DNA Status Research Articles

Downregulation of ACE, AGTR1, and ACE2 genes mediating SARS-CoV-2 pathogenesis by gut microbiota members and their postbiotics on Caco-2 cells

IntroductionCoronavirus disease-2019 (COVID-19) is a complex infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that can cause also gastrointestinal symptoms. There are various factors that determine the host susceptibility and severity of infection, including the renin-angiotensin system, the immune response, and the gut microbiota. In this regard, we aimed to investigate the gene expression of ACE, AGTR1, ACE2, and TMPRSS2, which mediate SARS-CoV-2 pathogenesis by Akkermansia muciniphila, Faecalibacterium prausnitzii, Bacteroides thetaiotaomicron, and Bacteroides fragilis on Caco-2 cells. Also, the enrichment analysis considering the studied genes was analyzed on raw data from the microarray analysis of COVID-19 patients. Materials and methodsCaco-2 cells were treated with live, heat-inactivated form and cell free supernatants of A. muciniphila, F. prausnitzii, B. thetaiotaomicron and B. fragilis for overnight. After RNA extraction and cDNA synthesis, the expression of studied genes was assessed by RT-qPCR. DNA methylation of studied genes was analyzed by Partek® Genomics Suite® software on the GSE174818 dataset. We used GSE164805 and GSE166552 datasets from COVID-19 patients to perform enrichment analysis by considering the mentioned genes via GEO2R, DAVID. Finally, the related microRNAs to GO terms concerned on the studied genes were identified by miRPath. ResultsThe downregulation of ACE, AGTR1, and ACE2 genes by A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis in live, heat-inactivated, and cell-free supernatants was reported for the first time. These genes had hypomethylated DNA status in COVID-19 patients' raw data. The highest fold enrichment in upregulated RAS pathways and immune responses belonged to ACE, AGTR1, and ACE2 by considering the protein-protein interaction network. The common miRNAs targeting the studied genes were reported as miR-124-3p and miR-26b-5p. ConclusionIn combination with our experimental data and bioinformatic analysis, we showed the potential of A. muciniphila, F. prausnitzii, B. thetaiotaomicron, and B. fragilis and their postbiotics to reduce ACE, ATR1, and ACE2 expression, which are essential genes that drive upregulated biological processes in COVID-19 patients.Accordingly, due to the potential of studied bacteria on the alteration of ACE, AGTR1, ACE2 genes expression, understanding their correlation with demonstrated miRNAs expression could be valuable. These findings suggest the importance of considering targeted gut microbiota intervention when designing the possible therapeutic strategy for controlling the COVID-19.

Expression of PD-L1 is HPV/P16-independent in oral squamous cell carcinoma

ObjectivesThis study aimed to investigate the expression of programmed cell death-ligand 1 (PD-L1) and its associations with human papillomavirus (HPV) 16/18 DNA status, p16 expression, demographic, clinicopathologic and risk parameters in patients with oral squamous cell carcinoma (OSCC). Study designA total of 85 formalin-fixed, paraffin-embedded OSCC specimens were collected. HPV16/18 DNA was detected by polymerase chain reaction. PD-L1 and p16 expressions were assessed using immunohistochemical technique. The immunostaining scores were calculated by combined positive score (CPS), previously described. The positive scoring value was determined at CPS ≥1, recommended by FDA. The associations between PD-L1 expression and HPV16/18 DNA status, p16 expression, demographic, clinicopathologic, and risk parameters were analyzed by Chi-square, Fisher's exact tests, and multivariate logistic regression. ResultsPD-L1 expression was detected in 22 out of 85 cases of OSCC (25.9%). 16.5% of all cases were HPV 16/18-positive and 62.4% were p16-positive. Statistically, there were no significant associations between PD-L1 expression in OSCC and HPV16/18 DNA status, p16 expression, demographic and, clinicopathologic parameters or risk behaviors. ConclusionApproximately one-fourth of OSCC cases were PD-L1-positive, suggesting candidacy for anti-PD-L1 immunotherapy. Furthermore, HPV infection and p16 expression were not involved with PD-L1 expression. Further clinical trials warrant the benefits of immunotherapy in patients with PD-L1-positive OSCC.

