Altered TLR7 Expression-Mediated Immune Modulation Is Supportive of Persistent Replication and Intrauterine Transmission of HBV.

Hepatitis B Virus (HBV) is posing as a serious public health threat mainly due to its asymptomatic nature of infection in pregnancy and vertical transmission. Viral sensing toll-like receptors (TLR) and Interleukins (IL) are important molecules in providing an antiviral state. The study aimed to assess the role of TLR7-mediated immune modulation, which might have an impact in the intrauterine transmission of HBV leading to mother to child transmission of the virus. We investigated the expression pattern of TLR7, IL-3, and IL-6 by RT-PCR in the placentas of HBV-infected pregnant women to see their role in the intrauterine transmission of HBV. We further validated the expression of TLR7 in placentas using Immunohistochemistry. Expression analysis by RT-PCR of TLR7 revealed significant downregulation among the Cord blood (CB) HBV DNA positive and negative cases with mean ± standard deviation (SD) of 0.43 ± 0.22 (28) and 1.14 ± 0.57 (44) with p = 0.001. IL-3 and IL-6 expression revealed significant upregulation in the CB HBV DNA-positive cases with p = 0.001. Multinomial logistic regression analysis revealed that TLR7 and IL-3 fold change and mother HBeAg status are important predictors for HBV mother to child transmission. Immunohistochemistry revealed the decreased expression of TLR7 in CB HBV DNA-positive cases. This study reveals that the downregulation of TLR7 in the placenta along with CB HBV DNA-positive status may lead to intrauterine transmission of HBV, which may lead to vertical transmission of HBV.

Occurrence of Existing BCR-ABL Baseline Mutations and Associated Haplotype (NmR) Among CML Patients with Diverse IM Response: A Hospital-based Study from North-East India.

Highly polymorphic BCR-ABL kinase domains have been reported to harbor more than a hundred mutations, and among these, 40-60% have been identified as influencers of imatinib mesylate (IM) resistance. The emergence of IM resistance poses a significant challenge in the management of Chronic Myeloid Leukemia (CML). M351T (rs121913457), E255K (rs387906517), and Y253H (rs121913461) are of particular clinical significance due to their association with high-level imatinib resistance. This study was conducted to investigate the potential role of three significant SNPs in CML progression due to IM resistance. During the study period from 2018 to 2022 (48 months), the blood samples from 219 Reverse transcriptase-PCR-confirmed CML patients following RNA extraction and cDNA preparation were subjected to M351T, E255K, and Y253H mutation analysis by PCR-RFLP. After agarose gel visualization, the samples were subjected to Sanger sequencing to confirm the nucleotide change at the polymorphic loci. The wild-type genotype of all three ABL1 SNPs under investigation exhibits a significant reduction in frequency among IM non-responders compared to the responder group. The CGT haplotype frequency exhibits a significant difference between IM responder (4.2%) and non-responder (11.8%) (p = 0.002 < 0.05). Further, CGC haplotype was observed solely among the imatinib non-responder patients with a frequency percentage of 3.3% (p = 0.004), whereas the said genotype was absent among the responder group. A reduced overall survival rate was observed with deviation from wild-type genotype (M351T loci (T > C) with 1.217 times, E255K (G > A) with 1.485 and Y253H (T > C) with 1.399 times increase in hazard ratio) thereby enhancing mortality risk due to disease progression. The significant increase in the frequency of M351T, E255K, and Y253H loci among the IM non-responder group indicated their probable association with the development of IM resistance among CML patients. A haplotype frequency distribution pattern analysis of ABL1 loci further identified the CGC haplotype as an independent predictor for IM resistance. As such the study highlights the importance of patient characteristics, genotype distribution, and haplotype frequency distribution in predicting the response to IM treatment and clinical outcomes of CML patients.

Epigenetic Modulation of DDIT3 and MGMT Expression Acts Synergistically with Resistance to Imatinib towards CML Disease Progression: A Hospital based Study.

