with Intrinsically Azole-Resistant Yeasts in the Elderly.

Microorganisms 2021 Jul 30;9(8). Epub 2021 Jul 30.

Institute for Medical Microbiology and Virology, University Medical Center Göttingen, 37075 Göttingen, Germany.

In elderly patients, several morbidities or medical treatments predisposing for fungal infections occur at a higher frequency, leading to high mortality and morbidity in this vulnerable patient group. Often, this is linked to an innately azole-resistant yeast species such as or . Additionally, host age per se and the wearing of dentures have been determined to influence the mix of colonizing species and, consequently, the species distribution of invasive fungal infections. Since both old age and the wearing of dentures are two tightly connected parameters, it is still unclear which of them is the main contributor. Here, we performed a cross-sectional study on a cohort ( = 274) derived from three groups of healthy elderly, diseased elderly, and healthy young controls. With increasing host age, the frequency of oral colonization by a non-  species, mainly by , also increased, and the wearing of dentures predisposed for colonization by irrespectively of host age. Physically diseased hosts, on the other hand, were more frequently orally colonized by than by other yeasts. For both and , isolates from the oral cavity did not generally display an elevated biofilm formation capacity. In conclusion, intrinsically azole-drug-resistant, non-  yeasts are more frequent in the oral cavities of the elderly, and fungal cells not contained in biofilms may predispose for subsequent systemic infection with these organisms. This warrants further exploration of diagnostic procedures, e.g., before undergoing elective abdominal surgery or when using indwelling devices on this patient group.
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http://dx.doi.org/10.3390/microorganisms9081627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8400291PMC
July 2021

Comparison of PHI and PHI Density for Prostate Cancer Detection in a Large Retrospective Caucasian Cohort.

Urol Int 2021 Aug 25:1-6. Epub 2021 Aug 25.

Department of Urology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Berlin, Germany.

Background: Beyond prostate-specific antigen (PSA), other biomarkers for prostate cancer (PCa) detection are available and need to be evaluated for clinical routine.

Objective: The aim of the study was to evaluate the Prostate Health Index (PHI) density (PHID) in comparison with PHI in a large Caucasian group >1,000 men.

Methods: PHID values were used from available patient data with PSA, free PSA, and [-2]pro-PSA and prostate volume from 3 former surveys from 2002 to 2014. Those 1,446 patients from a single-center cohort included 701 men with PCa and 745 with no PCa. All patients received initial or repeat biopsies. The diagnostic accuracy was evaluated by receiver operating characteristic (ROC) curves comparing area under the ROC curves (AUCs), precision-recall approach, and decision curve analysis (DCA).

Results: PHID medians differed almost 2-fold between PCa (1.12) and no PCa (0.62) in comparison to PHI (48.6 vs. 33; p always <0.0001). However, PHID and PHI were equal regarding the AUC (0.737 vs. 0.749; p = 0.226), and the curves of the precision-recall analysis also overlapped in the sensitivity range between 70 and 100%. DCA had a maximum net benefit of only ∼5% for PHID versus PHI between 45 and 55% threshold probability. Contrary, in the 689 men with a prostate volume ≤40 cm3, PHI (AUC 0.732) showed a significant larger AUC than PHID (AUC 0.69, p = 0.014).

Conclusions: Based on DCA, PHID had only a small advantage in comparison with PHI alone, while ROC analysis and precision-recall analysis showed similar results. In smaller prostates, PHI even outperformed PHID. The increment for PHID in this large Caucasian cohort is too small to justify a routine clinical use.
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http://dx.doi.org/10.1159/000517891DOI Listing
August 2021

A resampling strategy for studying robustness in virus detection pipelines.

Comput Biol Chem 2021 Oct 2;94:107555. Epub 2021 Aug 2.

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17p, 30559 Hannover, Germany. Electronic address:

Next-generation sequencing is regularly used to identify viral sequences in DNA or RNA samples of infected hosts. A major step of most pipelines for virus detection is to map sequence reads against known virus genomes. Due to small differences between the sequences of related viruses, and due to several biological or technical errors, mapping underlies uncertainties. As a consequence, the resulting list of detected viruses can lack robustness. A new approach for generating artificial sequencing reads together with a strategy of resampling from the original findings is proposed that can help to assess the robustness of the originally identified list of viruses. From the original mapping result in form of a SAM file, a set of statistical distributions are derived. These are used in the resampling pipeline to generate new artificial reads which are again mapped versus the reference genomes. By summarizing the resampling procedure, the analyst receives information about whether the presence of a particular virus in the sample gains or losses evidence, and thus about the robustness of the original mapping list but also that of individual viruses in this list. To judge robustness, several indicators are derived from the resampling procedure such as the correlation between original and resampling read counts, or the statistical detection of outliers in the differences of read counts. Additionally, graphical illustrations of read count shifts via Sankey diagrams are provided. To demonstrate the use of the new approach, the resampling approach is applied to three real-world data samples, one of them with laboratory-confirmed Influenza sequences, and to artificially generated data where virus sequences have been spiked into the sequencing data of a host. By applying the resampling pipeline, several viruses drop from the original list while new viruses emerge, showing robustness of those viruses that remain in the list. The evaluation of the new approach shows that the resampling approach is helpful to analyze the viral content of a biological sample, to rate the robustness of original findings and to better show the overall distribution of findings. The method is also applicable to other virus detection pipelines based on read mapping.
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http://dx.doi.org/10.1016/j.compbiolchem.2021.107555DOI Listing
October 2021

Sequential MAVS and MyD88/TRIF signaling triggers anti-viral responses of tick-borne encephalitis virus-infected murine astrocytes.

J Neurosci Res 2021 10 23;99(10):2478-2492. Epub 2021 Jul 23.

Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, is typically transmitted upon tick bite and can cause meningitis and encephalitis in humans. In TBEV-infected mice, mitochondrial antiviral-signaling protein (MAVS), the downstream adaptor of retinoic acid-inducible gene-I (RIG-I)-like receptor (RLR) signaling, is needed to induce early type I interferon (IFN) responses and to confer protection. To characterize the brain-resident cell subset that produces protective IFN-β in TBEV-infected mice, we isolated neurons, astrocytes, and microglia from mice and exposed these cell types to TBEV in vitro. Under such conditions, neurons showed the highest percentage of infected cells, whereas astrocytes and microglia were infected to a lesser extent. In the supernatant (SN) of infected neurons, IFN-β was not detectable, while infected astrocytes showed high and microglia low IFN-β expression. Transcriptome analyses of astrocytes implied that MAVS signaling was needed early after TBEV infection. Accordingly, MAVS-deficient astrocytes showed enhanced TBEV infection and significantly reduced early IFN-β responses. Nevertheless, at later time points, moderate amounts of IFN-β were detected in the SN of infected MAVS-deficient astrocytes. Transcriptome analyses indicated that MAVS deficiency negatively affected the induction of early anti-viral responses, which resulted in significantly increased TBEV replication. Treatment with MyD88 and TRIF inhibiting peptides reduced only late IFN-β responses of TBEV-infected WT astrocytes and blocked entirely IFN-β responses of infected MAVS-deficient astrocytes. Thus, upon TBEV exposure of brain-resident cells, astrocytes are important IFN-β producers showing biphasic IFN-β induction that initially depends on MAVS and later on MyD88/TRIF signaling.
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http://dx.doi.org/10.1002/jnr.24923DOI Listing
October 2021

Comparison of spotlighting monitoring data of European brown hare (Lepus europaeus) relative population densities with infrared thermography in agricultural landscapes in Northern Germany.

PLoS One 2021 9;16(7):e0254084. Epub 2021 Jul 9.

Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.

A successful wildlife management requires monitoring. Including non-scientific volunteers into monitoring actions is a common way for obtaining long-term and comprehensive data. Hunters present a valuable target group as they are spread out nationwide in Germany and additionally, they provide a know-how regarding game species. Since 1990s, various German hunting associations established monitoring programs and motivated hunters to join, in order to record population sizes of huntable game species under standardized census methods. The aim of this study was to compare instructed hunters performed spotlight counts of European brown hares with thermography in three federal states (Lower-Saxony, Saxony-Anhalt, North Rhine-Westphalia) in 2015-2018 in Northern Germany. Therefore, we modelled the number of hares counted by both methods with the associated observed area. Moreover, we performed repeated thermographic counts in selected areas and performed distance sampling to test the deviations of estimated population densities within a short time period. Repeated infrared thermographic counts on three consecutive nights show a coefficient of variation from 6.6% to 15.5% with deviations of 2.2-2.7 hares per 100 ha, while the method of distance sampling reveals minor deviations of 0.9-1.7 hares per 100 ha and a coefficient of variation from 3.1-7.4%. The coefficient of variation value between spotlight and infrared thermographic count lies between 0 to 21.4%. Our model confirmed no significant differences between the European brown hare density estimations based on a spotlight count and an infrared thermographic count on the following night. The results provide insight into the dimension of the error margin of density estimations performed by spotlight counts. Therefore, we recommend to take possible counting errors into account and to ideally perform repeated counts to assess the error margin for each counting site. This would help for example to quantify the uncertainty in the calculation of mortality rates. Additionally, our results show that monitoring data generated by instructed hunters can provide reliable and valid data, if implemented and conducted in a standardized scientific way.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0254084PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8270206PMC
November 2021

MyD88 signaling by neurons induces chemokines that recruit protective leukocytes to the virus-infected CNS.

Sci Immunol 2021 Jun;6(60)

Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, 30625 Hannover, Germany.

Viral encephalitis initiates a series of immunological events in the brain that can lead to brain damage and death. Astrocytes express IFN-β in response to neurotropic infection, whereas activated microglia produce proinflammatory cytokines and accumulate at sites of infection. Here, we observed that neurotropic vesicular stomatitis virus (VSV) infection causes recruitment of leukocytes into the central nervous system (CNS), which requires MyD88, an adaptor of Toll-like receptor and interleukin-1 receptor signaling. Infiltrating leukocytes, and in particular CD8 T cells, protected against lethal VSV infection of the CNS. Reconstitution of MyD88, specifically in neurons, restored chemokine production in the olfactory bulb as well as leukocyte recruitment into the infected CNS and enhanced survival. Comparative analysis of the translatome of neurons and astrocytes verified neurons as the critical source of chemokines, which regulated leukocyte infiltration of the infected brain and affected survival.
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http://dx.doi.org/10.1126/sciimmunol.abc9165DOI Listing
June 2021

Fourier transform infrared spectroscopy coupled with machine learning classification for identification of oxidative damage in freeze-dried heart valves.

Sci Rep 2021 06 10;11(1):12299. Epub 2021 Jun 10.

Biostabilization Laboratory, Lower Saxony Centre for Biomedical Engineering, Implant Research and Development, University of Veterinary Medicine Hannover, Stadtfelddamm 34, 30625, Hannover, Germany.

Freeze-drying can be used to ensure off-the-shelf availability of decellularized heart valves for cardiovascular surgery. In this study, decellularized porcine aortic heart valves were analyzed by nitroblue tetrazolium (NBT) staining and Fourier transform infrared spectroscopy (FTIR) to identify oxidative damage during freeze-drying and subsequent storage as well as after treatment with HO and FeCl. NBT staining revealed that sucrose at a concentration of at least 40% (w/v) is needed to prevent oxidative damage during freeze-drying. Dried specimens that were stored at 4 °C depict little to no oxidative damage during storage for up to 2 months. FTIR analysis shows that fresh control, freeze-dried and stored heart valve specimens cannot be distinguished from one another, whereas HO- and FeCl-treated samples could be distinguished in some tissue section. A feed forward artificial neural network model could accurately classify HO and FeCl treated samples. However, fresh control, freeze-dried and stored samples could not be distinguished from one another, which implies that these groups are very similar in terms of their biomolecular fingerprints. Taken together, we conclude that sucrose can minimize oxidative damage caused by freeze-drying, and that subsequent dried storage has little effects on the overall biochemical composition of heart valve scaffolds.
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http://dx.doi.org/10.1038/s41598-021-91802-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192956PMC
June 2021

Mining Protein Expression Databases Using Network Meta-Analysis.

