Sub-genomic analysis of Chikungunya virus E2 mutations in Pakistani isolates potentially modulating B-cell & T-Cell immune response.

BACKGROUND & OBJECTIVES: The Chikungunya virus (CHIKV) transmitted to the humans through Aedes species of the mosquitoes. In December 2016, a severe outbreak reported from Pakistan. However, there is no vaccine or anti-viral treatment currently available so host immune response against CHIKV gained significant interest. Therefore, this study was conducted to identify the mutations in CHIKV E2 region of currently circulating Pakistani strains & determine their potential immunogenicity in Pakistani population. METHODS: It was a cross sectional study in which a total of 60 CHIKV PCR positive samples were collected from Molecular Department of Pathology, Dow University of Health Sciences (DUHS), Karachi during November 2017 to February 2018. CHIKV E2 gene was amplified by PCR & sequenced. Sequences were analyzed by using bioinformatic tools followed by epitope prediction in E2 sequences by In-silico immunoinformatic approach. RESULTS: Several single nucleotide variations (SNVs) were identified in Pakistani isolates with six novel mutations in E2 sequences. Immunoinformatic analyses showed more proteasomal sites, CTL & B-Cell epitopes in Pakistani strains with respect to S27 prototype with 69.4% population coverage against these epitopes in Pakistan. The study also identified key mutations responsible for generation of unique epitopes and HLA restriction in Pakistani isolates. The strain specific mutations revealed the current outbreak was caused by ESCA.IOL lineage of CHIKV. CONCLUSION: The evolution of E2 protein in Pakistani strains has increased its immunogenicity in comparison to ancestral s27 strain. The identification of most immunogenic and conserved epitopes with high population coverage has high potential to be used in vaccine development against these local strains.

Serologic Evidence of Severe Fever with Thrombocytopenia Syndrome Virus and Related Viruses in Pakistan.

We describe the seroprevalence of severe fever with thrombocytopenia syndrome virus (SFTSV) and the association of antibody occurrence with location, sex, and age among the human population in Pakistan. Our results indicate substantial activity of SFTSV and SFTSV-related viruses in this country.

Prevalence of EBV, CMV, and HPV in oral squamous cell carcinoma patients in the Pakistani population.

Many studies have proposed an important role of viruses in the pathogenesis of oral cancer. The present study aimed to find out the prevalence of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and human papillomavirus (HPV) among patients with oral squamous cell carcinoma (OSCC) in a Pakistani cohort. We investigated tissue samples obtained from 58 patients with OSCC using the polymerase chain reaction assay. No sample was positive for HPV. EBV was identified in 15 patients (25.86%), and CMV in three patients (5.17%). Coinfection with one or more viruses was detected in two cases and was coinfection with EBV and CMV. These results suggest a low prevalence of these viruses in OSCC patients in the Pakistani population compared to most other countries where the prevalence of these viruses has been reported in the past. Nevertheless, further studies are necessary to determine the potential role of EBV and the possible importance of CMV as an infection cofactor in oral cancer.

Genomic characterization of lytic bacteriophages A¥L and A¥M infecting ESBL K. pneumoniae and its therapeutic potential on biofilm dispersal and in-vivo bacterial clearance.

BACKGROUND: The emerging carbapenem resistance and extended spectrum ß-lactamases (ESBL) producing strains of Klebsiella pneumoniae, are one of the critical pathogens for which novel therapeutic alternatives are required on urgent basis. Biofilm formation further aids in virulence due to impermeable nature. Bacteriophages are bacterial predators which due to their selective, and nontoxic nature can be used as an alternate to counter MDR infections. Hence, the current study was intended to isolate, characterize, and develop phage cocktail as a possible therapy against ESBL K. pneumoniae. MATERIAL AND METHOD: The two-novel bacteriophage A¥L and A¥M were isolated from environmental samples and characterized for host specificity and physicochemical stability (i.e., temperature and pH). Isolated phages alone or together as cocktail was further evaluated for in vitro biofilm eradication and infection clearance using in vivo murine model. Whole genome sequencing was performed for identification, evolutionary relationship, and bioinformatics analysis. RESULT: The isolated phage A¥L and A¥M belonged to Myoviridae and Siphoviridae family, respectively and showed good thermal (-20, 37, 45, and 60̊C) and pH (5, 7, 9, and 11) stability. At MOI of 0.001, both phages displayed short eclipse period of 5 and 10 min, respectively. Phages alone or together as cocktail showed 50-70% eradication of 48 h mature biofilm. Majority of the cells within biofilm was found dead as evinced from live dead staining. Atomic force and scanning electron microscopic analysis showed distorted biofilm with ruptured cells. The isolated phage or their cocktail significantly inhibited K. pneumoniae associated mortality in intraperitoneal inoculated mice model CONCLUSION: These findings imply that the phage might be a good option for eliminating K. pneumoniae infections and further studies could help in development of these phage as a bio-control product.

Possible Therapeutic Effects of Adjuvant Quercetin Supplementation Against Early-Stage COVID-19 Infection: A Prospective, Randomized, Controlled, and Open-Label Study.

