Genomic analysis of breast cancer patients from Kerala: A novel BRCA1 mutation detected.

BACKGROUND: Breast cancer is the most common cancer among females, with an incidence of 6,41,000 cases annually. The genetic makeup of the individuals, ethnicity, geographical location, lifestyle, and BMI are some well-described factors associated with breast cancer. It is well known that pathogenic variants in BRCA1 and BRCA2 are associated with a majority of hereditary breast cancer. Genome-wide association studies (GWAS) have identified more than 80 germline susceptibility loci responsible for hereditary breast cancer. METHODS: In the present study, analysis of 94 genes associated with hereditary cancer was performed using next generation sequencing (NGS) in twelve patients having breast cancer and suspected with hereditary association. RESULTS: Four out of twelve (33%) patients harbored pathogenic mutation of the BRCA1 gene. Two patients was identified p. E23Vfs*17 mutation in BRCA1, one patient had p.Glu1580Gln in BRCA1, and a novel frameshift variant p.T1456Ifs*9(c.4367Cdel) in one patient. CONCLUSION: In the present study, out of four detected mutations in the BRCA1 gene, three were known and one was a novel BRCA1 mutation. It is advised to perform NGS-based genome sequencing to identify the genetic predisposition in breast cancer patients.

Identification of rare atypical BCR-ABL1 transcript: A case report.

CML is characterized by the presence of a BCR-ABL1 fusion transcript. Several guidelines have been published for its detection and molecular monitoring. Here, a case is described of chronic myeloid leukemia presenting in the blast phase with a rare variant transcript, with a discussion on possible red flags in its detection and genetic testing and description of the patient's clinical characteristics. This case highlights the pitfalls of using real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) for diagnosis of CML, especially when the clinical picture and the test results are discordant.

New Frontiers in Three-Dimensional Culture Platforms to Improve Diabetes Research.

Diabetes mellitus is associated with defects in islet ß-cell functioning and consequent hyperglycemia resulting in multi-organ damage. Physiologically relevant models that mimic human diabetic progression are urgently needed to identify new drug targets. Three-dimensional (3D) cell-culture systems are gaining a considerable interest in diabetic disease modelling and are being utilized as platforms for diabetic drug discovery and pancreatic tissue engineering. Three-dimensional models offer a marked advantage in obtaining physiologically relevant information and improve drug selectivity over conventional 2D (two-dimensional) cultures and rodent models. Indeed, recent evidence persuasively supports the adoption of appropriate 3D cell technology in ß-cell cultivation. This review article provides a considerably updated view of the benefits of employing 3D models in the experimental workflow compared to conventional animal and 2D models. We compile the latest innovations in this field and discuss the various strategies used to generate 3D culture models in diabetic research. We also critically review the advantages and the limitations of each 3D technology, with particular attention to the maintenance of ß-cell morphology, functionality, and intercellular crosstalk. Furthermore, we emphasize the scope of improvement needed in the 3D culture systems employed in diabetes research and the promises they hold as excellent research platforms in managing diabetes.

Frequency of EGFR mutations in lung adenocarcinoma patients - A study from tertiary cancer center of South India.

BACKGROUND AND OBJECTIVES: Epidermal growth factor receptor (EGFR) mutation analysis has become an important part of the initial workup of non-squamous non-small cell lung cancer (NS-NSCLC) patients. This study is attempted as South Indians population is comprised of ethnic groups with diverse genetic makeup and only very limited data on EGFR mutation is available from south India. A detailed understanding of EGFR mutation profile will help in better planning of treatment strategies and resource allocation. METHODS: A retrospective analysis of EGFR mutation frequency in 350 patients diagnosed with adenocarcinoma of lung and its association with pathological characteristics was done. RESULTS: Out of 350 cases of pulmonary adenocarcinoma, within an age group ranging from 30 to 86 years. EGFR mutations were identified in 34.8% (n = 122) cases, out of which 35.24% (n = 43) were in non-smoker females (P = 0.001). Of the 14 cases with resistant type of EGFR mutations, nine were in smoker males and the remaining five in non-smoker females. INTERPRETATION AND CONCLUSION: Overall EGFR mutation frequency observed in our study was similar to other Indian studies. However, in our study, we observed that mutation in exon 21 was less frequent compared to other studies. A similar slightly increased frequency of rare mutations and double mutations were observed in our study. A detailed study of the molecular epidemiology of lung cancer and its association with different geographical zones of India is needed. This understanding will help in better planning of treatment strategies and resource allocation.

Testing for Microsatellite Instability in Colorectal Cancer - a Comparative Evaluation of Immunohistochemical and Molecular Methods.