INTRODUCTION: Imatinib Mesylate is an authenticated drug that aids in the treatment of Chronic Myeloid Leukaemia and Philadelphia patients which is recognized as a BCR-ABL tyrosine kinase inhibitor. Indeed, DNA Methylation occupies a key role in the stability of chromosomes. OBJECTIVE: Changes in the methylation status of genes may impart to the advancement of Chronic Myeloid Leukaemia. The present investigation aims to assess the role of expression analysis and methylation status of DDIT3 and MGMT genes in imatinib-resistant and nonresistant cases. METHODS: The Imatinib resistance was screened through RFLP. In this case maximum number of patients were recorded in the chronic phase belonging to the age group 40-59 and the accelerated and blast phase is more common in elderly patients showing the progressive nature of the disease with age. Hemoglobin and platelet count are found to be higher in cases where WBC count was minimal. A history of long-term alcohol consumption is found to be associated with the progression of the disease. RESULTS: The maximum level of expression of the DDIT3 gene was recorded in the chronic phase regardless of upstream (67.8%) and downstream (57.9%) regulation. The highest MGMT expression regulation was also observed in the case of chronic phase in both upstream (78.9%) and downstream (44%) regulation. Further, the MGMT gene showed the highest methylation of 6.6% and DDIT3 showed 3.3% in CML cases. CONCLUSION: In the present study notable depletion of survivality was established in the Imatinib resistance patients manifesting genetic malfunction of BCR-ABL transcripts among the North East Indian inhabitants and advocating for the expansion of the disease.

Association of Single Nucleotide Polymorphism in VDR, GC Globulin and CYP2R1 with the Risk of Esophageal Cancer.

BACKGROUND: The proactive role of vitamin D has been well determined in different cancers. The protein that encodes the components of the vitamin D metabolism could appear to play a pivotal role in vitamin D stability and its maintenance. A polymorphism in vitamin-D-receptor (VDR), carrier globulin/binding protein (GC) and cytochrome P-450 family 2, subfamily R, polypeptide 1 (CYP2R1) genes has been predicted to be associated with the development of cancer. This study was designed to detect the association of VDR, GC Globulin and CYP2R1 gene polymorphism with the risk of esophageal cancer in the North-east Indian population. METHODS: To carry out the study, a total of 100 patients diagnosed with esophageal cancer and 101 healthy controls were enrolled. In a case-control manner, all samples were subjected to do genotype testing for known SNPs on the VDR (rs1544410), GC (rs4588), and CYP2R1 (rs10741657) genes using Restriction-fragment length polymorphism (RFLP) followed by Sanger sequencing. The collected demographic and clinical data were analysed using the statistical software package SPSS v22.0. RESULTS: The VDR haplotype heterozygous TC was found strongly associated with the carcinoma group (OR:1.09, 95%CI:0.67-1.75). The risk factors analysis using the GC globulin rs4588 phenotype, found a positive correlation in terms of mutant AA's harmful influence on the cancer cohort (OR = 1.125, OR=1.125, 95% CI, 0.573-2.206). The influence of the CYP2R1 rs10741657 polymorphism on the malignant cohort revealed that the GG mutant had a significant negative influence on the carcinoma, has an influential role in disease severity ( OR:1.736, at 95% CI; 0.368-8.180). CONCLUSION: In conclusion, this study revealed the potential association of VDR gene polymorphism in the progression and development of esophageal cancer in north east Indian population cohort.

Altered Vitamin D Receptor Expression in Apa-I (rs7975232) Allelic Variants-A Probable Risk Factor for Susceptibility to Hepatitis B Virus Infection and Disease Progression.

Vitamin D exerts its antiviral effect through vitamin D receptor (VDR)/retinoid X receptor-mediated host immunomodulation. Besides the downregulation of VDR expression, its polymorphism was also observed among hepatitis B virus (HBV)-positive patients. To understand the possible link between VDR polymorphism and its altered expression during HBV infection and disease progression, VDR Apa-I [rs7975232 (C>A)] single nucleotide polymorphism (SNP) was analyzed in a case-control manner. VDR Apa-I (rs7975232, C>A) polymorphism was studied using 340 HBV patients and 102 healthy controls. Genotype analysis and gene expression study was performed using restriction fragment length polymorphism and quantitative polymerase chain reaction, respectively. Statistical analysis was performed using SPSS (IBM) considering p-value <0.05 as significant for comparing the differences between the groups. Significant mean difference in VDR expression was observed between HBV-positive patients (1.6 ± 0.94) and controls (0.69 ± 0.73). Furthermore, the mean fold change of Healthy control with CC genotype (1.92 ± 0.99) was found to be marginally significant compared with mutant genotype (CA/AA) (1.08 ± 0.43/0.59 ± 0.56, p = 0.045). In HBV+ patients, the mean fold change in the CC genotype was 0.88 ± 0.38, which exhibits a significant mean difference upon comparison with other genotypes (0.52 ± 0.49, 0.113 ± 0.34; p = 0.018, p = 0.048). However, the fold change value does not differ between CA and AA genotypes. Further comparative analysis of VDR expression between the control and case also exhibits significant differences (p = 0.001) among allelic variants. Observed genotype distribution frequency exhibits a significant association with disease type. The mutant genotype was found to be significantly associated with HBV infection and disease progression, (odds ratio = 0.730, 95% confidence interval = 0.462-1.152, p = 0.06). VDR SNP rs7975232 (C>A) may affect VDR expression by controlling several other variables and suggest that deviation from wild-type genotype (CC) is associated with downregulation of expression, which in turn involved in host immunomodulation in favor of HBV infection and disease progression.

