Supplementary Material for: Association of the –1072G/A Polymorphism in the <b><i>LTC4S</i></b> Gene with Asthma in an Indian Population

<b><i>Background:</i></b> Atopic asthma, the most common chronic disease affecting children and young adults, is a complex disorder with variable phenotypes. Cysteine leukotrienes (Cys-LTs) are powerful bronchoconstrictors and play a critical role in airway inflammation and remodeling that are characteristic of asthma. <b><i>Objective:</i></b> To investigate the association of <i>ALOX5</i>, <i>LTC4S </i>and<i> CysLTR2 </i>gene polymorphisms with atopic asthma in an Indian population. <b><i>Methods:</i></b> A total of 19 single nucleotide polymorphisms (SNPs) within these genes were genotyped in a family-based cohort (n = 239) and a case-control cohort (139 cases and 194 controls) followed by association analyses. <b><i>Results:</i></b> We found a significant association of the –1072G/A (rs3776944) SNP with atopic asthma in the family-based association analysis (p = 0.0004). These results were also replicated in the case-control cohort (p = 0.009). The allele A was negatively associated with atopic asthma. We also noted a significant association in the two-locus (rs3776944G/A and rs730012A/C) haplotypic analysis of this gene both in the family-based (p = 0.03) and the case-control (p = 0.02) analyses. <b><i>Conclusion:</i></b> This study supports the role of the <i>LTC4S </i>gene polymorphism in genetic susceptibility to atopic asthma in an Indian population

Supplementary Material for: Circulating tumor DNA testing overcomes limitations of comprehensive genomic profiling from tumor tissue

The “liquid biopsy” is an established technique for examining circulating tumor DNA (ctDNA) from a routine blood draw and detecting actionable biomarkers. Nonetheless, ctDNA testing is rarely utilized for patients with newly diagnosed metastatic colorectal cancer (CRC). We report a case in which ctDNA testing uncovered an actionable biomarker that was not detected by comprehensive genomic profiling of tumor tissue. An 81-year-old woman with a remote history of non-Hodgkin’s lymphoma presented with primary masses in the ascending colon and sigmoid colon. The ascending colon and sigmoid colon tumors were classified as microsatellite stable (MSS) and mismatch repair proficient (pMMR), and both ctDNA and tissue next-generation sequencing (NGS) from the ascending colon mass were ordered. Because tissue NGS results indicated that the ascending colon tumor was MSS, palliative 5-fluorouracil, leucovorin, and oxaliplatin (FOLFOX) chemotherapy was started. However, the ctDNA NGS results that arrived after the start of FOLFOX found high microsatellite instability (MSI-H) and mismatch repair deficient (dMMR) disease with a serine/threonine-protein kinase B-Raf (BRAFV600E) mutation. To treat both her MSS/pMMR ascending colon and sigmoid colon tumors and MSI-H/dMMR metastatic disease, the immunotherapy nivolumab was added to FOLFOX. After 8 months of combined nivolumab and chemotherapy, the patient’s metastatic disease had a complete clinical response. This case highlights the complementary role of ctDNA testing for biomarker identification. By performing simultaneous ctDNA testing at the time of diagnosis, an actionable biomarker was discovered that significantly altered this patient’s prognosis and treatment options. Orthogonal testing of key molecular alterations offers significant advantages for identifying actionable biomarkers and improving management of metastatic CRC

Supplementary Material for: HIV-1 Nef Interacts with HCV Core, Recruits TRAF2, TRAF5 and TRAF6, and Stimulates HIV-1 Replication in Macrophages

Tumor necrosis factor receptor-associated factor (TRAF) signaling plays a central role in many biological activities, such as the regulation of immune and inflammatory responses and control of apoptosis, which are key events in the pathogenesis of the human immunodeficiency virus (HIV)-1 and the hepatitis C virus (HCV) infections. Here we show that TRAF2, TRAF5 and TRAF6 interact with the HIV-1 Nef protein, an immunomodulatory viral protein expressed and released by cells infected by the virus. We also found that TRAF2 and TRAF5 interact with the HCV Core protein. Interestingly, we observed that HIV-1 Nef interacts with HCV Core. The activation of TRAF (2, 5, 6) - mediated by HIV-1 Nef and HCV Core - enhanced the activation of the nuclear factor-kappa B (NF-κB) and increased HIV-1 replication in monocyte- derived macrophages (MDMs). The knockdown of TRAF2, TRAF5 and TRAF6 resulted in decreased NF-κB activation and reduced HIV-1 replication in MDMs. Our results reveal a mechanism by which the activation of the TRAF pathway by HIV-1 Nef and HCV Core favors the replication of HIV-1 in macrophages and could be a critical factor for optimal replication of HIV-1 in macrophages of HIV-HCV-coinfected patients

Supplementary Material for: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) binds with spike protein and inhibits the entry of SARS-CoV-2 into host cells

Introduction: Coronavirus disease 2019 (COVID-19) caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of Spike (S) protein S1 subunit with angiotensin converting enzyme (ACE) present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. Method: Recombinant S protein, the receptor binding domain (RBD) and human GAPDH protein were used for solid phase binding assays and Biolayer interferometry (BLI). Pseudo virus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for Flow cytometry-based infectivity assays. Results: Pseudo-virus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD domain of SARS-CoV-2 with nano molar affinity. Conclusions: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells

Supplementary Material for: M235T Polymorphism in the <b><i>AGT</i></b> Gene and A/G^I8-83 Substitution in the <b><i>REN</i></b> Gene Correlate with End-Stage Renal Disease

<b><i>Background/Aims:</i></b> This study aimed at investigating if M235T polymorphism in the AGT gene and A/G^I8-83 polymorphism in the REN gene correlate with end-stage renal disease (ESRD). <b><i>Methods:</i></b> We analyzed 173 ESRD patients and 329 individuals with normal kidney function for differences in the genotype distribution of AGT-M235T and REN-A/G^I8-83 polymorphisms between the two groups. The data for cases and controls were compared using the χ² test. <b><i>Results:</i></b> We found significantly higher levels of serum creatinine and CRP in cases in comparison to controls (p < 0.0001). Data comparison showed a significant association of AGT M235T substitution with ESRD in the dominant model (p = 0.008) and in the comparison of the heterozygous substitution with the homozygous common genotype (p = 0.005). Similarly, REN A/G^I8-83 polymorphism showed a significant difference in the distribution of genotypes between cases and controls (p < 0.038) such that a heterozygous substitution was significantly more common in the ESRD cases in comparison to the homozygous common genotype (p = 0.023). <b><i>Conclusion:</i></b> We conclude that heterozygous substitutions at the AGT M235T and REN A/G^I8-83 loci correlate significantly with ESRD in a north Indian population