A new approach to describe the taxonomic structure of microbiome and its application to assess the relationship between microbial niches.

BACKGROUND: Data from microbiomes from multiple niches is often collected, but methods to analyse these often ignore associations between niches. One interesting case is that of the oral microbiome. Its composition is receiving increasing attention due to reports on its associations with general health. While the oral cavity includes different niches, multi-niche microbiome data analysis is conducted using a single niche at a time and, therefore, ignores other niches that could act as confounding variables. Understanding the interaction between niches would assist interpretation of the results, and help improve our understanding of multi-niche microbiomes. METHODS: In this study, we used a machine learning technique called latent Dirichlet allocation (LDA) on two microbiome datasets consisting of several niches. LDA was used on both individual niches and all niches simultaneously. On individual niches, LDA was used to decompose each niche into bacterial sub-communities unveiling their taxonomic structure. These sub-communities were then used to assess the relationship between microbial niches using the global test. On all niches simultaneously, LDA allowed us to extract meaningful microbial patterns. Sets of co-occurring operational taxonomic units (OTUs) comprising those patterns were then used to predict the original location of each sample. RESULTS: Our approach showed that the per-niche sub-communities displayed a strong association between supragingival plaque and saliva, as well as between the anterior and posterior tongue. In addition, the LDA-derived microbial signatures were able to predict the original sample niche illustrating the meaningfulness of our sub-communities. For the multi-niche oral microbiome dataset we had an overall accuracy of 76%, and per-niche sensitivity of up to 83%. Finally, for a second multi-niche microbiome dataset from the entire body, microbial niches from the oral cavity displayed stronger associations to each other than with those from other parts of the body, such as niches within the vagina and the skin. CONCLUSION: Our LDA-based approach produces sets of co-occurring taxa that can describe niche composition. LDA-derived microbial signatures can also be instrumental in summarizing microbiome data, for both descriptions as well as prediction.

Genome-wide siRNA screens identify RBBP9 function as a potential target in Fanconi anaemia-deficient head-and-neck squamous cell carcinoma.

Fanconi anaemia (FA) is a rare chromosomal-instability syndrome caused by mutations of any of the 22 known FA DNA-repair genes. FA individuals have an increased risk of head-and-neck squamous-cell-carcinomas (HNSCC), often fatal. Systemic intolerance to standard cisplatin-based protocols due to somatic-cell hypersensitivity underscores the urgent need to develop novel therapies. Here, we performed unbiased siRNA screens to unveil genetic interactions synthetic-lethal with FA-pathway deficiency in FA-patient HNSCC cell lines. We identified based on differential-lethality scores between FA-deficient and FA-proficient cells, next to common-essential genes such as PSMC1, PSMB2, and LAMTOR2, the otherwise non-essential RBBP9 gene. Accordingly, low dose of the FDA-approved RBBP9-targeting drug Emetine kills FA-HNSCC. Importantly both RBBP9-silencing as well as Emetine spared non-tumour FA cells. This study provides a minable genome-wide analyses of vulnerabilities to address treatment challenges in FA-HNSCC. Our investigation divulges a DNA-cross-link-repair independent lead, RBBP9, for targeted treatment of FA-HNSCCs without systemic toxicity.

A powerful global test for spliceQTL effects.

Statistical methods to test for effects of single nucleotide polymorphisms (SNPs) on exon inclusion exist but often rely on testing of associations between multiple exon-SNP pairs, with sometimes subsequent summarization of results at the gene level. Such approaches require heavy multiple testing corrections and detect mostly events with large effect sizes. We propose here a test to find spliceQTL (splicing quantitative trait loci) effects that takes all exons and all SNPs into account simultaneously. For any chosen gene, this score-based test looks for an association between the set of exon expressions and the set of SNPs, via a random-effects model framework. It is efficient to compute and can be used if the number of SNPs is larger than the number of samples. In addition, the test is powerful in detecting effects that are relatively small for individual exon-SNP pairs but are observed for many pairs. Furthermore, test results are more often replicated across datasets than pairwise testing results. This makes our test more robust to exon-SNP pair-specific effects, which do not extend to multiple pairs within the same gene. We conclude that the test we propose here offers more power and better replicability in the search for spliceQTL effects.