1710P Genomic landscape (GL) with potential of methylthioadenosine phosphorylase (MTAP) loss in clinically advanced breast cancer (CABC)

Homozygous deletion of MTAP causes intracellular arginine accumulation enabling anti-tumor effects of MTA2/PRMT5 inhibitors via a synthetic lethality mechanism. 7,301 cases of MBC underwent hybrid-capture based comprehensive genomic profiling (CGP). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and MSI status was determined on 114 loci. Tumor cell PD-L1 expression was determined by IHC (Dako 22C3). 208 (2.84%) of CABC featured MTAP loss. MTAP loss patients were younger (p=.002) and were more frequently ER- (30% v 50%; p<.0001), triple negative (TNBC) (47% v 27%; p<.0001) and less frequently HER2+ (2% v 8%; p=.0001) than MTAP intact MBC. Lobular histology and CDH1 mutations were more frequent in MTAP intact (14%) than MTAP loss MBC (p<.0001). CDKN2A (100%) and CDKN2B (97%) loss (9p21 co-deletion) were significantly associated with MTAP loss (p<.0001). Likely associated with the increased TNBC cases, BRCA1 mutation was also more frequent in MTAP loss MBC (10% v 4%; p<.0001). There was no clear pattern for predictive Immuno-Oncology (IO) drug biomarkers with higher TMB > 20 mut/Mb levels found in the MTAP intact MBC (p<.0001) and higher PD-L1 low expression (1-49%% TPS) in the MTAP loss MTAP (p=.002) CABC cases.Table: 1710PCases with MTAP intactCases with MTAP lossNumber7093208Mean age*57.854.5ER+/PR+ Status by IHC**70.0%/ 49.0%50.00%/29.90%HER2+ Amplification by CGP*7.80%1.92%TNBC Status*27.00%47.28%TP53*51.7061.30CDKN2A**3.10100.00CDKN2B**1.3096.70CDH1*14.300.90PTEN*13.1021.70PIK3CA**36.823.60BRCA1**3.709.90ERBB2 amplification*7.801.92ERBB2 sequence mutation*11.206.60EGFR *2.605.20MSI HighFrequency0.03%0.05%cases tested7077205TMB > 10%/>20%7.84%/7.40%5.32%/0.96%PD-L1 Positive IC expression (Dako 22C3)Low (1-49%)*11.45%42.90%High (> 50%)2.86%0.00%*Significant (P<0.05), **(P<0.0001). Open table in a new tab *Significant (P<0.05), **(P<0.0001). MTAP loss is associated with ER-, HER2- and TNBC status, features a distinctive GL with potential to impact both targeted and immunotherapies and enables emerging clinical trials testing MTA2 and PRMT5 inhibitors for patients with CABC.

Cellular analysis on the radiation induced bystander effects due to bismuth oxide nanoparticles with 6 MV photon beam radiotherapy

BackgroundThe therapeutic strategies in killing the cancerous tissue while keeping the normal healthy tissue uninterrupted can be further improved by introducing nanoparticles as radiosensitizer in radiotherapy. Nevertheless, healthy cells might still be affected by radiation-induced bystander effect (RIBE) response due the presence of nanoparticles. Thus, the potential negative effects in non-irradiated adjacent cells must be investigated. AimThe current study aims to explore the cellular effects of bismuth oxide nanoparticles (Bi2O3 NPs) on human breast cancer (MCF-7) and human foetal osteoblast (hFOB 1.19) cells due to the RIBE after irradiation with 6 MV clinical photon beam. Materials and methodsThe irradiated-cell conditioned medium (ICCM) treated with and without Bi2O3 NPs from the irradiated cells were collected and then transferred into the non-targeted cells. The ability of the bystander cells to proliferate and their ROS generation were analyzed. DNA status of the cells and the possibility of apoptosis were evaluated using FTIR spectroscopy and MUSE Cell Analyzer, respectively. ResultsThe current study revealed that MCF-7 and hFOB 1.19 bystander cells may continue to proliferate and survive following short and long-term incubation with ICCM treatment with Bi2O3 NPs. The survival rate of the treated bystander cells increased approximately 3%–8% compared to the untreated group. Although the bystander cells exhibited an increase in ROS and apoptosis, the addition of Bi2O3 NPs had no significant detrimental influence on the cells. Variation in the DNA vibrational amplitude detected by FTIR in bystander cells was less than 5% compared to the control group. ConclusionThis study discovered that the application of Bi2O3 NPs as a radiosensitizer did not give a negative impact on the bystander cells. This outcome may give an advantage to the application of Bi2O3 NPs as radiosensitizer in radiotherapy to enhance cancer cells death without introducing further detrimental effects to the non-targeted cells.

Prognostic value of systemic inflammation response index in nasopharyngeal carcinoma with negative Epstein-Barr virus DNA

