Genotypic distribution of human papillomavirus and phylogenetic analysis of E6 and E7 gene of HR-HPV variants isolated from Pakistani population.

High-risk-human papillomavirus (HR-HPV)-induced cervical cancer is the second most common cause of death among females worldwide. HPV16 is the most prevalent HR-HPV infection worldwide. This study found the genotypic distribution of HR-HPV in the local population and investigated the sequence variations among the E6 and E7 oncogenes of the local HPV16 genotype to the E6 and E7 oncogenes of the foreign HPV16 genotypes and constructed a phylogenetic relationship based on nucleotide sequence comparison among the variants identified in our study along with previously reported isolates that were obtained from different regions of the world. The samples were collected from patients with cervical cancer. Genomic DNA was extracted, and HR-HPV genotypes were determined using real-time PCR. The HPV16 E6 and E7 genes were amplified and sequenced. A HPV16 phylogenetic tree was constructed using the maximum likelihood method with MEGA 7. HPV16 was the most prevalent human papillomavirus (HPV) type identified in the present study. HPV16 isolates belonged to the A1 sublineage of the European branch. Twenty-one nucleotide sequences were included in this analysis. The first, second, and third codon positions are also included. The final dataset included 776 positions.

Molecular characterization of recombinant premembrane protein of dengue virus serotype-2 for development of diagnostic assay.

Dengue is an acute arboviral infection common in tropical and subtropical countries. Dengue has been highlighted as a public health concern in the last five decades, affecting almost 50% of the population in developing nations. Dengue infection results in a complex symptomatic disease that ranges from headache, fever, and skin rash to extreme hemorrhage fever and liver dysfunction. The diagnosis of the disease is essential for effective treatment. The early onset of the infection can be detected through viral structural peptides that act as markers for detection, including Pre-Membrane (Pre-M) protein. In the currently proposed research, the structural gene obtained from local isolates was targeted for studies. For this purpose, recombinant structural protein Pre-M was amplified, cloned, and expressed in the bacterial expression system. The expression of the structural protein (Pre-M) was scrutinized by Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and validated by western blot and dot blot, and afterwards, the antigen was purified. The purified Pre-M protein carries the potential for the development of in-house diagnostic assay as well as for vaccine production. This study aimed to develop a highly specific, sensitive, and cost-effective in-house enzyme-linked immunoassay (ELISA) for the detection of antibodies of Pakistani most prevalent dengue virus serotype 2 (DENV-2). The success of this research would also pave the way toward developing novel vaccines for the future prevention of dengue infection.

Hypomorphic GINS3 variants alter DNA replication and cause Meier-Gorlin syndrome.

The eukaryotic CDC45/MCM2-7/GINS (CMG) helicase unwinds the DNA double helix during DNA replication. The GINS subcomplex is required for helicase activity and is, therefore, essential for DNA replication and cell viability. Here, we report the identification of 7 individuals from 5 unrelated families presenting with a Meier-Gorlin syndrome-like (MGS-like) phenotype associated with hypomorphic variants of GINS3, a gene not previously associated with this syndrome. We found that MGS-associated GINS3 variants affecting aspartic acid 24 (D24) compromised cell proliferation and caused accumulation of cells in S phase. These variants shortened the protein half-life, altered key protein interactions at the replisome, and negatively influenced DNA replication fork progression. Yeast expressing MGS-associated variants of PSF3 (the yeast GINS3 ortholog) also displayed impaired growth, S phase progression defects, and decreased Psf3 protein stability. We further showed that mouse embryos homozygous for a D24 variant presented intrauterine growth retardation and did not survive to birth, and that fibroblasts derived from these embryos displayed accelerated cellular senescence. Taken together, our findings implicate GINS3 in the pathogenesis of MGS and support the notion that hypomorphic variants identified in this gene impaired cell and organismal growth by compromising DNA replication.

MicroRNA regulatory networks associated with abnormal muscle repair in survivors of critical illness.