Electronic Nose Sensor Drift Affects Diagnostic Reliability and Accuracy of Disease-Specific Algorithms.

Sensor drift is a well-known disadvantage of electronic nose (eNose) technology and may affect the accuracy of diagnostic algorithms. Correction for this phenomenon is not routinely performed. The aim of this study was to investigate the influence of eNose sensor drift on the development of a disease-specific algorithm in a real-life cohort of inflammatory bowel disease patients (IBD). In this multi-center cohort, patients undergoing colonoscopy collected a fecal sample prior to bowel lavage. Mucosal disease activity was assessed based on endoscopy. Controls underwent colonoscopy for various reasons and had no endoscopic abnormalities. Fecal eNose profiles were measured using Cyranose 320®. Fecal samples of 63 IBD patients and 63 controls were measured on four subsequent days. Sensor data displayed associations with date of measurement, which was reproducible across all samples irrespective of disease state, disease activity state, disease localization and diet of participants. Based on logistic regression, corrections for sensor drift improved accuracy to differentiate between IBD patients and controls based on the significant differences of six sensors (p = 0.004; p < 0.001; p = 0.001; p = 0.028; p < 0.001 and p = 0.005) with an accuracy of 0.68. In this clinical study, short-term sensor drift affected fecal eNose profiles more profoundly than clinical features. These outcomes emphasize the importance of sensor drift correction to improve reliability and repeatability, both within and across eNose studies.

Functional Screen for microRNAs Suppressing Anchorage-Independent Growth in Human Cervical Cancer Cells.

The progression of anchorage-dependent epithelial cells to anchorage-independent growth represents a critical hallmark of malignant transformation. Using an in vitro model of human papillomavirus (HPV)-induced transformation, we previously showed that acquisition of anchorage-independent growth is associated with marked (epi)genetic changes, including altered expression of microRNAs. However, the laborious nature of the conventional growth method in soft agar to measure this phenotype hampers a high-throughput analysis. We developed alternative functional screening methods using 96- and 384-well ultra-low attachment plates to systematically investigate microRNAs regulating anchorage-independent growth. SiHa cervical cancer cells were transfected with a microRNA mimic library (n = 2019) and evaluated for cell viability. We identified 84 microRNAs that consistently suppressed growth in three independent experiments. Further validation in three cell lines and comparison of growth in adherent and ultra-low attachment plates yielded 40 microRNAs that specifically reduced anchorage-independent growth. In conclusion, ultra-low attachment plates are a promising alternative for soft-agar assays to study anchorage-independent growth and are suitable for high-throughput functional screening. Anchorage independence suppressing microRNAs identified through our screen were successfully validated in three cell lines. These microRNAs may provide specific biomarkers for detecting and treating HPV-induced precancerous lesions progressing to invasive cancer, the most critical stage during cervical cancer development.

Distinct Patterns of Myeloid Cell Infiltration in Patients With hrHPV-Positive and hrHPV-Negative Penile Squamous Cell Carcinoma: The Importance of Assessing Myeloid Cell Densities Within the Spatial Context of the Tumor.