BackgroundInflammatory parameters and Epstein–Barr virus (EBV) DNA status have been confirmed to be associated with prognosis in nasopharyngeal carcinoma (NPC) patients. However, there are few in-depth studies on the prognosis of NPC patients with negative EBV DNA. Our study aimed to look for inflammatory biomarkers that can identify disease progression in NPC patients with negative EBV DNA.MethodsA total of 795 NPC patients were recruited, and ultimately 325 NPC patients with negative EBV DNA were included in this study (170 in training cohort and 155 in validation cohort). Kaplan–Meier method and log-rank test were used to analyze progression-free survival (PFS) and overall survival (OS). The multivariate analysis of Cox proportional hazards regression model was used to determine the independent prognostic factors. Receiver operating characteristic (ROC) curves were used to assess prognostic value. The logistic regression was used to evaluate the relationship between EBV DNA status and inflammatory parameters. The correlation between clinical characteristics was analyzed by the chi-squared test or the Fisher’s exact test.ResultsThe optimal cutoff point for the SIRI was 1.12. The EBV DNA-negative NPC patients with high SIRI level had worse PFS and OS (all p < 0.001). In multivariate Cox proportional hazard models analysis, SIRI was an independent prognostic factor for PFS and OS (all p < 0.05), and had higher prognostic value than other indicators. Above results were found in the training cohort and confirmed in the validation cohort. In addition, EBV DNA status was not associated with any inflammatory parameters.ConclusionsThe SIRI can provide more accurate risk stratification and better prognostic prediction for NPC patients with negative EBV DNA.

Analysis of uveal melanomas and paired constitutional DNA for exclusion of a BAP1-tumor predisposition syndrome

Uveal melanoma (UM) is a rare tumor originating from melanocytic cells in the eye. Familial aggregation of UM is rare and can occur as part of the tumor predisposition syndrome BAP1-TPDS. However, family history alone will only identify a subset of patients with BAP1-TPDS. In the present study, we used sequential testing of tumor and blood DNA from UM patients for differential diagnosis of BAP1-TPDS. The study group was an unselected prospective cohort of patients from whom UM tissue was available. First, chromosome 3 status in tumor DNA was determined in all 140 patients who consented to participate. As tumors with disomy 3 rarely show BAP1 alterations, sequence analysis of this gene was performed in the 72 tumors with monosomy 3 (M3) or partial M3 only. We identified oncogenic BAP1 alterations in 52 of these tumors (72%). Targeted sequencing of DNA from matched peripheral blood showed pathogenic variants in two patients (3.8%) thus proving BAP1-TPDS. Only one of these two patients also had a medical history suggestive of this syndrome. Conversely, in three patients known to have had additional tumors before diagnosis of UM, constitutional heterozygosity for a BAP1 mutation was excluded. Altogether, in 50 patients we could exclude BAP1-TPDS with high diagnostic certainty. The results of our study support that genetic testing for BAP1-TPDS should be offered to all patients with UM. Moreover, as genetic information from the tumor can help exclude heritable risk, the strategy for analysis should include efforts to obtain tumor samples for testing.

Integration of p16/HPV DNA Status with a 24-miRNA-Defined Molecular Phenotype Improves Clinically Relevant Stratification of Head and Neck Cancer Patients.

Simple SummaryHuman papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC), regarded as a distinct clinical entity, are characterized by a considerably favourable prognosis after radio(chemo)therapy and a not yet fully understood distinct molecular pathogenesis. We aimed to develop a miRNA-signature that identifies HPV-associated HNSCC according to their specific molecular pathogenesis, and to characterise the transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of n = 229 HPV characterized HNSCC specimens of patients treated by adjuvant radio(chemo) therapy. Using lasso-regression, a 24-miRNA signature predicting HPV-status was built in a multicentre cohort and validated in a single-centre cohort. Its combination with p16/HPV DNA status improved clinically relevant risk stratification, allowed the identification of an HPV-associated patient subgroup with impaired overall survival, and might be considered for future clinical decision-making. miRNA-transcriptome integration identified HPV-specific signaling pathways.Human papillomavirus (HPV)-driven head and neck squamous cell carcinomas (HNSCC) generally have a more favourable prognosis. We hypothesized that HPV-associated HNSCC may be identified by an miRNA-signature according to their specific molecular pathogenesis, and be characterized by a unique transcriptome compared to HPV-negative HNSCC. We performed miRNA expression profiling of two p16/HPV DNA characterized HNSCC cohorts of patients treated by adjuvant radio(chemo)therapy (multicentre DKTK-ROG n = 128, single-centre LMU-KKG n = 101). A linear model predicting HPV status built in DKTK-ROG using lasso-regression was tested in LMU-KKG. LMU-KKG tumours (n = 30) were transcriptome profiled for differential gene expression and miRNA-integration. A 24-miRNA signature predicted HPV-status with 94.53% accuracy (AUC: 0.99) in DKTK-ROG, and 86.14% (AUC: 0.86) in LMU-KKG. The prognostic values of 24-miRNA- and p16/HPV DNA status were comparable. Combining p16/HPV DNA and 24-miRNA status allowed patient sub-stratification and identification of an HPV-associated patient subgroup with impaired overall survival. HPV-positive tumours showed downregulated MAPK, Estrogen, EGFR, TGFbeta, WNT signaling activity. miRNA-mRNA integration revealed HPV-specific signaling pathway regulation, including PD−L1 expression/PD−1 checkpoint pathway in cancer in HPV-associated HNSCC. Integration of clinically established p16/HPV DNA with 24-miRNA signature status improved clinically relevant risk stratification, which might be considered for future clinical decision-making with respect to treatment de-escalation in HPV-associated HNSCC.