Methods Mol Biol 2021 ;2228:419-431

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.

Public databases featuring original, raw data from "Omics" experiments enable researchers to perform meta-analyses by combining either the raw data or the summarized results of several independent studies. In proteomics, high-throughput protein expression data is measured by diverse techniques such as mass spectrometry, 2-D gel electrophoresis or protein arrays yielding data of different scales. Therefore, direct data merging can be problematic, and combining the summarized data of the individual studies can be advantageous. A special form of meta-analysis is network meta-analysis, where studies with different settings of experimental groups can be combined. However, all studies must be linked by one experimental group that has to appear in each study. Usually that is the control group. Then, a study network is formed and indirect statistical inferences can also be made between study groups that appear not in each of the studies.In this chapter, we describe the working principle of and available software for network meta-analysis. The applicability to high-throughput protein expression data is demonstrated in an example from breast cancer research. We also describe the special challenges when applying this method.
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http://dx.doi.org/10.1007/978-1-0716-1024-4_29DOI Listing
June 2021

Impairment of the neurotrophic signaling hub B-Raf contributes to motoneuron degeneration in spinal muscular atrophy.

Proc Natl Acad Sci U S A 2021 05;118(18)

Institute of Neuroanatomy and Cell Biology, Hannover Medical School, Hannover 30625, Germany.

Spinal muscular atrophy (SMA) is a motoneuron disease caused by deletions of the ( and low SMN protein levels. SMN restoration is the concept behind a number of recently approved drugs which result in impressive yet limited effects. Since SMN has already been enhanced in treated patients, complementary SMN-independent approaches are needed. Previously, a number of altered signaling pathways which regulate motoneuron degeneration have been identified as candidate targets. However, signaling pathways form networks, and their connectivity is still unknown in SMA. Here, we used presymptomatic SMA mice to elucidate the network of altered signaling in SMA. The SMA network is structured in two clusters with AKT and 14-3-3 ζ/δ in their centers. Both clusters are connected by B-Raf as a major signaling hub. The direct interaction of B-Raf with 14-3-3 ζ/δ is important for an efficient neurotrophic activation of the MEK/ERK pathway and crucial for motoneuron survival. Further analyses in SMA mice revealed that both proteins were down-regulated in motoneurons and the spinal cord with B-Raf being reduced at presymptomatic stages. Primary fibroblasts and iPSC-derived motoneurons from SMA patients both showed the same pattern of down-regulation. This mechanism is conserved across species since a SMA model showed less expression of the B-Raf homolog Accordingly, motoneuron survival was rescued by a cell autonomous expression in a SMA model resulting in improved motor functions. This rescue was effective even after the onset of motoneuron degeneration and mediated by the MEK/ERK pathway.
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http://dx.doi.org/10.1073/pnas.2007785118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106332PMC
May 2021

Metabolic impact of weight variations in Icelandic horses.

PeerJ 2021 28;9:e10764. Epub 2021 Jan 28.

Clinic for Horses, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.

Background: Insulin dysregulation (ID) is an equine endocrine disorder, which is often accompanied by obesity and various metabolic perturbations. The relationship between weight variations and fluctuations of the insulin response to oral glucose tests (OGT) as well as the metabolic impact of ID have been described previously. The present study seeks to characterize the concomitant metabolic impact of variations in the insulin response and bodyweight during repeated OGTs using a metabolomics approach.

Methods: Nineteen Icelandic horses were subjected to five OGTs over one year and their bodyweight, insulin and metabolic response were monitored. Analysis of metabolite concentrations depending on time (during the OGT), relative bodyweight (rWeight; defined as the bodyweight at one OGT divided by the mean bodyweight across all OGTs) and relative insulin response (rAUC; defined accordingly from the area under the insulin curve during OGT) was performed using linear models. Additionally, the pathways significantly associated with time, rWeight and rAUC were identified by rotation set testing.

Results: The results suggested that weight gain and worsening of ID activate distinct metabolic pathways. The metabolic profile associated with weight gain indicated an increased activation of arginase, while the pathways associated with time and rAUC were consistent with the expected effect of glucose and insulin, respectively. Overall, more metabolites were significantly associated with rWeight than with rAUC.
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http://dx.doi.org/10.7717/peerj.10764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7847705PMC
January 2021

The Stable Matching Problem in TBEV Enzootic Circulation: How Important Is the Perfect Tick-Virus Match?

Microorganisms 2021 Jan 19;9(1). Epub 2021 Jan 19.

Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, 30559 Hanover, Germany.

Tick-borne encephalitis virus (TBEV), like other arthropod-transmitted viruses, depends on specific vectors to complete its enzootic cycle. It has been long known that ticks constitute the main vector for TBEV in Europe. In contrast to the wide distribution of the TBEV vector, the occurrence of TBEV transmission is focal and often restricted to a small parcel of land, whereas surrounding areas with seemingly similar habitat parameters are free of TBEV. Thus, the question arises which factors shape this focal distribution of TBEV in the natural habitat. To shed light on factors driving TBEV-focus formation, we used tick populations from two TBEV-foci in Lower Saxony and two TBEV-foci from Bavaria with their respective virus isolates as a showcase to analyze the impact of specific virus isolate-tick population relationships. Using artificial blood feeding and field-collected nymphal ticks as experimental means, our investigation showed that the probability of getting infected with the synonymous TBEV isolate as compared to the nonsynonymous TBEV isolate was elevated but significantly higher only in one of the four TBEV foci. More obviously, median viral RNA copy numbers were significantly higher in the synonymous virus-tick population pairings. These findings may present a hint for a coevolutionary adaptation of virus and tick populations.
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http://dx.doi.org/10.3390/microorganisms9010196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833397PMC
January 2021

PHI density prospectively improves prostate cancer detection.