Comprehensive analysis of tumor infiltrating myeloid cells in the tumor microenvironment of penile squamous cell carcinoma (PSCC) is lacking. In this retrospective study, for the first time, PSCC resection specimens (N = 103) were annotated into the following compartments: intratumoral tumor (IT Tumor), intratumoral stroma (IT Stroma), peritumoral tumor (PT Tumor) and peritumoral stroma (PT Stroma) compartments. We then quantified CD14+, CD68+ and CD163+ myeloid cells within these compartments using an image analysis software and assessed their association with various clinical parameters, including high-risk human papillomavirus (hrHPV) status. In the total cohort, hrHPV status, grade of differentiation, age and tumor size were associated with myeloid cell densities. hrHPV+ tumors had higher infiltration rates of CD14+, CD68+ and CD163+ myeloid cells in the IT tumor compartment (p < 0.001, for all) compared to hrHPV- tumors. Furthermore, when examining the association between compartment-specific infiltration and differentiation grade, increased myeloid cell densities in the IT tumor compartment were associated with a more advanced histological grade (p < 0.001, for all). This association remained significant when the hrHPV- cohort (N = 60) was analyzed (CD14+ p = 0.001; CD68+ p < 0.001; CD163+ p = 0.004). Subgroup analysis in the hrHPV+ group (N = 43) showed that high infiltration rates of CD68+ and CD163+ cells in the PT tumor compartment were associated with lymph node (LN) metastasis (p = 0.031 and p = 0.026, respectively). Regarding the association between myeloid cell densities and disease-specific survival, the risk of death was found to decrease slightly as the number of myeloid cells in the IT tumor compartment increased (CD14+ p = 0.04; CD68+ p = 0.05; CD163+ p = 0.02). However, after adjusting for hrHPV, no independent association between myeloid densities and disease-specific survival were found. Altogether, these findings demonstrate the importance of assessing myeloid cell densities within the spatial context of the tumor. Further studies are needed to unravel the specific phenotype of myeloid cells residing in the different compartments, their effect on clinical parameters and the impact of hrHPV on the recruitment of myeloid cell populations in PSCC.

Testing for coeliac disease rarely leads to a diagnosis: a population-based study.

BACKGROUND: Coeliac disease (CD) has an estimated prevalence of ∼1% in Europe with a significant gap between undiagnosed and diagnosed CD. Active case finding may help to bridge this gap yet the diagnostic yield of such active case finding in general practice by serological testing is unknown. OBJECTIVE: The aim of this study was to determine (1) the frequency of diagnosed CD in the general population, and (2) to investigate the yield of active case finding by general practitioners. METHODS: Electronic medical records of 207.200 patients registered in 49 general practices in The Netherlands in 2016 were analysed. An extensive search strategy, based on International Classification of Primary Care codes, free text and diagnostic test codes was performed to search CD- or gluten-related contacts. RESULTS: The incidence of CD diagnosis in general practice in 2016 was 0.01%. The prevalence of diagnosed CD reported in the general practice in the Netherlands was 0.19%, and considerably higher than previously reported in the general population. During the one year course of the study 0.95% of the population had a gluten-related contact with their GP; most of them (72%) were prompted by gastrointestinal complaints. Serological testing was performed in 66% (n = 1296) of these patients and positive in only 1.6% (n = 21). CONCLUSION: The number of diagnosed CD patients in the Netherlands is substantially higher than previously reported. This suggests that the gap between diagnosed and undiagnosed patients is lower than generally assumed. This may explain that despite a high frequency of gluten-related consultations in general practice the diagnostic yield of case finding by serological testing is low.Key pointsThe diagnostic approach of GPs regarding CD and the diagnostic yield is largely unknownCase finding in a primary health care practice has a low yield of 1.6%CD testing was mostly prompted by consultation for gastrointestinal symptomsThere is a heterogeneity in types of serological test performed in primary care.

Delta-Like Ligand-Notch1 Signaling Is Selectively Modulated by HPV16 E6 to Promote Squamous Cell Proliferation and Correlates with Cervical Cancer Prognosis.