BACKGROUND: Intensive care unit (ICU)-acquired weakness is characterized by muscle atrophy and impaired contractility that may persist after ICU discharge. Dysregulated muscle repair and regeneration gene co-expression networks are present in critical illness survivors with persistent muscle wasting and weakness. We aimed to identify microRNAs (miRs) regulating the gene networks and determine their role in the self-renewal of muscle in ICU survivors. METHODS: Muscle whole-transcriptome expression was assessed with microarrays in banked quadriceps biopsies obtained at 7 days and 6 months post-ICU discharge from critically ill patients (n = 15) in the RECOVER programme and healthy individuals (n = 8). We conducted an integrated miR-messenger RNA analysis to identify miR/gene pairs associated with muscle recovery post-critical illness and evaluated their impact on myoblast proliferation and differentiation in human AB1167 and murine C2C12 cell lines in vitro. Select target genes were validated with quantitative PCR. RESULTS: Twenty-two miRs were predicted to regulate the Day 7 post-ICU muscle transcriptome vs. controls. Thirty per cent of all differentially expressed genes shared a 3'UTR regulatory sequence for miR-424-3p/5p, which was 10-fold down-regulated in patients (P < 0.001) and correlated with quadriceps size (R = 0.86, P < 0.001), strength (R = 0.75, P = 0.007), and physical function (Functional Independence Measures motor subscore, R = 0.92, P < 0.001) suggesting its potential role as a master regulator of early recovery of muscle mass and strength following ICU discharge. Network analysis demonstrated enrichment for cellular respiration and muscle fate commitment/development related genes. At 6 months post-ICU discharge, a 14-miR expression signature, including miRs-490-3p and -744-5p, identified patients with muscle mass recovery vs. those with sustained atrophy. Constitutive overexpression of the novel miR-490-3p significantly inhibited AB1167 and C2C12 myoblast proliferation (cell count AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 7926 ± 4060 vs. 14 159 ± 3515 respectively, P = 0.006; proportion Ki67-positive nuclei AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 0.38 ± 0.07 vs. 0.54 ± 0.06 respectively, P < 0.001; proliferating cell nuclear antigen expression AB1167 miR-490-3p mimic or scrambled-miR transfected myoblasts 11.48 ± 1.97 vs. 16.75 ± 1.19 respectively, P = 0.040). Constitutive overexpression of miR-744-5p, a known regulator of myogenesis, significantly inhibited AB1167 and C2C12 myoblast differentiation (fusion index AB1167 miR-744-5p mimic or scrambled-miR transfected myoblasts 8.31 ± 7.00% vs. 40.29 ± 9.37% respectively, P < 0.001; myosin heavy chain expression miR-744-5p mimic or scrambled-miR transfected myoblasts 0.92 ± 0.39 vs. 13.53 ± 5.5 respectively, P = 0.01). CONCLUSIONS: Combined functional transcriptomics identified 36 miRs including miRs-424-3p/5p, -490-3p, and -744-5p as potential regulators of gene networks associated with recovery of muscle mass and strength following critical illness. MiR-490-3p is identified as a novel regulator of myogenesis.

Determinants of hardship financing in coping with out of pocket payment for care seeking of under five children in selected rural areas of Bangladesh.

BACKGROUND: Around 63% of total health care expenditure in Bangladesh is mitigated through out of pocket payment (OOP). Heavy reliance on OOP at the time of care seeking poses great threat for financial impoverishment of the households. Households employ different strategies to cope with the associated financial hardship. OBJECTIVE: The aim of this paper is to understand the determinants of hardship financing in coping with OOP adopted for health care seeking of under five childhood illnesses in rural setting of Bangladesh. METHODS: A community based cross sectional survey was conducted during August to October, 2014 in 15 low performing sub-districts of northern and north-east regions of Bangladesh. Of the 7039 mothers of under five children surveyed, 1895 children who suffered from illness and sought care for their illness episodes were reported in this study. Descriptive statistics and ordinal regression analysis were conducted. RESULTS: A total number of 7,039 under five children reported to have suffered illness by their mothers. Among these children 37% suffered from priority illness. Care was sought for 88% children suffering from illnesses. Among them 26% went to a public or private sector medically trained provider. 5% of households incurred illness cost more than 10% of the household's monthly expenditure. The need for assistance was higher among those compared to others (31% vs 13%). Different financing mechanisms adopted to meet OOP are loan with interest (6%), loan without interest (9%) and financial help from relatives (6%) Need for financial assistance varied from 19% among households in the lowest quintile to 9% in the highest wealth. Ordinal regression analysis revealed that burden of hardship financing increases by 2.17 times when care is sought from a private trained provider compared to care seeking from untrained provider (CI: 1.49, 3.17). Similarly, for families that incur a health care expenditure that is more than 10% of their total monthly expenditure (CI:1.46, 3.88), the probability of falling into more severe financial burden increases by 2.4 times. We also found severity of the hardship financing to be around half for households with monthly income of more than BDT 7500 (OR = 0.56, CI: 0.37, 0.86). The burden increased by 2.10 times for households with a deficit (CI: 1.53, 2.88) between their monthly income and expenditure. The interaction between family income and severity of illness showed to significantly affect the scale of hardship financing. Children suffering from priority illness belonging to poor households were found have two times (CI: 1.09, 3.47) higher risks of suffering from hardship financing. CONCLUSION AND POLICY IMPLICATIONS: Findings from this study will help the policy makers to identify the target groups and thereby design effective health financing programs.

Serum trace elements in diagnosis of breast malignancy.

BACKGROUND: Breast cancer is a highly heterogeneous disease globally. The absolute risk of breast cancer increases with age and is higher in postmenopausal women. This study tried to find out role of serum trace elements (cadmium, cobalt, copper and zinc) and biomarkers in diagnosis of breast malignancy. METHODS: Levels of serum trace elements were estimated by atomic absorption spectrophotometry in serum samples of 23 breast cancer patients of stage III. Analysis of protein pattern was observed by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: In menstruating women, the mean serum level of cadmium, copper and cobalt were significantly lowered as compared to control, however, zinc showed no significant difference. In premenopausal and postmenopausal women, the mean serum level of cadmium, copper, cobalt and zinc was decreased significantly in patients as compared to controls (p < 0.001). The ratio of Cu/Zn was also decreased in patients when compared to controls. Three major peptide bands with molecular weights of 33 kDa, 52 kDa and 185 kDa appeared in these samples when subjected to SDS PAGE. CONCLUSIONS: Serum trace elements are significantly lower in breast cancer patients compared to controls. The exact mechanism responsible for the alterations in trace element levels in patients with breast cancer is unclear and requires further evaluation. Gel electrophoresis may be important to find out breast cancer markers that may help in diagnosing the disease and response to treatment.