Human polyomaviruses JCPyV and MCPyV in urothelial cell carcinoma: a single institution experience.

Objective: Urothelial cell carcinoma (UCC) is the most common type of urinary bladder. JCPyV and BKPyV have been detected in the urine and tissue of urothelial cell carcinomas (UCC) in immunocompetent patients. Here, we investigated the presence of several HPyVs in UCC samples using diverse molecular techniques to study the prevalence of HPyVs in UCC. Methods: A large single-institution database of urine cytology specimens (UCS; n = 22.867 UCS) has previously been searched for decoy cells (n = 30), suggesting polyomavirus infection. The available urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC patients were tested for the presence of JCPyV-LTAg expression by immunohistochemistry (IHC) labeled with SV40-LTAg antibody (clone: PAb416) and subsequent PCR followed by sequencing. In addition, the presence of the oncogenic Merkel cell polyomavirus (MCPyV) and the presence of human polyomavirus 6 (HPyV6) and 7 (HPyV7) DNA were tested with DNA PCR or IHC. Results: Of the 30 patients harboring decoy cells, 14 were diagnosed with UCC of the urinary bladder (14/30; 46.6%) before presenting with decoy cells in the urine. The SV40-LTAg IHC was positive in all 14 UCC urine sediments and negative in the FFPE tissues. JCPyV-DNA was identified in all five available UCS and in three FFPE samples of UCC (three of 14; 21.4%). Two UCC cases were positive for MCPyV-DNA (two of 14; 14.3%), and one of them showed protein expression by IHC (one of 14; 7.1%). All specimens were HPyV6 and HPyV7 negative. Conclusion: Our findings show the presence of JCPyV in the urine and UCC of immunocompetent patients. Moreover, MCPyV was detected in two UCC cases. In total, five UCC cases showed the presence of either JCPyV or MCPyV. The evidence here supports the hypothesis that these viruses might sporadically be associated with UCC. Further studies are needed to confirm the relevance of JCPyV or MCPyV as a possible risk factor for UCC development.

Association of mutation and low expression of the CTCF gene with breast cancer progression.

BACKGROUND: CTCF encodes 11-zinc finger protein which is implicated in multiple tumors including the carcinoma of the breast. The Present study investigates the association of CTCF mutations and their expression in breast cancer cases. METHODS: A total of 155 breast cancer and an equal number of adjacent normal tissue samples from 155 breast cancer patients were examined for CTCF mutation(s) by PCR-SSCP and automated DNA sequencing. Immunohistochemistry (IHC) method was used to analyze CTCF expression. Molecular findings were statistically analyzed with various clinicopathological features to identify associations of clinical relevance. RESULTS: Of the total, 16.1% (25/155) cases exhibited mutation in the CTCF gene. Missense mutations Gln > His (G > T) in exon 1 and silent mutations Ser > Ser (C > T) in exon 4 of CTCF gene were analyzed. A significant association was observed between CTCF mutations and some clinicopathological parameters namely menopausal status (p = 0.02) tumor stage (p = 0.03) nodal status (p = 0.03) and ER expression (p = 0.04). Protein expression analysis showed 42.58% samples having low or no expression (+), 38.0% with moderate (++) expression and 19.35% having high (+++) expression for CTCF. A significant association was found between CTCF protein expression and clinicopathological parameters include histological grade (p = 0.04), tumor stage (p = 0.04), nodal status (p = 0.03) and ER status (p = 0.04). CONCLUSIONS: The data suggest that CTCF mutations leading to its inactivation significantly contribute to the progression of breast cancer.

Inappropriate Dental Antibiotic Prescriptions: Potential Driver of the Antimicrobial Resistance in Albaha Region, Saudi Arabia.

PURPOSE: The aim of this study was to analyze and evaluate dental antibiotic prescriptions in Albaha Region, Saudi Arabia. PATIENTS AND METHODS: A two-year retrospective cohort study was conducted between September 1, 2017 and September 1, 2019 in children and adults. Data collected from the patients' medical records were analyzed using SPSS. The Z-test with Bonferroni correction and descriptive proportions were utilized to compare several levels of categorical variables. RESULTS: Of the 43,255 dental visits, antibiotics were provided during 12,573 (29.1%). The commonly prescribed antibiotics were amoxicillin and amoxicillin combined with metronidazole (56.3% and 16.9%, respectively). Alarmingly, antibiotics were provided in several conditions for which they are medically neither recommended nor indicated; together, they represented 27.8% of those consultations in which antibiotics were prescribed. Female dentists prescribed more antibiotics than male dentists (30%, P = < 0.000), with male patients receiving more antibiotics than female patients (36%, P = <0.0001). CONCLUSION: Unnecessary prescription of antibiotics was observed in the present study. Improving knowledge and awareness of Saudi dentists on dental antibiotic prescription is warranted.

Characterization of In vitro inhibitory effects of consensus short interference RNAs against non-structural 5B gene of hepatitis C virus 1a genotype.

PURPOSE: Chronic hepatitis C has infected approximately 170 million people worldwide. The novel direct-acting antivirals have proven their clinical efficacy to treat hepatitis C infection but still very expensive and beyond the financial range of most infected patients in low income and even resource replete nations. This study was conducted to establish an in vitro stable human hepatoma 7 (Huh-7) cell culture system with consistent expression of the non-structural 5B (NS5B) protein of hepatitis C virus (HCV) 1a genotype and to explore inhibitory effects of sequence-specific short interference RNA (siRNA) targeting NS5B in stable cell clones, and against viral replication in serum-inoculated Huh-7 cells. MATERIALS AND METHODS: In vitro stable Huh-7 cells with persistent expression of NS5B protein was produced under gentamycin (G418) selection. siRNAs inhibitory effects were determined by analysing NS5B expression at mRNA and protein level through reverse transcription-polymerase chain reaction (PCR), quantitative real-time PCR, and Western blot, respectively. Statistical significance of data (NS5B gene suppression) was performed using SPSS software (version 16.0, SPSS Inc.). RESULTS: siRNAs directed against NS5B gene significantly decreased NS5B expression at mRNA and protein levels in stable Huh-7 cells, and a vivid decrease in viral replication was also exhibited in serum-infected Huh-7 cells. CONCLUSIONS: Stable Huh-7 cells persistently expressing NS5B protein should be helpful for molecular pathogenesis of HCV infection and development of anti-HCV drug screening assays. The siRNA was effective against NS5B and could be considered as an adjuvant therapy along with other promising anti-HCV regimens.

In vitro inhibitory analysis of consensus siRNAs against NS3 gene of hepatitis C virus 1a genotype.

OBJECTIVE: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs (small interference RNA) against NS3 gene of hepatitis C virus (HCV) 1a genotype in stable Huh-7 (human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. METHODS: Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin (G418) selection. The cell clones resistant to 1000 µg antibiotic concentration (G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. RESULTS: RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested (50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24 (80%, P = 0.013) and 48 h (75%, P = 0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70% (P < 0.05) viral RNA reduction as compared to NS3-is33, which showed a 64% (P < 0.05) decrease in viral copy number. siRNA synergism (NS3-is33 + NS3-is44) decreased viral load by 84% (P < 0.05) as compared to individual inhibition by each siRNA (i.e., 64%-70% (P < 0.05)) in serum-inoculated cells. Synthetic siRNAs mixture (NS5B-is88 + NS3-is33) targeting different region of HCV genome (NS5B and NS3) also decreased HCV viral load by 85% (P < 0.05) as compared to siRNA inhibitory effects alone (70% and 64% respectively, P < 0.05). CONCLUSIONS: siRNAs directed against NS3 gene significantly decreased mRNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.