Human papillomavirus (HPV) drives high-grade intraepithelial neoplasia and cancer; for unknown reasons, this occurs most often in the cervical transformation zone. Either mutation or HPV E6-driven inhibition of Notch1 can drive neoplastic development in stratified squamous epithelia. However, the contribution of Notch1 and its Delta-like ligands (DLL) to site susceptibility remains poorly understood. Here, we map DLL1/DLL4 expression in cell populations present in normal cervical biopsies by immunofluorescence. In vitro keratinocyte 2D monolayer models, growth assays, and organotypic raft cultures were used to assess the functional role of DLL-Notch signaling in uninfected cells and its modulation by HPV16 in neoplasia. An RNA sequencing-based gene signature was used to suggest the cell of origin of 279 HPV-positive cervical carcinomas from The Cancer Genome Atlas and to relate this to disease prognosis. Finally, the prognostic impact of DLL4 expression was investigated in three independent cervical cancer patient cohorts. Three molecular cervical carcinoma subtypes were identified, with reserve cell tumors the most common and linked to relatively good prognosis. Reserve cells were characterized as DLL1-/DLL4+, a proliferative phenotype that is temporarily observed during squamous metaplasia and wound healing but appears to be sustained by HPV16 E6 in raft models of low-grade and, more prominently, high-grade neoplasia. High expression of DLL4 was associated with an increased likelihood of cervical cancer-associated death and recurrence. Taken together, DLL4-Notch1 signaling reflects a proliferative cellular state transiently present during physiologic processes but inherent to cervical reserve cells, making them strongly resemble neoplastic tissue even before HPV infection has occurred. SIGNIFICANCE: This study investigates cervical cancer cell-of-origin populations and describes a DLL-Notch1 phenotype that is associated with disease prognosis and that might help identify cells that are susceptible to HPV-induced carcinogenesis.

Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells.

Germline mutations in the Folliculin (FLCN) tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. FLCN is a conserved, essential gene linked to diverse cellular processes but the mechanism by which FLCN prevents kidney cancer remains unknown. Here, we show that disrupting FLCN in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.

Expression of Oncolytic Adenovirus-Encoded RNAi Molecules Is Most Effective in a pri-miRNA Precursor Format.

Oncolytic adenoviruses are being developed as new anti-cancer agents. Their efficacy can be improved by incorporating RNA interference (RNAi) molecules. RNAi molecules can be expressed in various precursor formats. The aim of this study was to determine the most effective format. To this end, we constructed three Δ24-type oncolytic adenoviruses, with human microRNA-1 (miR-1) expression cassettes in short hairpin RNA (shRNA), precursor microRNA (pre-miRNA), and primary miRNA (pri-miRNA) format, respectively. The viruses were compared for virus replication, mature miR-1 expression, and target gene silencing in cancer cells. Incorporation of the cassettes had only minor effects on virus replication. Mature miR-1 expression from the pri-miRNA format reached on average 100-fold higher levels than from the other two formats. This expression remained stable upon long-term virus propagation. Infection with the pri-miR-1-expressing virus silenced the validated miR-1 targets FOXP1 and MET. Drosha knockout almost completely abrogated mature miR-1 expression, confirming that processing of adenovirus-encoded pri-miR-1 was dependent on the host cell miRNA machinery. Using simple in vitro recombination cloning, a similar virus expressing miR-26b was made and shown to silence the validated miR-26b target PTGS2. We thus provide a platform for construction of oncolytic adenoviruses with high expression of RNAi molecules of choice.

Chemopreventive targeted treatment of head and neck precancer by Wee1 inhibition.

HPV-negative head and neck squamous cell carcinomas (HNSCCs) develop in precancerous changes in the mucosal lining of the upper-aerodigestive tract. These precancerous cells contain cancer-associated genomic changes and cause primary tumors and local relapses. Therapeutic strategies to eradicate these precancerous cells are very limited. Using functional genomic screens, we identified the therapeutic vulnerabilities of premalignant mucosal cells, which are shared with fully malignant HNSCC cells. We screened 319 previously identified tumor-lethal siRNAs on a panel of cancer and precancerous cell lines as well as primary fibroblasts. In total we identified 147 tumor-essential genes including 34 druggable candidates. Of these 34, 13 were also essential in premalignant cells. We investigated the variable molecular basis of the vulnerabilities in tumor and premalignant cell lines and found indications of collateral lethality. Wee1-like kinase (WEE1) was amongst the most promising targets for both tumor and precancerous cells. All four precancerous cell lines were highly sensitive to Wee1 inhibition by Adavosertib (AZD1775), while primary keratinocytes tolerated this inhibitor. Wee1 inhibition caused induction of DNA damage during S-phase followed by mitotic failure in (pre)cancer cells. In conclusion, we uncovered Wee1 inhibition as a promising chemopreventive strategy for precancerous cells, with comparable responses as fully transformed HNSCC cells.