BACKGROUND: Quercetin, a well-known naturally occurring polyphenol, has recently been shown by molecular docking, in vitro and in vivo studies to be a possible anti-COVID-19 candidate. Quercetin has strong antioxidant, anti-inflammatory, immunomodulatory, and antiviral properties, and it is characterized by a very high safety profile, exerted in animals and in humans. Like most other polyphenols, quercetin shows a very low rate of oral absorption and its clinical use is considered by most of modest utility. Quercetin in a delivery-food grade system with sunflower phospholipids (Quercetin Phytosome®, QP) increases its oral absorption up to 20-fold. METHODS: In the present prospective, randomized, controlled, and open-label study, a daily dose of 1000 mg of QP was investigated for 30 days in 152 COVID-19 outpatients to disclose its adjuvant effect in treating the early symptoms and in preventing the severe outcomes of the disease. RESULTS: The results revealed a reduction in frequency and length of hospitalization, in need of non-invasive oxygen therapy, in progression to intensive care units and in number of deaths. The results also confirmed the very high safety profile of quercetin and suggested possible anti-fatigue and pro-appetite properties. CONCLUSION: QP is a safe agent and in combination with standard care, when used in early stage of viral infection, could aid in improving the early symptoms and help in preventing the severity of COVID-19 disease. It is suggested that a double-blind, placebo-controlled study should be urgently carried out to confirm the results of our study.

Pan-Cancer Multiomics Analysis of TC2N Gene Suggests its Important Role(s) in Tumourigenesis of Many Cancers.

BACKGROUND: Role of TC2N in carcinogenesis has been largely unfathomed until recently when it was identified as a novel oncogene in lung cancer. Subsequently, a tumour suppressor role of TC2N was reported in breast cancer. It is therefore highly relevant to investigate TC2N molecular partners/mechanisms on a larger scale including a wider range of tumour types. METHODS: We investigated TC2N mRNA expression, its promoter methylation levels, effects of TC2N transcription on overall patient survival, somatic mutations in TC2N gene and correlation between TC2N mRNA expression and other cancer genes in pan-cancer by using data available from the Cancer Genome Atlas (TCGA) and the Genotype Tissue Expression (GTEx) databases. RESULTS: TC2N mRNA expression was differentially regulated in 9/33 TCGA tumour types. Of these 9 tumours, 5 tumour types (cholangiocarcinoma, ovarian-serous-cystadenocarcinoma, rectal-adenocarcinoma, stomach-adenocarcinoma and thymoma) had significantly higher TC2N mRNA expression while 4 (pheochromocytoma-and-paraganglioma, skin-cutaneous-melanoma, thyroid-carcinoma and uterine-carcinosarcoma) had significantly lower TC2N mRNA expression compared to matched and normal controls. TC2N promoter was hypermethylated in most cancers while hypomethylated in head-and-neck-squamous-cell-carcinoma and kidney-renal-clear-cell carcinoma. TC2N transcription was positively correlated with transcription of several other cancer genes including genes from Myc, cell-cycle, Nrf2, Wnt, PI3K, Hippo, Notch, TGFß and RAS/RTK pathways. Poor prognosis was associated with higher TC2N mRNA levels in pancreatic-adenocarcinoma and brain-lower-grade-glioma and lower TC2N mRNA levels in kidney-renal-clear-cell-carcinoma, mesothelioma, sarcoma and skin-cutaneous melanoma. Functional protein partners of TC2N were identified as STX2, SMEK1, SMEK2, STXBP5, SCARA5, MMRN1, CATSPER2, CATSPERB, CLEC4M and STAB2. Many of these proteins are key players in carcinogenesis of various cancers. Highest pathogenic somatic mutation rates in TC2N were found in skin-cutaneous-melanoma, uterine-corpus-endometrial-carcinoma, colon-endocervical-adenocarcinoma, bladder-urothelial-carcinoma and breast-invasive-carcinoma. CONCLUSION: Our findings unravel several un-explored avenues related to the role of TC2N in tumourigenesis of several cancers, suggesting TC2N as an important player and a potential candidate for tumour-therapy.

Factors Influencing Quality of Life in Patients with Gastroesophageal Reflux Disease in a Tertiary Care Hospital in Pakistan.

OBJECTIVE: To assess the impact of gastroesophageal reflux disease (GERD) on quality of life (QOL) in local population. STUDY DESIGN: Cross-sectional, descriptive study. PLACE AND DURATION OF STUDY: Hepatogastroenterology Department, Sindh Institute of Urology and Transplantation, Karachi, from June 2016 to December 2017. METHODOLOGY: Patients with GERD visiting the outpatients' clinics were enrolled. Those who had history of dysphagia, malignant disease, anemia, weight loss, cardiovascular diseases, renal failure, cirrhosis, rheumatologic diseases and patients taking non-steroidal anti-inflammatory drugs (NSAIDS) or steroids during the last two weeks were exluded. A predesigned questionnaire was used to calculate GERD impact scale (GIS) score, with a value of >18 being considered as impaired QOL. Chi-square test was used to identify factors associated with impaired QOL. RESULTS: A total of 782 patients with mean age of 37.3 ±8.99 years (range: 18-65 years) were studied. Majority (505, 64.5%) were younger than 40 years of age. Predominant study population were females, i.e. 413 (52.8%). Among these, 127 (16.2%) patients had symptoms lasting more than a year, 132 (16.9%) were smokers, 82 (10.5%) had diabetes mellitus (DM), and 63 (8.1%) had hypertension. A total of 297 (38%) patients had impaired QOL. On logistic regression analysis, the impaired QOL was associated with age greater than 40 years (p=0.001), body mass index (BMI) >25 Kg/m2 (p= 0.001), smoking (p=0.001), hypertension (p=0.001) and diabetes mellitus (p=0.001). CONCLUSION: A significant proportion of patients had impaired QOL due to GERD. Factors affecting QOL in such patients were higher age, increased BMI, history of smoking, hypertension and DM.