The Divergence of Chromosome Structures and 45S Ribosomal DNA Organization in Cucumis debilis Inferred by Comparative Molecular Cytogenetic Mapping.

Cucumis debilis W.J.de Wilde & Duyfjes is an annual and monoecious plant. This species is endemic to Southeast Asia, particularly Vietnam. However, C. debilis is rarely studied, and no detailed information is available regarding its basic chromosome number, 45S ribosomal DNA (rDNA) status, and divergence among other Cucumis species. In this study, we characterized the morphological characters and determined and investigated the basic chromosome number and chromosomal distribution of 45S rDNA of C. debilis using the fluorescent in situ hybridization (FISH) technique. A maximum likelihood tree was constructed by combining the chloroplast and internal transcribed spacer of 45S rDNAs to infer its relationship within Cucumis. C. debilis had an oval fruit shape, green fruit peel, and protrusion-like white spots during the immature fruit stage. FISH analysis using 45S rDNA probe showed three pairs of 45S rDNA loci located at the terminal region in C. debilis, similar to C. hystrix. Meanwhile, two, two, and five pairs of 45S rDNA loci were observed for C. melo, C. metuliferus, and C. sativus, respectively. One melon (P90) and cucumber accessions exhibited different chromosomal localizations compared with other members of Cucumis. The majority of Cucumis species showed the terminal location of 45S rDNA, but melon P90 and cucumber exhibited terminal–interstitial and all interstitial orientations of 45S rDNA loci. Based on molecular cytogenetics and phylogenetic evidence, C. debilis is more closely related to cucumber than melon. Therefore, C. debilis may serve as a potential parental accession for genetic improvement of cucumber through interspecific hybridization.

Effect of Condom Use after CIN Treatment on Cervical HPV Biomarkers Positivity: Prolonged Follow Up Study.

Simple SummaryThe definite effects of consistent condom use following cervical local excisional procedures on preventing recurrent preinvasive disease as well as in biomarkers of HPV expression have not been studied so far. We embarked on a prospective observational study enrolling over 200 individuals who received strong advice for consistent condom use after undergoing local surgical CIN treatment. We assessed in the mid and long term (six and twenty-four-month post-op intervals, respectively) rates of CIN recurrence and the expression of HPV dependent and other biomarkers (HPV DNA, HPV mRNA E6 & E7, p16) in correlation with consistency of use. A favorable effect of routine condom use in rates of CIN relapse and biomarker expression was evident at the 6-month follow up; this was more pronounced at the 2-year assessment. In conclusion, consistent condom use following cervical local excisional treatment might influence favorably rates of CIN recurrence and biomarkers of HPV expression.Background: Several factors contribute in the cervical healing process following local surgical treatment; in a previous work our group has documented a beneficial mid-term role of regular condom use immediately postoperatively in terms of CIN relapse prevention and expression of active viral biomarkers. Materials and Methods: Aiming to investigate whether the favorable contribution of consistent condom use could be extrapolated in the longer term, we conducted a prospective single center observational study including women scheduled to undergo conservative excisional treatment for CIN (LLETZ procedure). In all women a strong recommendation for consistent use for the first 6 months was given. For 204 women who underwent the procedure and completed successfully the two-year follow up a complete dataset of HPV biomarkers’ results obtained six months and two years postoperatively was available. Patients were asked to complete a questionnaire to assess condom use compliance. A 90% compliance rate represented the threshold for consistent use. An LBC sample was obtained and tested for HPV genotyping, E6 & E7 mRNA by NASBA technique as well as flow cytometry, and p16 at 0 (pre-treatment), 6 and 24 months. HPV DNA and other related biomarkers status at 6 and 24 months, treatment failures at 24 months and condom use compliance rates represented study outcomes. Results: Six months post-operatively we documented a reduction in the rates of HPV DNA positivity, which was detected in only 23.2% of compliant condom users in comparison to 61.9% in the non-compliant group (p < 0.001, OR: 0.19, 95%CI: 0.1–0.36). For the HPV mRNA test, either assessed with the NASBA method or with flow cytometry, reduced positivity percentages were observed in the compliant group, in particular 1.6% vs. 8% for NASBA and 7.1% vs. 16.4% using flow cytometry, although these differences were not statistically significant (p = 0.1039 and 0.0791, respectively). Finally, reduced p16 positivity rates were documented in the compliant group. At the two year follow up, a more pronounced difference in HPV DNA positivity rates was observed, specifically only 13% positivity among the compliant women compared with 71% of the non-compliant (p < 0.0001); this illustrates a further decreasing trend compared with the 6th month in the compliant group as opposed to an increasing tendency in the non-compliant group, respectively (difference: 9.0%, 95% CI: 0% to 20.6%, p = 0.1523). At that time, 80% of the failed treatments were HPV mRNA positive compared to 10% positivity for the cases treated successfully (OR: 34, 95%CI: 6.8–173, p < 0.0001), a finding indicative that HPV mRNA E6 & E7 positivity accurately predicts treatment failure; p16 positivity was also observed at higher rates in cases with treatment failure. Conclusions: Consistent condom use following conservative excisional CIN treatment appears to significantly reduce rates of CIN recurrence and biomarkers of HPV expression. Additional HPV vaccination at the time of treatment could further enhance the positive effect of consistent condom use.