Functional Genomic Screen in Mesothelioma Reveals that Loss of Function of BRCA1-Associated Protein 1 Induces Chemoresistance to Ribonucleotide Reductase Inhibition.

Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR < 0.05) that were more cytotoxic to BAP1-proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (BAP1 +/-) overexpressing BAP1 C91A (catalytically dead mutant) was more resistant to RNR inhibition, while BAP1 knockdown in the BAP1-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in BAP1-proficient cell line-derived spheroids compared with BAP1 deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in BAP1 mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with BAP1-WT but not with BAP1 C91A. Upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.

Neoadjuvant cisplatin and paclitaxel modulate tumor-infiltrating T cells in patients with cervical cancer.

Resistance to chemotherapy is widely recognized as one of the major factors limiting therapeutic efficacy and influences clinical outcomes in patients with cancer. Many studies on various tumor types have focused on combining standard-of-care chemotherapy with immunotherapy. However, for cervical cancer, the role of neoadjuvant chemotherapy (NACT) on the local immune microenvironment is largely unexplored. We performed a pilot study on 13 primary cervical tumor samples, before and after NACT, to phenotype and enumerate tumor-infiltrating T-cell subpopulations using multiplex immunohistochemistry (CD3, CD8, FoxP3, Ki67, and Tbet) and automated co-expression analysis software. A significant decrease in proliferating (Ki67+) CD3+CD8- T cells and FoxP3+(CD3+CD8-) regulatory T cells was observed in the tumor stroma after cisplatin and paclitaxel treatment, with increased rates of cytotoxic CD8+ T cells, including activated and CD8+Tbet+ T cells. No effect was observed on the number of tumor-infiltrating T cells in the cervical tumor microenvironment after treatment with cisplatin only. Therefore, we conclude that patients treated with cisplatin and paclitaxel had more tumor-infiltrating T-cell modulation than patients treated with cisplatin monotherapy. These findings enhance our understanding of the immune-modulating effect of chemotherapy and warrant future combination of the standard-of-care therapy with immunotherapy to improve clinical outcome in patients with cervical cancer.

High-throughput RNAi screening reveals cancer-selective lethal targets in the RNA spliceosome.

Novel therapeutic strategies for non-small-cell lung cancer (NSCLC) are urgently needed. RNA splicing, orchestrated by the spliceosome, is deregulated in many forms of cancer, including NSCLC. Here, we performed high-throughput screening with a small interfering RNA library targeting all annotated human spliceosome proteins to identify cancer-selective lethal targets in the RNA splicing machinery. Silencing of several spliceosome proteins reduced cell viability in a panel of NSCLC cell lines, but not in non-malignant lung fibroblasts and epithelial cells. Interestingly, the cancer-selective lethal target set comprised all seven Sm proteins that, together with small nuclear RNA, form the core structure of most spliceosome subunits. Interfering with Sm protein expression induced apoptosis in NSCLC cells, but not in non-malignant cells. In silico analysis revealed that Sm proteins are frequently upregulated in NSCLC. For several Sm proteins, increased expression showed a positive correlation with disease severity. Together, our results suggest that the Sm proteins represent particularly useful novel targets for selective treatment of NSCLC.

Fetal fraction evaluation in non-invasive prenatal screening (NIPS).

