Dominant roles of BRCA1 in cellular tolerance to a chain-terminating nucleoside analog, alovudine.

Alovudine is a chain-terminating nucleoside analog (CTNA) that is frequently used as an antiviral and anticancer agent. Generally, CTNAs inhibit DNA replication after their incorporation into nascent DNA during DNA synthesis by suppressing subsequent polymerization, which restricts the proliferation of viruses and cancer cells. Alovudine is a thymidine analog used as an antiviral drug. However, the mechanisms underlying the removal of alovudine and DNA damage tolerance pathways involved in cellular resistance to alovudine remain unclear. Here, we explored the DNA damage tolerance pathways responsible for cellular tolerance to alovudine and found that BRCA1-deficient cells exhibited the highest sensitivity to alovudine. Moreover, alovudine interfered with DNA replication in two distinct mechanisms: first: alovudine incorporated at the end of nascent DNA interfered with subsequent DNA synthesis; second: DNA replication stalled on the alovudine-incorporated template strand. Additionally, BRCA1 facilitated the removal of the incorporated alovudine from nascent DNA, and BRCA1-mediated homologous recombination (HR) contributed to the progressive replication on the alovudine-incorporated template. Thus, we have elucidated the previously unappreciated mechanism of alovudine-mediated inhibition of DNA replication and the role of BRCA1 in cellular tolerance to alovudine.

Impact of vitamin D binding protein (GC) and vitamin D receptor (VDR) gene polymorphism on the risk of developing preeclampsia.

Objective: Preeclampsia is a multifactorial disease characterized by high blood pressure and protein in the urine. In this study, we investigated the association of vitamin D binding protein (GC) and vitamin D receptor (VDR) gene polymorphism with the risk of developing preeclampsia. Methods: 25-hydroxyvitamin D was measured using High-performance Liquid Chromatography. Vitamin D binding protein and vitamin D receptor gene polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. Results: The control subjects have significant higher level of 25-hydroxyvitamin D (33.5 ± 1.194 ng/mL) relative to patients (23.97 ± 1.604 ng/mL) (p < 0.05). Vitamin D receptor rs1544410 and rs2228570 dominant model (GA + AA; TC + CC) showed significant higher risk of developing Preeclampsia (OR = 4.11, 95% CI = 0.62-27.09, p < 0.01; OR = 3.58, 95%CI = 0.78-16.38, p < 0.001 respectively). Similarly, vitamin D binding protein rs7041 and rs4588, dominant model (TG + GG; CA + AA) showed higher risk of preeclampsia development compared to control people (OR = 1.69, 95%CI = 0.35-8.19, p < 0.05; OR = 1.06, 95%CI = 0.25-4.44, p < 0.05 respectively). AA genotype of rs4588 of GC gene was significantly associated with 25-hydroxyvitamin D level in serum relative to CC and CA (p < 0.05). Conclusion: From our study, we can conclude that a low level of 25-hydroxyvitamin D, GC (rs1544410 and rs2228570), and VDR (rs4588 and rs7041) gene polymorphism is linked with an increased risk of developing preeclampsia.

CTF18-RFC contributes to cellular tolerance against chain-terminating nucleoside analogs (CTNAs) in cooperation with proofreading exonuclease activity of DNA polymerase ε.

Chemotherapeutic nucleoside analogs, such as cytarabine (Ara-C), are incorporated into genomic DNA during replication. Incorporated Ara-CMP (Ara-cytidine monophosphate) serves as a chain terminator and inhibits DNA synthesis by replicative polymerase epsilon (Polε). The proofreading exonuclease activity of Polε removes the misincorporated Ara-CMP, thereby contributing to the cellular tolerance to Ara-C. Purified Polε performs proofreading, and it is generally believed that proofreading in vivo does not need additional factors. In this study, we demonstrated that the proofreading by Polε in vivo requires CTF18, a component of the leading-strand replisome. We found that loss of CTF18 in chicken DT40 cells and human TK6 cells results in hypersensitivity to Ara-C, indicating the conserved function of CTF18 in the cellular tolerance of Ara-C. Strikingly, we found that proofreading-deficient POLE1D269A/-, CTF18-/-, and POLE1D269A/-/CTF18-/- cells showed indistinguishable phenotypes, including the extent of hypersensitivity to Ara-C and decreased replication rate with Ara-C. This observed epistatic relationship between POLE1D269A/- and CTF18-/- suggests that they are interdependent in removing mis-incorporated Ara-CMP from the 3' end of primers. Mechanistically, we found that CTF18-/- cells have reduced levels of chromatin-bound Polε upon Ara-C treatment, suggesting that CTF18 contributes to the tethering of Polε on fork at the stalled end and thereby facilitating the removal of inserted Ara-C. Collectively, these data reveal the previously unappreciated role of CTF18 in Polε-exonuclease-mediated maintenance of the replication fork upon Ara-C incorporation.