World J Urol 2021 Sep 20;39(9):3273-3279. Epub 2021 Jan 20.

Department of Urology, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Charitéplatz 1, 10117, Berlin, Germany.

Purpose: To evaluate the Prostate Health Index (PHI) density (PHID) in direct comparison with PHI in a prospective large cohort.

Methods: PHID values were calculated from prostate-specific antigen (PSA), free PSA and [- 2]proPSA and prostate volume. The 1057 patients included 552 men with prostate cancer (PCa) and 505 with no evidence of malignancy (NEM). In detail, 562 patients were biopsied at the Charité Hospital Berlin and 495 patients at the Sana Hospital Offenbach. All patients received systematic or magnetic resonance imaging (MRI)/ultrasound fusion-guided biopsies. The diagnostic accuracy was evaluated by receiver operating characteristic (ROC) curves comparing areas under the ROC-curves (AUC). The decision curve analysis (DCA) was performed with the MATLAB Neural Network Toolbox.

Results: PHID provided a significant larger AUC than PHI (0.835 vs. 0.801; p = 0.0013) in our prospective cohort of 1057 men from 2 centers. The DCA had a maximum net benefit of ~ 5% for PHID vs. PHI between 35 and 65% threshold probability. In those 698 men within the WHO-calibrated PSA grey-zone up to 8 ng/ml, PHID was also significantly better than PHI (AUC 0.819 vs. 0.789; p = 0.0219). But PHID was not different from PHI in the detection of significant PCa.

Conclusions: Based on ROC analysis and DCA, PHID had an advantage in comparison with PHI alone to detect any PCa but PHI and PHID performed equal in detecting significant PCa.
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http://dx.doi.org/10.1007/s00345-020-03585-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8510982PMC
September 2021

The discriminative ability of Prostate Health Index to detect prostate cancer is enhanced in combination with miR-222-3p.

Cancer Biomark 2021 ;30(4):381-393

Department of Urology, Charité - Universitätsmedizin Berlin, Berlin, Germany.

Background: There is an urgent need for better prostate cancer (PCa) biomarkers due to the low specificity of prostate specific antigen (PSA).

Objective: Prostate Health Index (PHI) is an advanced PSA-based test for early detection of PCa. The present study aim was to investigate the potential improvement of diagnostic accuracy of PHI by its combination with suitable discriminative microRNAs (miRNAs).

Methods: A two-phase study was performed. In a discovery phase, a panel of 177 miRNAs was measured in ten men with biopsy proven PCa and ten men with histologically no evidence of malignancy (NEM). These results were validated in a second phase including 25 patients in each group. The patients of all groups were matched regarding their PSA values and PHI were measured.

Results: Based on data in the discovery phase, four elevated miRNAs were selected as potential miRNA candidates for further validation. A combination of miR-222-3p as the best discriminative miRNA with PHI extended the diagnostic accuracy of PHI from an AUC value of 0.690 to 0.787 and resulted in a sensitivity of 72.0% and a specificity of 84.0%.

Conclusion: Circulating microRNAs show useful diagnostic potential in combination with common used biomarkers to enhance their diagnostic power.
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http://dx.doi.org/10.3233/CBM-201600DOI Listing
November 2021

Metabolic changes induced by oral glucose tests in horses and their diagnostic use.

J Vet Intern Med 2021 Jan 5;35(1):597-605. Epub 2020 Dec 5.

Clinic for Horses, University of Veterinary Medicine Hannover, Foundation, Hanover, Germany.

Background: Little is known about the implications of hyperinsulinemia on energy metabolism, and such knowledge might help understand the pathophysiology of insulin dysregulation.

Objectives: Describe differences in the metabolic response to an oral glucose test, depending on the magnitude of the insulin response.

Animals: Twelve Icelandic horses in various metabolic states.

Methods: Horses were subjected to 3 oral glucose tests (OGT; 0.5 g/kg body weight glucose). Basal, 120 and 180 minutes samples were analyzed using a combined liquid chromatography tandem mass spectrometry and flow injection analysis tandem mass spectrometry metabolomic assay. Insulin concentrations were measured using an ELISA. Analysis was performed using linear models and partial least-squares regression.

Results: The kynurenine : tryptophan ratio increased over time during the OGT (adjusted P-value = .001). A high insulin response was associated with lower arginine (adjusted P-value = .02) and carnitine (adjusted P-value = .03) concentrations. A predictive model using only baseline samples performed well with as few as 7 distinct metabolites (sensitivity, 86%; 95% confidence interval [CI], 81%-90%; specificity, 88%; 95% CI, 84%-92%).

Conclusions And Clinical Importance: Our results suggest induction of low-grade inflammation during the OGT. Plasma arginine and carnitine concentrations were lower in horses with high insulin response and could constitute potential therapeutic targets. Development of screening tools to identify insulin-dysregulated horses using only baseline blood sample appears promising.
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http://dx.doi.org/10.1111/jvim.15992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848347PMC
January 2021

Measuring reproducibility of virus metagenomics analyses using bootstrap samples from FASTQ-files.

Bioinformatics 2021 05;37(8):1068-1075

Institute for Animal Breeding and Genetics, University of Veterinary Medicine Hannover, Hannover D-30559, Germany.

Motivation: High-throughput sequencing data can be affected by different technical errors, e.g. from probe preparation or false base calling. As a consequence, reproducibility of experiments can be weakened. In virus metagenomics, technical errors can result in falsely identified viruses in samples from infected hosts. We present a new resampling approach based on bootstrap sampling of sequencing reads from FASTQ-files in order to generate artificial replicates of sequencing runs which can help to judge the robustness of an analysis. In addition, we evaluate a mixture model on the distribution of read counts per virus to identify potentially false positive findings.