Loss of adipose TET proteins enhances β-adrenergic responses and protects against obesity by epigenetic regulation of β3-AR expression

β-adrenergic receptor (β-AR) signaling plays predominant roles in modulating energy expenditure by triggering lipolysis and thermogenesis in adipose tissue, thereby conferring obesity resistance. Obesity is associated with diminished β3-adrenergic receptor (β3-AR) expression and decreased β-adrenergic responses, but the molecular mechanism coupling nutrient overload to catecholamine resistance remains poorly defined. Ten-eleven translocation (TET) proteins are dioxygenases that alter the methylation status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine and further oxidized derivatives. Here, we show that TET proteins are pivotal epigenetic suppressors of β3-AR expression in adipocytes, thereby attenuating the responsiveness to β-adrenergic stimulation. Deletion of all three Tet genes in adipocytes led to increased β3-AR expression and thereby enhanced the downstream β-adrenergic responses, including lipolysis, thermogenic gene induction, oxidative metabolism, and fat browning invitro and invivo. In mouse adipose tissues, Tet expression was elevated after mice ate a high-fat diet. Mice with adipose-specific ablation of all TET proteins maintained higher levels of β3-AR in both white and brown adipose tissues and remained sensitive to β-AR stimuli under high-fat diet challenge, leading to augmented energy expenditure and decreased fat accumulation. Consequently, they exhibited improved cold tolerance and were substantially protected from diet-induced obesity, inflammation, and metabolic complications, including insulin resistance and hyperlipidemia. Mechanistically, TET proteins directly repressed β3-AR transcription, mainly in an enzymatic activity-independent manner, and involved the recruitment of histone deacetylases to increase deacetylation of its promoter. Thus, the TET-histone deacetylase-β3-AR axis could be targeted to treat obesity and related metabolic diseases.

Fetal DNA Causes Sex-Specific Inflammation From Human Fetal Membranes.

Inflammation is central to the mechanisms of parturition, but the lack of understanding of how it is controlled in normal parturition hampers our ability to understand how it may diverge resulting in preterm birth. Cell-free fetal DNA is found in the amniotic fluid, and it is thought to be able to activate inflammation as a danger-associated molecular pattern. Although its levels increases with gestational age, its effect has not been studied on the human fetal membranes. Thus, the aim of this study was to determine if the fetal DNA can trigger inflammation in the human fetal membranes and, thus, potentially contribute to the inflammatory load. Isolated human amniotic epithelial cells and fetal membrane explants were treated apically with fetal DNA causing the translocation of NF-KB into the nucleus of cells and throughout the cells of the explant layers with time. Fetal membrane explants were treated apically with either small or larger fragments of fetal DNA. IL-6, TNFα, and GM-CSF secretion was measured by ELISA, and pro-MMP2 and pro-MMP9 activity was measured by zymography from apical and basal media. Increased apical IL-6 secretion and basal pro-MMP2 activity was seen with small fragments of fetal DNA. When the data were disaggregated based on fetal sex, males had significant increases in IL-6 secretion and basal increased activity in pro-MMP2 and 9, whereas females had significantly increased basal secretion of TNFα. This was caused by the smaller fragments of fetal DNA, whereas the larger fragments did not cause any significant increases. Male fetal DNA had significantly lower percentages of methylation than females. Thus, when the cytokine and pro-MMP activity data were correlated with methylation percentage, IL-6 secretion significantly correlated negatively, whereas GM-CSF secretion positively correlated. These data support the role of fetal DNA as an inflammatory stimulus in the FM, as measured by increased NF-κB translocation, cytokine secretion, and increased pro-MMP activity. However, the data also suggested that the responses are different from FM tissues of male and female fetuses, and both the fragment size and methylation status of the fetal DNA can influence the magnitude and type of molecule secreted.