Genomic and evolutionary study from SARS-CoV-2 virus isolates from Bangladesh during the early stage of pandemic strongly correlate with European origin and not with China.

The goal of this study was to identify the genomic variants and determine molecular epidemiology of SARS-CoV-2 virus during the early pandemic stage in Bangladesh. Viral RNA was extracted, converted to cDNA, and amplified using Ion AmpliSeq™ SARS-CoV-2 Research Panel. 413 unique mutants from 151 viral isolates were identified. 80% of cases belongs to 8 mutants: 241C toT, 1163A toT, 3037C toT, 14408C toT, 23403A toG, 28881G toA, 28,882 G toA, and 28883G toC. Observed dominance of GR clade variants that have strong presence in Europe, suggesting European channel a possible entry route. Among 37 genomic mutants significantly associated with clinical symptoms, 3916CtoT (associated with sore-throat), 14408C to T (associated with cough-protection), 28881G to A, 28882G to A, and 28883G to C (associated with chest pain) were notable. These findings may inform future research platforms for disease management and epidemiological study.

Assessment of angiotensin converting enzyme gene polymorphism in preeclampsia mothers of Bangladesh.

The study aimed to assess the association of ACE insertion/deletion (I/D) polymorphisms with the susceptibility for preeclampsia in Bangladesh. It was a case-control study involving 220 subjects (100 preeclamptic and 120 normal pregnant women). The ACE (I/D) genotyping was done using the conventional PCR method. Overall, the frequency of ACE II genotypes was significantly (p<.05) higher in normal pregnant women than the preeclamptic women. The pregnant mother with DD genotype was at 3.43-fold higher risk (OR = 3.43; p<.01) of developing preeclampsia while pregnant mother with ID genotype was at lower risk (OR = 1.32; p>.05). On the other hand, patients having either DD or ID genotypes showed 1.9 fold (OR = 1.90; p>.05) increased risk of developing preeclampsia compared to the control group but not statistically significant. This study suggested that ACE (DD) genotypes may have strong associations with the occurrence of preeclampsia.Impact StatementWhat is already known on this subject? The study was conducted among 100 preeclamptic and 120 normal pregnant women. The preeclamptic patients were diagnosed by protein in urine and high blood pressure. The normal pregnant women were selected with no known complications.What the results of this study add? Overall, the pregnant mothers with DD genotype were at 3.43-fold higher risk of developing preeclampsia while pregnant mothers with ID or II genotypes were at a lower risk.What the implications are of these findings for clinical practice and/or further research? ACE (I/D) gene would be a biomarker of early diagnosis of preeclampsia and also be helpful to intervene in personalised medicine and gene therapy as a novel treatment of preeclampsia.

Association of glutathione S-transferase theta 1 and glutathione S-transferase mu 1 gene polymorphism with the risk of pre-eclampsia during pregnancy in Bangladesh.

AIM: In this study, we analyzed the risk of developing pre-eclampsia with respect to glutathione S-transferase theta 1 (GSTT1) and glutathione S-transferase mu 1 (GSTM1) genotypes. We also tried to find relationship between genotypes and biochemical parameter change in pre-eclampsia patients. METHODS: In total, 104 pre-eclampsia patients and 200 healthy controls were recruited for the study. Peripheral venous blood was drawn from study subjects and DNA was extracted from whole blood and multiplex polymerase chain reaction method was used to identify genotypes of GSTT1 and GSTM1 gene. All biochemical parameters were measured using colorimetric method. RESULTS: Serum glutamic pyruvic transaminase level was significantly higher (P < 0.01) and hemoglobin level was significantly lower (P < 0.001) in pre-eclampsia patients compared to control subjects. Significant association was found in GSTM1 null genotype with pre-eclampsia (P < 0.001) with an odds ratio (OR) analysis showing more than four-fold increased risk (OR = 4.75; 95% CI = 2.17-10.39; P <0.001). But for GSTT1 gene, null genotype was not associated with increased risk of developing pre-eclampsia (P > 0.05). In case of GSTT1 and GSTM1, the patients having both null genotypes for GSTT1 and GSTM1 showed significant (P < 0.001) higher risk of developing pre-eclampsia (OR = 7.64; 95% CI = 2.38-24.60; P < 0.001). CONCLUSION: GSTM1 null genotype increases the risk of pre-eclampsia. Combined GSTT1 and GSTM1 null genotype, the risk was even higher.

Lead Molecule Prediction and Characterization for Designing MERS-CoV 3C-like Protease Inhibitors: An In silico Approach.