Results: The evaluation of our approach on an artificially generated dataset with known viral sequence content shows in general a high reproducibility of uncovering viruses in sequencing data, i.e. the correlation between original and mean bootstrap read count was highly correlated. However, the bootstrap read counts can also indicate reduced or increased evidence for the presence of a virus in the biological sample. We also found that the mixture-model fits well to the read counts, and furthermore, it provides a higher accuracy on the original or on the bootstrap read counts than on the difference between both. The usefulness of our methods is further demonstrated on two freely available real-world datasets from harbor seals.

Availability And Implementation: We provide a Phyton tool, called RESEQ, available from https://github.com/babaksaremi/RESEQ that allows efficient generation of bootstrap reads from an original FASTQ-file.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa926DOI Listing
May 2021

Endocytosis-Mediated Replenishment of Amino Acids Favors Cancer Cell Proliferation and Survival in Chromophobe Renal Cell Carcinoma.

Cancer Res 2020 12 28;80(24):5491-5501. Epub 2020 Oct 28.

Max Planck Institute for Molecular Genetics, Berlin, Germany.

Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in . chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca/protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca/PKC axis as a potential therapeutic target in patients with chRCC. SIGNIFICANCE: This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-1998DOI Listing
December 2020

Circular RNAs and Their Linear Transcripts as Diagnostic and Prognostic Tissue Biomarkers in Prostate Cancer after Prostatectomy in Combination with Clinicopathological Factors.

Int J Mol Sci 2020 Oct 22;21(21). Epub 2020 Oct 22.

Department of Urology, Charité-Universitätsmedizin Berlin, 10117 Berlin, Germany.

As new biomarkers, circular RNAs (circRNAs) have been largely unexplored in prostate cancer (PCa). Using an integrative approach, we aimed to evaluate the potential of circRNAs and their linear transcripts (linRNAs) to act as (i) diagnostic biomarkers for differentiation between normal and tumor tissue and (ii) prognostic biomarkers for the prediction of biochemical recurrence (BCR) after radical prostatectomy. In a first step, eight circRNAs (circ, circ, circ, circ, circ, circ, circ, and circ) were identified as differentially expressed via a genome-wide circRNA-based microarray analysis of six PCa samples. Additional bioinformatics and literature data were applied for this selection process. In total, 115 malignant PCa and 79 adjacent normal tissue samples were examined using robust RT-qPCR assays specifically established for the circRNAs and their linear counterparts. Their diagnostic and prognostic potential was evaluated using receiver operating characteristic curves, Cox regressions, decision curve analyses, and C-statistic calculations of prognostic indices. The combination of circ and lin showed a high discriminative ability between malignant and adjacent normal tissue PCa. The combination of lin, lin, and lin proved to be the best predictive RNA-signature for BCR. The combination of this RNA signature with five established reference models based on only clinicopathological factors resulted in an improved predictive accuracy for BCR in these models. This is an encouraging study for PCa to evaluate circRNAs and their linRNAs in an integrative approach, and the results showed their clinical potential in combination with standard clinicopathological variables.
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http://dx.doi.org/10.3390/ijms21217812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672590PMC
October 2020

Prognostic Role of Survivin and Macrophage Infiltration Quantified on Protein and mRNA Level in Molecular Subtypes Determined by RT-qPCR of , , and in Muscle-Invasive Bladder Cancer Treated by Adjuvant Chemotherapy.

Int J Mol Sci 2020 Oct 8;21(19). Epub 2020 Oct 8.

STRATIFYER Molecular Pathology GmbH, DE-50935 Cologne, Germany.

Objectives: Bladder cancer is a heterogeneous malignancy. Therefore, it is difficult to find single predictive markers. Moreover, most studies focus on either the immunohistochemical or molecular assessment of tumor tissues by next-generation sequencing (NGS) or PCR, while a combination of immunohistochemistry (IHC) and PCR for tumor marker assessment might have the strongest impact to predict outcome and select optimal therapies in real-world application. We investigated the role of proliferation survivin/ and macrophage infiltration (CD68, MAC387, CLEVER-1) on the basis of molecular subtypes of bladder cancer (KRT5, KRT20, ERBB2) to predict outcomes of adjuvant treated muscle-invasive bladder cancer patients with regard to progression-free survival (PFS) and disease-specific survival (DSS).

Materials And Methods: We used tissue microarrays (TMA) from n = 50 patients (38 males, 12 female) with muscle-invasive bladder cancer. All patients had been treated with radical cystectomy followed by adjuvant triple chemotherapy. Median follow-up time was 60.5 months. CD68, CLEVER-1, MAC387, and survivin protein were detected by immunostaining and subsequent visual inspection. , , , , and mRNAs were detected by standardized RT-qPCR after tissue dot RNA extraction using a novel stamp technology. All these markers were evaluated in three different centers of excellence.

Results: Nuclear staining rather than cytoplasmic staining of survivin predicted DSS as a single marker with high levels of survivin being associated with better PFS and DSS upon adjuvant chemotherapy ( = 0.0138 and = 0.001, respectively). These results were validated by the quantitation of mRNA by PCR ( = 0.0004 and = 0.0508, respectively). Interestingly, nuclear staining of survivin protein was positively associated with mRNA, while cytoplasmic staining was inversely related, indicating that the translocation of survivin protein into the nucleus occurred at a discrete, higher level of its mRNA. Combining survivin/ levels based on molecular subtype being assessed by expression improved the predictive value, with tumors having low survivin/ and mRNA levels having the best survival (75% vs. 20% vs. 10% 5-year DSS, = 0.0005), and these values were independent of grading, node status, and tumor stage in multivariate analysis ( = 0.0167). Macrophage infiltration dominated in basal tumors and was inversely related with the luminal subtype marker gene expression. The presence of macrophages in survivin-positive or -positive tumors was associated with worse DSS.