The effect of HPV DNA and p16 status on the prognosis of patients with hypopharyngeal carcinoma: a meta-analysis

ObjectiveTo evaluate whether the presence of human papillomavirus (HPV) DNA and p16 might be associated with better prognosis in patients with hypopharyngeal carcinoma (HPC), especially on overall survival (OS) and disease-free survival (DFS).MethodPubMed, the Cochrane Library, the Web of Science and EMBASE were searched from inception to April 2021 to search for HPV DNA- and p16-related prognostic articles on HPC. Meta-analysis was performed on the selected articles according to the inclusion and exclusion criteria. Publication bias was assessed for the included studies with Egger’s test. All studies were analyzed by using Stata 16.0 statistical software.ResultsA total of 18 studies were included, including 12 HPV DNA studies and 11 p16 studies. Meta-analysis showed that HPV DNA positivity was a strong prognostic factor for improved OS in patients with HPC, with a pooled hazard ratio (HR) of 0.61 (95% CI, 0.54–0.69), but there was no statistically significant difference in DFS (HR, 0.60; 95% CI, 0.31–1.16). Patients with p16-positive tumors had better OS (HR, 0.66; 95% CI, 0.49–0.89) and DFS (HR, 0.59; 95% CI, 0.44–0.78) than patients with p16-negative tumors.ConclusionsThis study suggests that the presence of HPV DNA leads to better OS in patients with HPC, and the presence of p16 also corresponds to better OS and DFS. Our results provide up-to-date evidence to clinicians and researchers. Larger studies adjusting for prognostic factors are needed in subsequent studies.

Abstract 3823: Viral transcript and tumor immune microenvironment-based transcriptomic profiling of HPV-associated head and neck cancer identifies subtypes associated with prognosis

Abstract Human papillomavirus (HPV)-associated Head and Neck Squamous Cell Carcinoma (HPV+HNSCC) is now the most common HPV-associated malignancy in the United States. Current treatments can be associated with severe side-effects or lack of efficacy yet prognostic biomarkers are limited, slowing efforts to personalize treatment in HPV+HNSCC. Here, we describe the use of a transcriptomic-based analytical platform to analyze expression patterns of viral transcripts, the tumor microenvironment (TME), and viral genome integration, and associate these features with overall survival. Functional gene expression signatures were analyzed on publicly available HPV+HNSCC expression data (n=266). Unsupervised clustering analysis revealed 5 distinct and novel TME types across patients (immune-enriched non-fibrotic, immune-enriched fibrotic, fibrotic, immune-desert, immune-enriched luminal). These microenvironment subtypes were highly correlated with both overall survival and patient prognosis. Tumors with an immune-enriched microenvironment showed the highest survival rates, whereas fibrotic TME types were associated with poor survival (p &amp;lt; 0.05). Unsupervised clustering of a HPV+HNSCC cohort from The Cancer Genome Atlas (TCGA) (n=53), based on HPV transcript expression, revealed 4 HPV-related subtypes. Each subtype was enriched for distinct viral transcripts: E2/E5, E6/E7, E1/E4 and L1/L2. We then validated TME and HPV transcript-related classifications on an independent HPV+HNSCC cohort (n=132). Utilizing both viral transcript and TME subtypes, we found that the E2/E5 HPV subtype was associated with an immune-enriched TME and had a higher overall survival compared to the other subtypes. The E2/E5 subtype was also enriched for samples without HPV-genome integration, suggesting that HPV episomal DNA status and E2/E5 expression pattern may drive an inflamed microenvironment and improved prognosis. In contrast, E6/E7 subtype samples were associated with the fibrotic and depleted TME types, with lower values of T-cell and B-cell gene expression signatures and a lower survival rate. Both E1/E4 and L1/L2 subtypes were associated with the immune-enriched luminal TME types. These findings suggest that HPV-transcript expression patterns may drive modulation of the TME, and hence impact prognosis. Further validation of the relationships between viral gene expression, TME, and prognosis is warranted to understand if such subtypes could aid in the development of prognostic biomarkers for treatment selection. Citation Format: Daria Kiriy, Dmitry Tychinin, Nikita Kotlov, Olga Kudryashova, Anastasia Nikitina, Andrey Tyshevich, Naira Samarina, Ksenia Demina, Sandrine Degryse, Susan Raju Paul, Mark Poznansky, Krystle Lang Kuhs, James S. Lewis, Robert L. Ferris, Xiaowei Wang, Alexander Bagaev, Nathan Fowler, Lori Wirth, Daniel Faden. Viral transcript and tumor immune microenvironment-based transcriptomic profiling of HPV-associated head and neck cancer identifies subtypes associated with prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3823.

HBV pgRNA profiles in Chinese HIV/HBV coinfected patients under pre- and posttreatment: a multicentre observational cohort study.