BACKGROUND: 3C-like protease also called the main protease is an essential enzyme for the completion of the life cycle of Middle East Respiratory Syndrome Coronavirus. In our study we predicted compounds which are capable of inhibiting 3C-like protease, and thus inhibit the lifecycle of Middle East Respiratory Syndrome Coronavirus using in silico methods. METHODS: Lead like compounds and drug molecules which are capable of inhibiting 3C-like protease was identified by structure-based virtual screening and ligand-based virtual screening method. Further, the compounds were validated through absorption, distribution, metabolism and excretion filtering. RESULTS: Based on binding energy, ADME properties, and toxicology analysis, we finally selected 3 compounds from structure-based virtual screening (ZINC ID: 75121653, 41131653, and 67266079) having binding energy -7.12, -7.1 and -7.08 Kcal/mol, respectively and 5 compounds from ligandbased virtual screening (ZINC ID: 05576502, 47654332, 04829153, 86434515 and 25626324) having binding energy -49.8, -54.9, -65.6, -61.1 and -66.7 Kcal/mol respectively. All these compounds have good ADME profile and reduced toxicity. Among eight compounds, one is soluble in water and remaining 7 compounds are highly soluble in water. All compounds have bioavailability 0.55 on the scale of 0 to 1. Among the 5 compounds from structure-based virtual screening, 2 compounds showed leadlikeness. All the compounds showed no inhibition of cytochrome P450 enzymes, no blood-brain barrier permeability and no toxic structure in medicinal chemistry profile. All the compounds are not a substrate of P-glycoprotein. CONCLUSION: Our predicted compounds may be capable of inhibiting 3C-like protease but need some further validation in wet lab.

Association of ATP binding cassette transporter 1 (ABCA 1) gene polymorphism with type 2 diabetes mellitus (T2DM) in Bangladeshi population.

AIMS: ATP-binding cassette transporter 1 (ABCA1) gene polymorphism has been reported as one of the genetic risk factors for T2DM in various populations with conflicting results. The aim of this study is to investigate the association of ABCA1 C69T polymorphism and lipid profile with T2DM in Bangladeshi population. MATERIALS AND METHODS: A total of 102 T2DM subjects and 98 healthy controls were recruited and their genotypes for ABCA1 gene polymorphisms were determined based on the PCR-RFLP technique. Serum lipid profiles (total cholesterol, HDL-C, LDL-C and TG) were also estimated by using standard methods. RESULTS: ABCA 1 (C69T) genotypes frequencies were estimated. The percentages of CC, CT and TT genotypes at 69 position of ABCA1 gene were 31.63%, 58.16% and 10.21% in control as well as 22.54%, 69.60% and 7.86% in diabetes group respectively. Significant association was not found between ABCA1 (C69T) genotypes and T2DM in Bangladeshi population (Odd Ratio: 1.67; 95% Confidence Interval: 0.88 to 3.19 for CT genotype and Odd Ratio: 1.07; Confidence Interval: 0.36 to 3.16 for TT genotype; p > 0.05). Serum lipid profiles were not associated with T2DM. CONCLUSION: ABCA1 gene polymorphism might not be a genetic risk factor for T2DM subjects among Bangladeshis. We did not find a relationship between genotypes and lipid concentrations in our two groups. Study with a larger sample size will help us to understand the relationship of ABCA1 C69T genotype and lipid profile with T2DM.

Association of vitamin D and vitamin D binding protein (DBP) gene polymorphism with susceptibility of type 2 diabetes mellitus in Bangladesh.

AIMS: Polymorphism in vitamin D binding protein gene may have an impact on serum vitamin D transport and thus may have relation with type 2 diabetes mellitus. In our study, we investigated the association of serum vitamin D level and vitamin D-binding protein gene polymorphism with the onset of type 2 diabetes mellitus. MATERIALS AND METHODS: Blood samples were collected from 104 type 2 diabetic patients and 107 healthy volunteers. Serum vitamin D was measured by high-performance liquid chromatography. Genetic analysis of vitamin D-binging protein gene was carried out by polymerase chain reaction - restriction fragment length polymorphism method. RESULTS: We found significantly (p<0.001) lower level of vitamin D in type 2 diabetic patients compared to control subjects. A significantly negative correlation (r=-0.25, p<0.05) between vitamin D level and fasting blood glucose level was found among type 2 diabetic subjects. The Glu/Glu at codon 416 (rs7041) (p<0.05) and Lys/Lys at codon 420 (rs4588) (p<0.01) variants of vitamin D binding protein gene was significantly higher in type 2 diabetic subjects than controls. The patients with Glu/Glu and Lys/Lys genotypes respectively at codon 416 (odds ratio=2.87; 95% confidence interval=1.19 to 6.95) and 420 (odds ratio=8.9; 95% confidence interval=1.89 to 41.99) were at high risk of developing type 2 diabetes. CONCLUSIONS: Our present study strongly suggests that there might have an association of vitamin D, and vitamin D-binding protein gene (codon 416 & 420) polymorphisms with the occurrence of type 2 diabetes mellitus.