Conclusions: For muscle-invasive bladder cancer patients, the proliferative activity as determined by the nuclear staining of survivin or RT-qPCR on the basis of molecular subtype characteristics outperforms single marker detections and single technology approaches. Infiltration by macrophages detected by IHC or PCR is associated with worse outcome in defined subsets of tumors. The limitations of this study are the retrospective nature and the limited number of patients. However, the number of molecular markers has been restricted and based on predefined assumptions, which resulted in the dissection of muscle-invasive disease into tumor-biological axes of high prognostic relevance, which warrant further investigation and validation.
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http://dx.doi.org/10.3390/ijms21197420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582791PMC
October 2020

Tick populations from endemic and non-endemic areas in Germany show differential susceptibility to TBEV.

Sci Rep 2020 09 23;10(1):15478. Epub 2020 Sep 23.

Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, Hanover, Germany.

Tick-borne encephalitis virus (TBEV) is endemic in twenty-seven European countries, transmitted via the bite of an infected tick. TBEV is the causative agent of one of the most important viral diseases of the central nervous system (CNS). In Germany, 890 human cases were registered between the years 2018-2019. The castor bean tick, Ixodes ricinus, is the TBEV vector with the highest importance in Central Europe, including Germany. Despite the nationwide distribution of this tick species, risk areas of TBEV are largely located in Southern Germany. To increase our understanding of TBEV-tick interactions, we collected ticks from different areas within Germany (Haselmühl/Bavaria, Hanover/Lower Saxony) and infected them via an in vitro feeding system. A TBEV isolate was obtained from an endemic focus in Haselmühl. In two experimental series conducted in 2018 and 2019, ticks sampled in Haselmühl (TBEV focus) showed higher artificial feeding rates, as well as higher TBEV infections rates than ticks from the non-endemic area (Hanover). Other than the tick origin, year and month of the infection experiment as well as co-infection with Borrelia spp., had a significant impact on TBEV Haselmühl infection rates. Taken together, these findings suggest that a specific adaptation of the tick populations to their respective TBEV virus isolates or vice versa, leads to higher TBEV infection rates in those ticks. Furthermore, co-infection with other tick-borne pathogens such as Borrelia spp. can lower TBEV infection rates in specific populations.
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http://dx.doi.org/10.1038/s41598-020-71920-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511395PMC
September 2020

Impact of tourism on habitat use of black grouse (Tetrao tetrix) in an isolated population in northern Germany.

PLoS One 2020 4;15(9):e0238660. Epub 2020 Sep 4.

Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.

As many other wild living species, black grouse Tetrao tetrix has to cope with anthropogenic disturbances in many habitats. Impacts of tourism and outdoor recreation on grouse species Tetraoninae have been subject to several studies in mountainous habitats in Central Europe and the United Kingdom. The geographically isolated and critically endangered black grouse population of Lower Saxony (North Germany) has been consistently monitored but beyond that poorly studied. This also applies to the key habitats of the nature reserve Lüneburg Heath (Lüneburger Heide) which, in turn, serves as a recreational area as well. In this study, the impact of tourism activity on habitat use of black grouse was investigated using data of GPS-tracked individuals. Additionally, visitor numbers on public and (usually undisturbed) closed routes were monitored using infrared light barriers. The spatio-temporal distribution of locations and the recreational activity were evaluated by linear mixed-effects models. Tagged individuals avoided the vicinity of public routes and avoiding distances were directly related to intensity of human activity. There was no seasonal change of black grouse habitat use alongside public routes. However, towards closed routes, significantly higher distances appeared during peak phases of visitor numbers (August and September), implying temporary increased disturbance levels within a key refuge area. Diurnal adaptation of habitat use was strongly dependent on the route density within the home range. Individuals used the vicinity of public trails at night and dawn but evaded these habitats during peak human activity around noon and afternoon. Recreational disturbances appeared to significantly affect the effective habitat availability for black grouse in the nature reserve. Visual cover by vegetation, however, seemed to diminish negative effects emerging from hiking trails. This provides an effective protective measure which requires minimal effort for the local conservation management.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0238660PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473583PMC
October 2020

Instability of circular RNAs in clinical tissue samples impairs their reliable expression analysis using RT-qPCR: from the myth of their advantage as biomarkers to reality.

Theranostics 2020 23;10(20):9268-9279. Epub 2020 Jul 23.

Department of Urology, Charité - University Medicine, 10117 Berlin, Germany.

Circular RNAs (circRNAs) are a new class of RNAs with medical significance. Compared to that of linear mRNA transcripts, the stability of circRNAs against degradation owing to their circular structure is considered advantageous for their use as biomarkers. As systematic studies on the stability of circRNAs depending on the RNA integrity, determined as RNA integrity number (RIN), in clinical tissue samples are lacking, we have investigated this aspect in the present study under model and clinical conditions. Total RNA isolated from kidney cancer tissue and cell lines (A-498 and HEK-293) with different RIN after thermal degradation was used in model experiments. Further, RNA isolated from kidney cancer and prostate cancer tissue collected under routine surgical conditions, representing clinical samples with RIN ranging from 2 to 9, were examined. Quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) analysis of several circRNAs (, and ), their corresponding linear counterparts, tissue-specific reference genes, and three microRNAs (as controls) was performed. The quantification cycles were converted into relative quantities and normalized to the expression of specific reference genes for the corresponding tissue. The effect of RIN on the expression of different RNA entities was determined using linear regression analysis, and clinical samples were classified into two groups based on RIN greater or lesser than 6. The results of model experiments and clinical sample analyses showed that all relative circRNA expression gradually decreased with reduction in RIN values. The adverse effect of RIN was partially compensated after normalizing the data and limiting the samples to only those with RIN values > 6. Our results suggested that circRNAs are not stable in clinical tissue samples, but are subjected to degradative processes similar to mRNAs. This has not been investigated extensively in circRNA expression studies, and hence must be considered in future for obtaining reliable circRNA expression data. This can be achieved by applying the principles commonly used in mRNA expression studies.
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http://dx.doi.org/10.7150/thno.46341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415809PMC
May 2021

Decreased Mitochondrial DNA Content Drives OXPHOS Dysregulation in Chromophobe Renal Cell Carcinoma.