Altered expression of endosomal Toll-like receptors and HBeAg seropositivity may act synergistically towards the vertical transmission of HBV.

PROBLEM: Hepatitis B is one of the leading causes of mortality in India. Despite the mass vaccination programme, the burden of the infection is still increasing due to its vertical transmission. Asymptomatic nature of hepatitis B virus (HBV) infection owing to immune tolerance among pregnant women is a major issue in this regard. METHOD OF STUDY: As such, this study aims to investigate the potential role of altered Toll-like receptor (TLR) expression (TLR-3, 7 and 9) along with peripheral blood HBeAg status in attaining differential cord blood (CB) HBV DNA status. RESULT: Expression analysis reveals an overall downregulation of expression with mean ± SD value 1.14 ± 1.05, 0.86 ± 0.5 and 0.71 ± 0.4 (TLR 3, 7 and 9, respectively) upon comparison with healthy women. Further stratification based on CB HBV DNA status; the downregulation of expression was found to be significantly (p < .05) associated with positive CB HBV DNA status apart from peripheral HBeAg status. One hundred percent HBeAg positive parturiting women exhibit positive CB HBV DNA. Pearson's correlation analysis reveals a positive correlation between CB HBV DNA status and altered TLR expression, HBeAg status and mother HBV DNA status and as such can be associated with the potential risk of HBV vertical transmission. CONCLUSION: This study suggests that the downregulation of TLR 3, 7 and 9 may be a risk factor for potential vertical transmission of HBV.

Upregulation of anaphase promoting complex (APC) 7 as a prognostic marker for esophageal squamous cell carcinoma: A hospital based study.

Esophageal cancer is the sixth leading cause of cancer death, and esophageal squamous cell carcinoma (ESCC) is the most prevalent type worldwide, with a poor prognosis due to late diagnosis. The search for new molecular prognostic biomarkers revealed that dysregulation of anaphase promoting complex/cyclosome (APC/C) activation due to altered expression of APC molecules might lead to perturbed mitotic progression leading to malignancy. We analyzed the expression of the four different subunits of the APC/C complex-APC3, APC4, APC5 and APC7-by Real Time Polymerase Chain Reaction (RT-PCR). The findings were then correlated with clinicopathological parameters and different lifestyle factors. Significant upregulation of APC7 (tissue and blood: N = 50; 3.72 ± 1.21 and 4.45 ± 1.18, respectively) and APC3 (tissue and blood: N = 52 and 55 and 4.50 ± 1.41 and 4.58 ± 1.06, respectively) suggests their role in uncontrolled cell proliferation. In addition to their association with increasing age, their significant association with tumor size, node stage (only APC7 (p < 0.05)), and dysphagia grade supports a potential role in tumorigenic transformation in ESCC. Furthermore, several exclusive lifestyle-associated factors play a crucial supporting role in the development of ESCC in the Northeast Indian population. Various lifestyle factors, such as the duration of smoking, tobacco and betel nut consumption, and the duration of alcohol consumption, are significantly associated with the expression of APC. Analysis based on Pearson's correlation coefficient indicated a positive correlation among the gene expression levels ofAPC3 (both blood and tissue), APC5 (tissue) and APC3 (tissue), APC7 (tissue) and APC3 (tissue), and APC7 (tissue) and APC3 (blood). Additionally, a positive correlation was found between APC7 expression in blood and tissue samples. However, no significant correlation was found between APC 7 expression and APC4 and APC5 expression in either blood or tissue samples.

In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity.

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.