ICRC mouse with congenital mega-esophagus as a model to study esophageal tumorigenesis

ICRC mouse, an inbred strain, developed at the Cancer Research Institute, Bombay, exhibits mega-esophagus with markedly hyperplastic mucosa. Diethylnitrosamine (DEN) when given in drinking water at the dose of 4 mg/kg body weight/day, induced esophageal papillomas consistently in 100% of the animals, in a short period of 12 weeks. Further, tumors were produced, even at a very low cumulative dose of 28 mg/kg body weight. Development of the esophageal papillomas was dose dependent. DEN even induces esophageal tumors transplacentally in the ICRC F1 progeny. Tobacco acts predominantly as a promoter in this system. ICRC mouse thus provides a much needed animal model to study esophageal tumorigenesis, including the two-stage carcino genesis. An interesting feature of the study is that initiation could be induced by exposure to low doses of DEN in the intra-uterine life. Tumors develop in such F1 animals only if they are fed tobacco, a predominant promoter, post-natally

Primary hyperparathyroidism as first manifestation in multiple endocrine neoplasia type 2A: an international multicenter study.

Multiple endocrine neoplasia type 2A (MEN 2A) is a rare syndrome caused by RET germline mutations and has been associated with primary hyperparathyroidism (PHPT) in up to 30% of cases. Recommendations on RET screening in patients with apparently sporadic PHPT are unclear. We aimed to estimate the prevalence of cases presenting with PHPT as first manifestation among MEN 2A index cases and to characterize the former cases. An international retrospective multicenter study of 1085 MEN 2A index cases. Experts from MEN 2 centers all over the world were invited to participate. A total of 19 centers in 17 different countries provided registry data of index cases followed from 1974 to 2017. Ten cases presented with PHPT as their first manifestation of MEN 2A, yielding a prevalence of 0.9% (95% CI: 0.4-1.6). 9/10 cases were diagnosed with medullary thyroid carcinoma (MTC) in relation to parathyroid surgery and 1/10 was diagnosed 15 years after parathyroid surgery. 7/9 cases with full TNM data were node-positive at MTC diagnosis. Our data suggest that the prevalence of MEN 2A index cases that present with PHPT as their first manifestation is very low. The majority of index cases presenting with PHPT as first manifestation have synchronous MTC and are often node-positive. Thus, our observations suggest that not performing RET mutation analysis in patients with apparently sporadic PHPT would result in an extremely low false-negative rate, if no other MEN 2A component, specifically MTC, are found during work-up or resection of PHPT.S D received a national grant (AZV 16-32665A).S

Single-cell polymerase chain reaction-based pre-implantation genetic diagnosis using fragment analysis for β-thalassemia in an Indian couple with β-globin gene mutations

Despite advances in diagnostic techniques, approximately 10,000 babies with β-thalassemia major are born annually in India. Pre-implantation genetic diagnosis (PGD), an alternative to prenatal diagnosis, helps in negative selection of affected embryos prior to implantation. Hereby, we report the first successful β-thalassemia PGD pregnancy in an Indian carrier couple. β-Thalassemia mutation analysis by Amplification-Refractory Mutation Sequence (ARMS)-polymerase chain reaction (PCR) in the parents, followed by PGD for β-thalassemia mutation in embryos in two consequent in vitro fertilization (IVF) cycles, with transfer for three β-thalassemia minor embryos, resulted in singleton successful pregnancy, the results of which were confirmed on prenatal diagnosis. With advances in assisted reproductive techniques and molecular diagnosis, PGD for monogenic diseases is feasible in high-risk couples. The methodology in the current study included two rounds of PCR using fluorescently labeled primers, fragment analysis using the ABI 3100 nucleotide sequencer and the GeneMapper software, purification, and concentration of PCR product, which enabled distinct clear peaks making the analysis and interpretation non-ambiguous

Global Transcriptional Profiling of Longitudinal Clinical Isolates of <em>Mycobacterium tuberculosis</em> Exhibiting Rapid Accumulation of Drug Resistance