Cancer Res 2020 09 21;80(18):3830-3840. Epub 2020 Jul 21.

Max Planck Institute for Molecular Genetics, Berlin, Germany.

Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are closely related, rare kidney tumors. Mutations in complex I (CI)-encoding genes play an important role in dysfunction of the oxidative phosphorylation (OXPHOS) system in renal oncocytoma, but are less frequently observed in chRCC. As such, the relevance of OXPHOS status and role of CI mutations in chRCC remain unknown. To address this issue, we performed proteome and metabolome profiling as well as mitochondrial whole-exome sequencing to detect mitochondrial alterations in chRCC tissue specimens. Multiomic analysis revealed downregulation of electron transport chain (ETC) components in chRCC that differed from the expression profile in renal oncocytoma. A decrease in mitochondrial (mt)DNA content, rather than CI mutations, was the main cause for reduced OXPHOS in chRCC. There was a negative correlation between protein and transcript levels of nuclear DNA- but not mtDNA-encoded ETC complex subunits in chRCC. In addition, the reactive oxygen species scavenger glutathione (GSH) was upregulated in chRCC due to decreased expression of proteins involved in GSH degradation. These results demonstrate that distinct mechanisms of OXPHOS exist in chRCC and renal oncocytoma and that expression levels of ETC complex subunits can serve as a diagnostic marker for this rare malignancy. SIGNIFICANCE: These findings establish potential diagnostic markers to distinguish malignant chRCC from its highly similar but benign counterpart, renal oncocytoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0754DOI Listing
September 2020

Histological Comparison of Testicular Needle Biopsy and En Bloc Samples in Abattoir Calves.

Animals (Basel) 2020 May 25;10(5). Epub 2020 May 25.

Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, 30173 Hannover, Germany.

the aim of this study was to test whether a single testicular needle biopsy could provide histological results comparable to en bloc resection histology and whether one biopsy was sufficient to reflect the histology of an entire pair of testicles. Two methods of sample collection were tested on 32 bull calves aged five to eight months to compare histological parameters of needle biopsy with those of en bloc resection samples. One testicular needle biopsy of the right and three en bloc samples of both testicles were collected and compared for the number of tubular cross sections, tubules with elongated spermatids (ES), outer/inner diameter of tubules, thickness of tubular wall, and number of Sertoli cells (SC). Additionally, animal data were considered. No significant differences were found between the left and right testis or among the individual locations of en bloc samples. However, histologically significant differences (Bonferroni-adjusted significance level: 0.05/6 = 0.0083) were found between the needle biopsy and en bloc resection regarding the tubular cross sections per visual field ( 0.05), the outer ( 0.01) and inner diameter and the thickness of the tubular wall (both 0.01). In the SOX9 immunohistochemical staining, no significant differences ( 0.05) could be observed for SC numbers between needle biopsy and en bloc samples. In conclusion, results of testicular needle biopsy do not have the same validity as the en bloc resection histology. Furthermore, one biopsy is insufficient to reflect the histology of the entire testicular pair.
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http://dx.doi.org/10.3390/ani10050918DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278371PMC
May 2020

Ovine C-type lectin receptor hFc-fusion protein library - A novel platform to screen for host-pathogen interactions.

Vet Immunol Immunopathol 2020 Apr 18;224:110047. Epub 2020 Apr 18.

Immunology Unit & Research Center for Emerging Infections and Zoonoses (RIZ), University for Veterinary Medicine Hannover, Foundation. Hannover, Germany. Electronic address:

C-type lectin receptors (CTLRs) are pattern recognition receptors which are important constituents of the innate immunity. However, their role has mostly been studied in humans and in mouse models. To bridge the knowledge gap concerning CTLRs of veterinary relevant species, a novel ovine CTLR hFc-fusion protein library which allows in vitro ligand identification and pathogen binding studies has been established. Its utility was tested with known ligands of corresponding murine CTLRs in ELISA- and flow cytometry based binding studies. The ovine CTLR-hFc library was subsequently used in a proof-of-principle pathogen binding study with the ruminant pathogen Mycoplasma mycoides subsp. capri. Some ovine CTLRs, such as Dendritic Cell Immunoreceptor (DCIR, Clec4a), Macrophage C-Type Lectin (MCL, Clec4d) and Myeloid Inhibitory C-Type Lectin-Like Receptor (MICL, Clec12a) were identified as possible candidate receptors whose role in Mycoplasma recognition can now be unraveled in further studies. This study thus shows the utility of this novel ovine CTLR-hFc fusion protein library to screen for CTLR/pathogen interactions.
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http://dx.doi.org/10.1016/j.vetimm.2020.110047DOI Listing
April 2020

Loss of Cx43 in Murine Sertoli Cells Leads to Altered Prepubertal Sertoli Cell Maturation and Impairment of the Mitosis-Meiosis Switch.

Cells 2020 03 10;9(3). Epub 2020 Mar 10.

Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, 30173 Hannover, Lower Saxony, Germany.