Data on hepatitis B virus (HBV) pregenomic (pgRNA) levels in HIV/HBV coinfected patients pre‐ and post‐combined antiretroviral therapy (cART) are limited. This study aimed to evaluate the distribution of HBV pgRNA levels in treatment‐naive coinfected patients and explore the changes that occur after the initiation of cART by examining patients from multicentre cohort studies performed in China. We included HIV/HBV coinfected subjects from the China AIDS Clinical Trial cohorts established from 2008 to 2014. Clinical and serological markers of HIV and HBV infection and biochemical data were acquired at baseline and after 96 and 240–480 weeks of cART. The correlations between HBV pgRNA and HBV DNA levels as well as HBsAg levels were calculated using Spearman's bivariate correlation analysis, and multivariate regression analysis was performed to determine factors associated with undetectable HBV pgRNA levels before cART and HBeAg loss after cART. A total of 132 HIV/HBV coinfected patients were enrolled, and 100 individuals were HBeAg‐negative. A total of 34.4% (32/93) of patients were positive for HBV pgRNA, and the median HBV pgRNA level was 4.92 (IQR: 4.21–6.12) log10 copies/mL before cART. The median HBV pgRNA level was significantly lower in HBeAg‐negative individuals than in HBeAg‐positive individuals (4.22 (IQR: 2.70–4.84) log10 copies/mL vs. 5.77 (IQR: 4.63–6.55) log10 copies/mL, p = 0.002). HBV pgRNA was moderately correlated with HBsAg (r = 0.594, p = 0.001), and positively associated with HBV DNA (r = 0.445, p = 0.011). The factors independently associated with undetectable HBV pgRNA level before cART were HBV DNA (OR: 5.61, 95% CI: 1.50–20.96, p = 0.01) and HBeAg status (OR: 5.95, 95% CI: 1.52–23.25, p = 0.01). A total of 87.5% (28/32) of patients were followed for a median duration of 138 (IQR: 54–240) weeks, and the HBV pgRNA levels became undetectable in seven patients. The 132 patients were observed for 695.5 person‐years, and no HBsAg loss occurred. Thirteen individuals achieved HBeAg loss, four patients had undetectable levels of HBV pgRNA pre‐cART, and the level of six individuals became undetectable during the 48‐week (IQR: 48–264) follow‐up period. HBeAg status was significantly associated with HBV pgRNA level in HIV/HBV coinfected patients pre‐ and post‐cART. Additionally, undetectable HBV pgRNA level may be associated with HBeAg loss after cART.

Effect of an Antioxidant Supplement Combination on Boar Sperm.

Simple SummaryBoar spermatozoa can be exposed to many harmful factors. The low antioxidant capacity of spermatozoa makes them susceptible to reactive oxygen species. The protection of spermatozoa from these harmful effects lies in the seminal plasma, which is enriched by antioxidant enzymes. This study aimed to measure the antioxidant status of boar seminal plasma, and to determine spermatozoa DNA status, and the total number of motile spermatozoa after the concurrent joint application in the boars’ diet of two commercial products with antioxidative potential.The study was conducted on a commercial pig farm located in Serbia. Thirty Duroc or Landrace breed boars were randomly selected for this study. The experimental group was fed a compound feed with added organic selenium and Oxynat 3D. The antioxidant status parameters of boar seminal plasma were evaluated using a biochemical analyzer and commercial Randox kits. The sperm chromatin structure assay (SCSA) using flow cytometry (FC) provided information about spermatozoa’s DNA status. Additionally, the total number of motile spermatozoa and spermatozoa kinematic parameters were measured using the computer-assisted sperm analysis (CASA) system. The aim of this study was to improve the parameters of semen by combining two preparations that have a potential antioxidant effect, but also to establish the level of various antioxidant enzymes in native sperm. There was no statistically significant difference in total antioxidant capacity and glutathione peroxidase activity in the seminal plasma obtained from the experimental and control groups of boars. Regarding the superoxide dismutase activity, the research results showed a difference in the control group compared to the experimental one. Moreover, spermatozoa DNA fragmentation and the total number of motile spermatozoa showed statistically significant lower and higher values, respectively, in experimental compared to the control groups. The combination of these two preparations shows significantly enhanced vital parameters of semen. To the best of our knowledge, this study is the first in which the ejaculate parameters were examined after the application of a combination of these two antioxidant supplements.

Evaluation of DNA fragmentation in dog sperm using the sperm chromatin dispersion test.

The study's objective was to adapt the Sperm Chromatin Dispersion (SCD) protocol to evaluate sperm DNA fragmentation and implement a fragmentation control in dogs. Correlation between DNA status and routine sperm parameters was also analysed. To adapt the SCD, two different mercaptoethanol (ME) concentrations were assayed (2.5% and 5%) in fourteen ejaculates from seven dogs and semen incubation with 0.3M NaOH for 15min at room temperature was assayed as a control for sperm DNA fragmentation. Data were analysed using a Mann-Whitney test and either Pearson's or Spearman's correlation. The selected ME concentration to use in the SCD test was 5%, as it produced the largest DNA dispersion halo while preserving the core nucleus structure. Four DNA halo patterns were identified as follows: large dispersion halos, medium halos, small halos and nuclei without halos. Semen incubated with NaOH showed 100% sperm without halos (damaged DNA). A significant positive correlation was observed between sperm with fragmented DNA and sperm with coiled tails. Thus, it was possible to adapt the SCD protocol to evaluate dog sperm DNA fragmentation in raw semen without using a commercial kit and establish incubation with NaOH as a DNA fragmentation control. Only coiled tails showed correlation with DNA fragmentation.