<div><p>The identification of multidrug resistant (MDR), extensively and totally drug resistant Mycobacterium <em>tuberculosis</em> (<em>Mtb</em>), in vulnerable sites such as Mumbai, is a grave threat to the control of tuberculosis. The current study aimed at explaining the rapid expression of MDR in Directly Observed Treatment Short Course (DOTS) compliant patients, represents the first study comparing global transcriptional profiles of 3 pairs of clinical <em>Mtb</em> isolates, collected longitudinally at initiation and completion of DOTS. While the isolates were drug susceptible (DS) at onset and MDR at completion of DOTS, they exhibited identical DNA fingerprints at both points of collection. The whole genome transcriptional analysis was performed using total RNA from H37Rv and 3 locally predominant spoligotypes viz. MANU1, CAS and Beijing, hybridized on MTBv3 (BuG@S) microarray, and yielded 36, 98 and 45 differentially expressed genes respectively. Genes encoding transcription factors (<em>sig</em>, <em>rpoB</em>), cell wall biosynthesis (<em>emb</em> genes), protein synthesis (<em>rpl</em>) and additional central metabolic pathways (<em>ppdK, pknH, pfkB)</em> were found to be down regulated in the MDR isolates as compared to the DS isolate of the same genotype. Up regulation of drug efflux pumps, ABC transporters, trans-membrane proteins and stress response transcriptional factors (<em>whiB</em>) in the MDR isolates was observed. The data indicated that <em>Mtb</em>, without specific mutations in drug target genes may persist in the host due to additional mechanisms like drug efflux pumps and lowered rate of metabolism. Furthermore this population of <em>Mtb</em>, which also showed reduced DNA repair activity, would result in selection and stabilization of spontaneous mutations in drug target genes, causing selection of a MDR strain in the presence of drug pressures. Efflux pump such as <em>drrA</em> may play a significant role in increasing fitness of low level drug resistant cells and assist in survival of <em>Mtb</em> till acquisition of drug resistant mutations with least fitness cost.</p> </div

In-silico identification of small molecules targeting H-Ras and in-vitro cytotoxicity with caspase-mediated apoptosis in carcinoma cells

H-Ras oncogene plays a critical role in the transformation of normal cells to a malignant phenotype through constitutive activation of the GTP bound protein leading to uncontrolled cell proliferation in several human cancers. Thus, H-Ras oncoprotein serves as an excellent target for anticancer drug discovery. To identify novel H-Ras inhibitors, we performed structure-based virtual screening of the Maybridge HitFinder (TM) library using Schrodinger suite. Thirty ligands from the chemical library were identified as they showed preferential in silico binding initially to H-Ras proteins with Gly12Val, Gly13Asp, and Gly12Val-Gly13Asp mutations. Absorption, distribution, metabolism, excretion, and toxicity profile confirmed drug-like properties of the compounds. Three representative molecules were tested for antiproliferative effect on T24 urinary bladder carcinoma cell line, MCF-7 breast cancer cell line and HDF-7 normal dermal fibroblast cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Two compounds (Cmpds) showed antiproliferative activity exclusively in the cancer cell lines with minimal effect on the control HDF-7 cells. The effect of compound treatment on cell cycle progression, assessed by propidium iodide (PI) staining, depicted increased arrest of T24 cell line in the sub G1 phase. Further, Annexin-V PI dual staining and pan caspase inhibitor Z-VAD-fmk indicated caspase-dependent apoptotic activity of Cmpds 1 and 3. Our findings demonstrate caspase-dependent apoptotic activity of Cmpds 1 and 3 selectively against Gly12Val mutated T24 cancer cell line implicating a potential for treatment of bladder cancer. We envisage that these molecules may be promising candidates with potential therapeutic value in H-Ras mutation-associated cancers

Hierarchical clustering.

<p>Hierarchical clustering of genes in (A) MANU1; (B) CAS (C) Beijing.</p

Genes with significant differential expression (p<0.005).

<p>Genes with significant differential expression (p<0.005).</p

H-ras-1 restriction fragment length polymorphism in normal individuals and oral cancer patients in India

Restriction fragment length polymorphism (RFLP) of the human H-ras-l gene has been indicated as a marker for detection of individuals at high risk of cancer. We have investigated the association of RFLP at the H-ras-l locus and susceptibility to oral cancer by Southern hybridization analysis in 77 primary oral tumors and 99 healthy donors. The frequency distribution of the Bum HI fragments of H-ras-l revealed homozygous or heterozygous alleles in the two sub-populations. The heterozygous genotype occurred more frequently in the normal subjects (53%) as compared to the cancer patients (36%). Four common alleles Cl to C4, were noted predominantly in both groups, with rare alleles detected at a lower frequency. The common allele with 7.6 kb BamHI fragment was significantly higher in normals (10%) than in the tumor population (4%) (P≤0.05). However, a similar distribution of rare alleles in both groups indicated that the presence of rare alleles is not indicative of predisposition to oral cancer