Mutations in the IRGM gene have been associated with Crohn's disease in several populations but have not been explored in Indian patients with this disease. This study examined the association of IRGM mutations with ulcerative colitis and Crohn's disease in Indian patients with inflammatory bowel disease. The IRGM gene was amplified in four segments and Sanger-sequenced in 101 participants (42 Crohn's disease, 39 ulcerative colitis, and 20 healthy controls). Ten single nucleotide polymorphisms (SNP) were genotyped in 1200 participants (352 Crohn's disease, 400 ulcerative colitis, and 448 healthy controls) using Sequenom MassARRAY iPLEX. Disease associations were evaluated for each of the ten SNPs. Thirty one mutations were identified in the IRGM gene, of which two had not hitherto been reported (150226250- ss947429272 & 150227858- ss947429273). Ten SNPs (6 from the above and 4 from the literature) were evaluated. Significant associations with Crohn's disease were noted with the T allele of rs1000113 (OR 1.46, 95% CI 1.12–1.90), T allele of rs9637876 (OR 1.25, 95% CI 1.005–1.561) and C allele of rs 13361189 (OR 1.33, 95% CI 1.07–1.669). Two SNPs – rs11747270 and rs180802994 – did not exhibit Hardy-Weinberg equilibrium but were associated with both Crohn's disease and ulcerative colitis in this population. The remaining SNPs did not show significant associations with either Crohn's disease or ulcerative colitis. Association of IRGM gene SNPs with Crohn's disease is reported for the first time in Indian patients. We also report, for the first time, an association of rs 9637876 in the IRGM gene with Crohn's disease.

The health benefits of dietary amylase resistant starch (RS) arise from intestinal microbial fermentation and generation of short chain fatty acids (SCFA). We compared the intestinal fermentative capability of stunted and nonstunted (‘healthy’) children in southern India using two types of RS: high amylose maize starch (HAMS) and acetylated HAMS (HAMSA). Twenty children (10 stunted and 10 healthy) aged 2 to 5 years were fed biscuits containing HAMS (10 g/day) for two weeks followed by a 2-week washout and then HAMSA biscuits (10 g/day) for 2 weeks. Fecal samples were collected at 3-4 day intervals and pH and SCFA analyzed. At entry, stunted children had lower SCFA concentrations compared to healthy children. Both types of RS led to a significant decrease in fecal pH and increase in fecal acetate and propionate in both healthy and stunted children. However, while HAMS increased fecal butyrate in both groups of children, HAMSA increased butyrate in healthy but not stunted children. Furthermore, healthy children showed a significantly greater increase than stunted children in both acetate and butyrate when fed either RS. No adverse effects were reported with either RS. Stunted children have impaired capacity to ferment certain types of RS which has implications for choice of RS in formulations aimed at improving microbial function in stunted children.

Doublecortin like kinase 1 (DCLK1) plays a crucial role in several cancers including colon and pancreatic adenocarcinomas. However, its role in squamous cell carcinoma (SCC) remains unknown. To this end, we examined DCLK1 expression in head and neck squamous cell carcinoma (HNSCC) and anal squamous cell carcinoma (ASCC). We found that DCLK1 is elevated in patient SCC tissue, which correlated with cancer progression and poorer overall survival. Furthermore, DCLK1 expression is significantly elevated in HPV negative cancer tissues, which are typically aggressive with poor responses to radiation therapy. To understand the role of DCLK1 in tumorigenesis, we used specific shRNA to suppress DCLK1 expression. This significantly reduced tumor growth, spheroid formation, and migration of HNSCC cancer cells. To further the translational relevance of our studies, we sought to identify a selective DCLK1 inhibitor. Current attempts to target DCLK1 using pharmacologic approaches have relied on non-specific suppression of DCLK1 kinase activity. Here, we demonstrate that DiFiD [3,5-bis (2,4-difluorobenzylidene)-4-piperidone] binds to DCLK1 with high selectivity. Moreover, DiFiD mediated suppression of DCLK1 led to G2/M arrest and apoptosis and significantly suppressed tumor growth of HNSCC xenografts and ASCC patient derived xenografts, supporting that DCLK1 is critical for SCC growth.

Myeloid-derived suppressor cells (MDSCs) contribute to immune suppression in cancer through arginase-1 (ARG1), which limits T-cell activation. Conventional assays such as ELISA or qPCR cannot determine the cellular source of ARG1 in blood. Recognizing the importance of ARG1 in immunosuppression, we developed and validated a standardized flow cytometry method using a commercially available ARG1 antibody to measure intracellular ARG1 in MDSC subsets. This method includes detailed instructions, including experimental optimization using positive and negative controls, to help other researchers perform flow cytometric measurement of ARG1 expressing cells. In this method, peripheral blood mononuclear cells were stained for lymphoid and myeloid markers (CD3, CD20, CD56, HLA-DR, CD33, CD11b, CD14, and CD15), fixed, and permeabilized before intracellular ARG1 staining. Antibody specificity was verified using neutrophils as positive and CD4⁺ T cells as negative controls. The method was applied to blood samples from patients with bladder cancer (n = 23) and healthy volunteers (n = 10), demonstrating reliable detection of intracellular ARG1 in MDSC subpopulations. This accessible protocol provides an adaptable method for quantifying cell-specific ARG1 expression and studying MDSC-mediated immunosuppression. • The flow cytometry method described here was developed by validating the arginase1 antibody binding specificity using cells expressing high and very low levels of arginase-1. • Arginase-1-expressing cells of the peripheral blood monocytes were identified using this method. • This method can be adapted to any cell type and will be a valuable tool in future immunosuppression studies.