Genetic origin of sporadic cases and RNA toxicity in neuronal intranuclear inclusion disease

BackgroundGGC repeat expansion in NOTCH2NLC has been recently linked to neuronal intranuclear inclusion disease (NIID) via unknown disease mechanisms. Herein, we explore the genetic origin of the sporadic cases and...

β2-Integrins - Regulatory and Executive Bridges in the Signaling Network Controlling Leukocyte Trafficking and Migration.

Leukocyte trafficking is an essential process of immunity, occurring as leukocytes travel within the bloodstream and as leukocyte migration within tissues. While it is now established that leukocytes can utilize the mesenchymal migration mode or amoeboid migration mode, differences in the migratory behavior of leukocyte subclasses and how these are realized on a molecular level in each subclass is not fully understood. To outline these differences, first migration modes and their dependence on parameters of the extracellular environments will be explained, as well as the intracellular molecular machinery that powers migration in general. Extracellular parameters are detected by adhesion receptors such as integrins. β2-integrins are surface receptors exclusively expressed on leukocytes and are essential for leukocytes exiting the bloodstream, as well as in mesenchymal migration modes, however, integrins are dispensable for the amoeboid migration mode. Additionally, the balance of different RhoGTPases – which are downstream of surface receptor signaling, including integrins – mediate formation of membrane structures as well as actin dynamics. Individual leukocyte subpopulations have been shown to express distinct RhoGTPase profiles along with their differences in migration behavior, which will be outlined. Emerging aspects of leukocyte migration include signal transduction from integrins via actin to the nucleus that regulates DNA status, gene expression profiles and ultimately leukocyte migratory phenotypes, as well as altered leukocyte migration in tumors, which will be touched upon.

Widespread of Potential Pathogen-Derived Extracellular Vesicles Carrying Antibiotic Resistance Genes in Indoor Dust.

Extracellular vesicles (EVs) are newly recognized as important vectors for carrying and spreading antibiotic resistance genes (ARGs). However, the ARGs harbored by EVs in ambient environments and the transfer potential are still unclear. In this study, the prevalence of ARGs and mobile genetic elements (MGEs) in EVs and their microbial origins were studied in indoor dust from restaurants, kindergarten, dormitories, and vehicles. The amount of EVs ranged from 3.40 × 107 to 1.09 × 1011 particles/g dust. The length of EV-associated DNA fragments was between 21 bp and 9.7 kb. Metagenomic sequencing showed that a total of 241 antibiotic ARG subtypes encoding resistance to 16 common classes were detected in the EVs from all four fields. Multidrug, quinolone, and macrolide resistance genes were the dominant types. 15 ARG subtypes were exclusively carried and even enriched in EVs compared to the indoor microbiome. Moreover, several ARGs showed co-occurrence with MGEs. The EVs showed distinct taxonomic composition with their original dust microbiota. 30.23% of EV-associated DNA was predicted to originate from potential pathogens. Our results indicated the widespread of EVs carrying ARGs and virulence genes in daily life indoor dust, provided new insights into the status of extracellular DNA, and raised risk concerns on their gene transfer potential.

Global 5mC and 5hmC DNA Levels in Human Sperm Subpopulations with Differentially Protaminated Chromatin in Normo- and Oligoasthenozoospermic Males

Epigenetic modifications play a special role in the male infertility aetiology. Published data indicate the link between sperm quality and sperm chromatin protamination. This study aimed to determine the relationship between methylation (5mC) and hydroxymethylation (5hmC) in sperm DNA, with respect to sperm chromatin protamination in three subpopulations of fertile normozoospermic controls and infertile patients with oligo-/oligoasthenozoospermia. For the first time, a sequential staining protocol was applied, which allowed researchers to analyse 5mC/5hmC levels by immunofluorescence staining, with a previously determined chromatin protamination status (aniline blue staining), using the same spermatozoa. TUNEL assay determined the sperm DNA fragmentation level. The 5mC/5hmC levels were diversified with respect to chromatin protamination status in both studied groups of males, with the highest values observed in protaminated spermatozoa. The linkage between chromatin protamination and 5mC/5hmC levels in control males disappeared in patients with deteriorated semen parameters. A relationship between 5mC/5hmC and sperm motility/morphology was identified in the patient group. Measuring the 5mC/5hmC status of sperm DNA according to sperm chromatin integrity provides evidence of correct spermatogenesis, and its disruption may represent a prognostic marker for reproductive failure.