An important factor in quality control of non-invasive prenatal screening (NIPS) or testing (NIPT) is a sufficient percentage of fetal DNA to avoid false-negative results. Here we evaluate 14,379 shallow whole-genome sequenced diagnostic NIPS samples, as well as negative controls, for both technical and biological factors that can influence fetal fraction and its assessment. Technically, bioinformatics analyses can have a profound impact on fetal fraction determination. We found best performance for fetal fraction determination with the Y chromosome based tool DEFRAG for male fetuses and the count based tool SeqFF for female fetuses. Biologically, gestational age of up to 21 weeks and maternal age had no influence on fetal fraction, while an increase in weight and BMI had a negative influence on fetal fraction. While a trend was observed, no statistically significant difference in fetal fraction was found between trisomy and normal samples. Overall, these results confirm the influence of biological factors and give insight into technical factors that can affect fetal fractions in NIPS.

Improvement of menopausal symptoms and the impact on work ability: A retrospective cohort pilot study.

OBJECTIVE: In this study we aimed to pilot test the hypothesis that in women who are severely bothered by their menopausal complaints, improvement of menopausal symptoms is associated with an improvement in self-perceived work ability. STUDY DESIGN: This retrospective cohort study assessed the work ability of first-time attendees (n = 31) of a menopause clinic at baseline (T0) and 3-9 months follow-up (T1). All patients received care as usual according to local protocol, no interventions were applied by the researchers. Self-reported questionnaire data assessing work ability (Work Ability Index; WAI) and menopausal symptoms (Greene Climacteric Scale; GCS) were used. MAIN OUTCOME MEASURES: Multiple linear regression was used in an exploratory analysis to examine the relationship between change in WAI score (ΔWAI) and change in menopausal symptoms (ΔGCS), after adjustment for potential confounders. Additional exploratory univariate linear regression analyses were performed to assess the associations of change in WAI score with change in the different GCS domains and with type of treatment. RESULTS: Twenty-seven out of 31 women reported improvement in work ability at follow-up (T1) (M = 30.73, SD = 6.42 respectively, M = 34.86, SD = 5.98). All women reported to be less bothered by their menopausal symptoms at T1 (M = 26.57, SD = 8.69 respectively, M = 14.73, SD = 6.36). Multivariate linear regression demonstrated a significant association between the WAI and GCS change scores after correction for confounders (beta ΔGCS = 0.283, p = 0.014). After additional adjustment for WAI at baseline, this association was no longer significant (beta ΔGCS = 0.172, p = 0.164). Change in GCS depression domain (ΔGCS depression) was significantly associated with ΔWAI, although after correction for WAI at baseline the effect of ΔGCS depression was no longer significant (beta = 0.855, p = 0.113). The WAI and GCS change scores were highly correlated, as a result their coefficients were not statistically significant separately. CONCLUSIONS: Treatment aimed at alleviating menopausal symptoms in symptomatic women could lead to improvement of menopausal symptoms along with improvement in work ability. Improvement of depressive symptoms seem particularly important for this outcome.

HLA class II expression on tumor cells and low numbers of tumor-associated macrophages predict clinical outcome in oropharyngeal cancer.

BACKGROUND: Human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) is a highly immunogenic tumor and differences in tumor microenvironment might contribute to the improved survival of HPV-positive OPSCC patient. METHODS: A comprehensive multivariate analysis with clinical and immune variables (human leukocyte antigen [HLA] I/II, programmed death ligand 1 (PD-L1), programmed death receptor 1 (PD1), T cells, and macrophages) was performed in 142 OPSCC patients. RESULTS: We found an inverse correlation between the expression of HLA class II molecules on tumor cells and CD68+ CD163+ tumor-associated macrophages (TAMs). High HLA-DP/DQ/DR expression and low number of TAMs were associated with longer disease-specific survival and disease-free survival (DFS). Furthermore, a new population of CD8+ FoxP3+ T cells was correlated with shorter DFS in multivariate analysis. CONCLUSIONS: \We identified new prognostic markers for patients with oropharyngeal cancer, which can be used for selecting patients that can benefit from immunotherapy.