Male factor infertility is a problem in today's society but many underlying causes are still unknown. The generation of a conditional Sertoli cell (SC)-specific connexin 43 (Cx43) knockout mouse line (SCCx43KO) has provided a translational model. Expression of the gap junction protein Cx43 between adjacent SCs as well as between SCs and germ cells (GCs) is known to be essential for the initiation and maintenance of spermatogenesis in different species and men. Adult SCCx43KO males show altered spermatogenesis and are infertile. Thus, the present study aims to identify molecular mechanisms leading to testicular alterations in prepubertal SCCx43KO mice. Transcriptome analysis of 8-, 10- and 12-day-old mice was performed by next-generation sequencing (NGS). Additionally, candidate genes were examined by qRT-PCR and immunohistochemistry. NGS revealed many significantly differentially expressed genes in the SCCx43KO mice. For example, GCspecific genes were mostly downregulated and found to be involved in meiosis and spermatogonial differentiation (e.g., , ). In contrast, SC-specific genes implicated in SC maturation and proliferation were mostly upregulated (e.g., , ). In conclusion, Cx43 in SCs appears to be required for normal progression of the first wave of spermatogenesis, especially for the mitosis-meiosis switch, and also for the regulation of prepubertal SC maturation.
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http://dx.doi.org/10.3390/cells9030676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140672PMC
March 2020

Urine E-cadherin: A Marker for Early Detection of Kidney Injury in Diabetic Patients.

J Clin Med 2020 Feb 27;9(3). Epub 2020 Feb 27.

Clinic for Nephrology and Rheumatology, University Medical Center Göttingen 1, 37075 Göttingen, Germany.

Diabetic nephropathy (DN) is the main reason for end-stage renal disease. Microalbuminuria as the non-invasive available diagnosis marker lacks specificity and gives high false positive rates. To identify and validate biomarkers for DN, we used in the present study urine samples from four patient groups: diabetes without nephropathy, diabetes with microalbuminuria, diabetes with macroalbuminuria and proteinuria without diabetes. For the longitudinal validation, we recruited 563 diabetic patients and collected 1363 urine samples with the clinical data during a follow-up of 6 years. Comparative urinary proteomics identified four proteins Apolipoprotein A-I (APOA1), Beta-2-microglobulin (B2M), E-cadherin (CDH1) and Lithostathine-1-alpha (REG1A), which differentiated with high statistical strength ( < 0.05) between DN patients and the other groups. Label-free mass spectrometric quantification of the candidates confirmed the discriminatory value of E-cadherin and Lithostathine-1-alpha ( < 0.05). Immunological validation highlighted E-cadherin as the only marker able to differentiate significantly between the different DN stages with an area under the curve (AUC) of 0.85 (95%-CI: [0.72, 0.97]). The analysis of the samples from the longitudinal study confirmed the prognostic value of E-cadherin, the critical increase in urinary E-cadherin level was measured 20 ± 12.5 months before the onset of microalbuminuria and correlated significantly ( < 0.05) with the glomerular filtration rate measured by estimated glomerular filtration rate (eGFR).
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http://dx.doi.org/10.3390/jcm9030639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141221PMC
February 2020

Inhibiting WNT and NOTCH in renal cancer stem cells and the implications for human patients.

Nat Commun 2020 02 17;11(1):929. Epub 2020 Feb 17.

Cancer Research Program, Max Delbrueck Center for Molecular Medicine (MDC) in the Helmholtz Association, Berlin, Germany.

Current treatments for clear cell renal cell cancer (ccRCC) are insufficient because two-thirds of patients with metastases progress within two years. Here we report the identification and characterization of a cancer stem cell (CSC) population in ccRCC. CSCs are quantitatively correlated with tumor aggressiveness and metastasis. Transcriptional profiling and single cell sequencing reveal that these CSCs exhibit an activation of WNT and NOTCH signaling. A significant obstacle to the development of rational treatments has been the discrepancy between model systems and the in vivo situation of patients. To address this, we use CSCs to establish non-adherent sphere cultures, 3D tumor organoids, and xenografts. Treatment with WNT and NOTCH inhibitors blocks the proliferation and self-renewal of CSCs in sphere cultures and organoids, and impairs tumor growth in patient-derived xenografts in mice. These findings suggest that our approach is a promising route towards the development of personalized treatments for individual patients.
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http://dx.doi.org/10.1038/s41467-020-14700-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026425PMC
February 2020

Limited utility of qPCR-based detection of tumor-specific circulating mRNAs in whole blood from clear cell renal cell carcinoma patients.

BMC Urol 2020 Feb 4;20(1). Epub 2020 Feb 4.

Department of Urology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.

Background: RNA sequencing data is providing abundant information about the levels of dysregulation of genes in various tumors. These data, as well as data based on older microarray technologies have enabled the identification of many genes which are upregulated in clear cell renal cell carcinoma (ccRCC) compared to matched normal tissue. Here we use RNA sequencing data in order to construct a panel of highly overexpressed genes in ccRCC so as to evaluate their RNA levels in whole blood and determine any diagnostic potential of these levels for renal cell carcinoma patients.

Methods: A bioinformatics analysis with Python was performed using TCGA, GEO and other databases to identify genes which are upregulated in ccRCC while being absent in the blood of healthy individuals. Quantitative Real Time PCR (RT-qPCR) was subsequently used to measure the levels of candidate genes in whole blood (PAX gene) of 16 ccRCC patients versus 11 healthy individuals. PCR results were processed in qBase and GraphPadPrism and statistics was done with Mann-Whitney U test.

Results: While most analyzed genes were either undetectable or did not show any dysregulated expression, two genes, CDK18 and CCND1, were paradoxically downregulated in the blood of ccRCC patients compared to healthy controls. Furthermore, LOX showed a tendency towards upregulation in metastatic ccRCC samples compared to non-metastatic.

Conclusions: This analysis illustrates the difficulty of detecting tumor regulated genes in blood and the possible influence of interference from expression in blood cells even for genes conditionally absent in normal blood. Testing in plasma samples indicated that tumor specific mRNAs were not detectable. While CDK18, CCND1 and LOX mRNAs might carry biomarker potential, this would require validation in an independent, larger patient cohort.
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http://dx.doi.org/10.1186/s12894-019-0542-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6998103PMC
February 2020
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