Oxidative stress induced sperm DNA damage, a possible reason for male infertility.

BACKGROUND: Sperm DNA damage is an important factor in the etiology of male infertility. OBJECTIVE: The aim of the study was to evaluate the association of oxidative stress induced sperm DNA damage with the pathogenesis of male infertility. MATERIALS AND METHODS: The study comprised a total of 66 subjects, including fertile men (n=25) and infertile men (n=41) matched by age. Seminal malondialdehyde (MDA), phospholipid hydroperoxide (PHP), superoxide dismutase (SOD), total antioxidant status (TAS) and 8-hydroxy-2'-deoxy guanosine (8-OHdG) were estimated by spectrophotometric and ELISA based methods and the association with the sperm parameters was assessed. RESULTS: The percentages of motile and morphologically normal cells were significantly lower (p < 0.001, p <0.001, respectivly) in infertile men. Seminal levels of MDA, PHP and 8-OHdG were significantly higher (p < 0.001, p < 0.001, and p=0. 02, respectively) while the SOD and TAS were significantly lower (p=0. 0003, p< 0.001, respectively) in infertile men. Sperm parameters were negatively correlated with MDA, PHP and 8-OHdG while positively correlated with SOD and TAS. A positive correlation of 8-OHdG with MDA and PHP and a negative correlation with TAS and SOD were also found. CONCLUSION: These results suggested that oxidative stress induced sperm DNA damage might have a critical effect on the etiology of infertility. Therefore, evaluation of oxidative status, antioxidant defense systems and DNA damage, together with sperm parameters might be a useful tool for diagnosis and treatment of male infertility.

Association of TP53 gene polymorphisms with susceptibility of bladder cancer in Bangladeshi population.

TP53 is considered to be the most frequently mutated gene in every forms of human cancer. The objective of this study was to evaluate the association of TP53 codon 72 and 248 polymorphisms with the susceptibility and severity of bladder cancer in Bangladeshi population. A case-control study on 102 bladder cancer patients and 140 control subjects was conducted. The genotype analysis was done by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method. The patients with Pro/Pro genotypes at 72 position were at high risk (odds ratio (OR) = 3.02; 95 % confidence interval (95 % CI) = 1.42 to 6.40) of developing bladder cancer. The cigarette smokers with Pro/Pro genotypes at 72 position were found to have a 3.91-fold increased risk to develop bladder cancer (OR = 3.91; 95 % CI = 1.33 to 11.5). There was no significant association of codon 248 polymorphisms with bladder cancer in the study population. Taken together, these findings indicate an association between p53 codon72 polymorphism and bladder cancer risk in Bangladeshi population.

N-acetyltransferase 2 gene polymorphism as a biomarker for susceptibility to bladder cancer in Bangladeshi population.

AIM: To investigate the association between the three most common single nucleotide polymorphisms of the N-acetyltransferase 2 gene together with cigarette smoking and the risk of developing bladder cancer and its aggressiveness. METHODS: A case-control study on 102 bladder cancer patients and 140 control subjects was conducted. The genomic DNA was extracted from peripheral white blood cells and N-acetyltransferase 2 alleles were differentiated by polymerase chain reaction-based restriction fragment length polymorphism methods. RESULTS: Bladder cancer risk was estimated as odds ratio and 95% confidence interval using binary logistic regression models adjusting for age and gender. Overall, N-acetyltransferase 2 slow genotypes were associated with bladder cancer risk (odds ratio=4.45; 95% confidence interval=2.26-8.77). The cigarette smokers with slow genotypes were found to have a sixfold increased risk to develop bladder cancer (odds ratio=6.05; 95% confidence interval=2.23-15.82). Patients with slow acetylating genotypes were more prone to develop high-grade (odds ratio=6.63; 95% confidence interval=1.15-38.13; P<0.05) and invasive (odds ratio=10.6; 95% confidence interval=1.00-111.5; P=0.05) tumor. CONCLUSION: N-acetyltransferase 2 slow genotype together with tobacco smoking increases bladder cancer risk. Patients with N-acetyltransferase 2 slow genotypes were more likely to develop a high-grade and invasive tumor. N-acetyltransferase 2 slow genotype is an important genetic determinant for bladder cancer in Bangladesh population.