Vitamin D Attenuates Endothelial Dysfunction in Uremic Rats and Maintains Human Endothelial Stability.

Background Dysfunctional endothelium may contribute to the development of cardiovascular complications in chronic kidney disease ( CKD ). Supplementation with active vitamin D has been proposed to have vasoprotective potential in CKD , not only by direct effects on the endothelium but also by an increment of α-Klotho. Here, we explored the capacity of the active vitamin D analogue paricalcitol to protect against uremia-induced endothelial damage and the extent to which this was dependent on increased α-Klotho concentrations. Methods and Results In a combined rat model of CKD with vitamin D deficiency, renal failure induced vascular permeability and endothelial-gap formation in thoracic aorta irrespective of baseline vitamin D, and this was attenuated by paricalcitol. Downregulation of renal and serum α-Klotho was found in the CKD model, which was not restored by paricalcitol. By measuring the real-time changes of the human endothelial barrier function, we found that paricalcitol effectively improved the recovery of endothelial integrity following the addition of the pro-permeability factor thrombin and the induction of a wound. Furthermore, immunofluorescence staining revealed that paricalcitol promoted vascular endothelial-cadherin-based cell-cell junctions and diminished F-actin stress fiber organization, preventing the formation of endothelial intracellular gaps. Conclusions Our results demonstrate that paricalcitol attenuates the CKD -induced endothelial damage in the thoracic aorta and directly mediates endothelial stability in vitro by enforcing cell-cell interactions.

Rscreenorm: normalization of CRISPR and siRNA screen data for more reproducible hit selection.

BACKGROUND: Reproducibility of hits from independent CRISPR or siRNA screens is poor. This is partly due to data normalization primarily addressing technical variability within independent screens, and not the technical differences between them. RESULTS: We present "rscreenorm", a method that standardizes the functional data ranges between screens using assay controls, and subsequently performs a piecewise-linear normalization to make data distributions across all screens comparable. In simulation studies, rscreenorm reduces false positives. Using two multiple-cell lines siRNA screens, rscreenorm increased reproducibility between 27 and 62% for hits, and up to 5-fold for non-hits. Using publicly available CRISPR-Cas screen data, application of commonly used median centering yields merely 34% of overlapping hits, in contrast with rscreenorm yielding 84% of overlapping hits. Furthermore, rscreenorm yielded at most 8% discordant results, whilst median-centering yielded as much as 55%. CONCLUSIONS: Rscreenorm yields more consistent results and keeps false positive rates under control, improving reproducibility of genetic screens data analysis from multiple cell lines.

A functional siRNA screen identifies RhoGTPase-associated genes involved in thrombin-induced endothelial permeability.

Thrombin and other inflammatory mediators may induce vascular permeability through the disruption of adherens junctions between adjacent endothelial cells. If uncontrolled, hyperpermeability leads to an impaired barrier, fluid leakage and edema, which can contribute to multi-organ failure and death. RhoGTPases control cytoskeletal dynamics, adhesion and migration and are known regulators of endothelial integrity. Knowledge of the precise role of each RhoGTPase, and their associated regulatory and effector genes, in endothelial integrity is incomplete. Using a combination of a RNAi screen with electrical impedance measurements, we quantified the effect of individually silencing 270 Rho-associated genes on the barrier function of thrombin-activated, primary endothelial cells. Known and novel RhoGTPase-associated regulators that modulate the response to thrombin were identified (RTKN, TIAM2, MLC1, ARPC1B, SEPT2, SLC9A3R1, RACGAP1, RAPGEF2, RHOD, PREX1, ARHGEF7, PLXNB2, ARHGAP45, SRGAP2, ARHGEF5). In conclusion, with this siRNA screen, we confirmed the roles of known regulators of endothelial integrity but also identified new, potential key players in thrombin-induced endothelial signaling.