BACKGROUND: Introduction: The majority of colorectal cancers (CRC) develop through the chromosomal instability pathway and approximately 15% display microsatellite instability (MSI) as a carcinogenic event. CRCs with microsatellite instability status have a characteristic phenotype. We aimed to assess the clinico-pathological and MSI profiles of sixty-one cases of CRC through immunohistochemical (IHC) staining for the mismatch repair(MMR) proteins and DNA based Polymerase Chain Reaction (PCR) assay for microsatellite markers. PATIENTS & METHODS: Haematoxylin & Eosin stained sections of the tumor were evaluated for various histopathologic features. Immunohistochemistry was performed for the four MMR proteins, MLH1, MSH2, MSH6 and PMS2. NCI recommended panel of five nucleotide repeat markers was amplified from tumor DNA. RESULTS: The majority of the patients were males above fifty years of age. Around 61% of tumors were in the leftsided colon. Adenocarcinoma NOS (55, 90%) was the most common histological type. A total of 18 (29.5 %) cases showed dMMR by immunohistochemistry. Loss of PMS2 protein and combined loss of MSH2 & MSH6 were the most common findings in low and high MSI respectively. Of the 13 cases selected for PCR analysis, nine cases had high MSI (at least two markers unstable) and four cases had low MSI (one marker unstable) Results of PCR based DNA assay showed good concordance with IHC. No significant statistical association could be identified between the status of MSI by either methods and sociodemographic or clinical features. DISCUSSION: MSI constitutes 12%-20% and 6%-13% of CRCs in Western and Eastern countries respectively. In our series IHC staining revealed that 29.5% of cases showed dMMR. This was similar to other Indian studies which reported a prevalence of 22-27%. The combined loss of MSH2 & MSH6 (78%) was the most common type of dMMR. There was good concordance between IHC and PCR results. The issue of heterogenous or weak staining is a limiting factor in IHC interpretation and few cases of dMMR may be missed. CONCLUSION: To conclude, IHC can be a very useful screening tool to detect microsatellite instability and triage cases of dMMR for MSI biomarker testing. The MSI status also serves as a prognostic and predictive tool. KEY WORDS: Colorectal cancer, microsatellite instability, immunohistochemistry, Polymerase chain reaction.

Deciphering the Cold Adaptive Mechanisms in Pseudomonas psychrophila MTCC12324 Isolated from the Arctic at 79° N.

Psychrophiles, host of cold environments, have been successfully undergoing the process of evolution by which they have acquired innate adaptations to withstand the unfavorable effects of low temperature. Psychrophiles renders immense opportunity to explore the underlying mechanisms of cold adaptation. The present study focused to explore the cold adaptive mechanisms of Pseudomonas psychrophila MTCC12324, a facultative psychrophilic bacterium isolated from the Ny-Alesund, an island in the Svalbard Archipelago (79°55' N, 11°56' E) in the Arctic. Whole genome sequencing of P. psychrophila MTCC12324 and its analysis revealed the redundant nature of genome and identified several cold acclimation genes including cold shock proteins, and chaperones involved in the adaptive mechanism to thrive in the cold environment. Comparative proteome analysis of P. psychrophila MTCC12324 at 4 °C and 25 °C has thrown lights on the metabolic pathways and cellular processes adopted to withstand the cold environment. Basic survival pathways and factors involved in energy metabolism were found to be unaltered whereas stress response factors, enzymes involved in fatty acid elongation and cold-adapted chaperones were found to be enhanced towards cold stress. The present study facilitates recognition of crucial factors including polyunsaturated fatty acid biosynthesis, mRNA chaperones, and other cold-inducible proteins which favors the bacteria in conferring cold adaptation.

Comparative label-free lipidomic analysis of Mycobacterium tuberculosis during dormancy and reactivation.

Mycobacterium tuberculosis employs several strategies to combat and adapt to adverse conditions encountered inside the host. The non-replicative dormant state of the bacterium is linked to drug resistance and slower response to anti-tubercular therapy. It is known that alterations in lipid content allow dormant bacteria to acclimatize to cellular stress. Employing comparative lipidomic analysis we profiled the changes in lipid metabolism in M. tuberculosis using a modified Wayne's model of hypoxia-induced dormancy. Further we subjected the dormant bacteria to resuscitation, and analyzed their lipidomes until the lipid profile was similar to that of normoxially grown bacteria. An enhanced degradation of cell wall-associated and cytoplasmic lipids during dormancy, and their gradual restoration during reactivation, were clearly evident. This study throws light on distinct lipid metabolic patterns that M. tuberculosis undergoes to maintain its cellular energetics during dormancy and reactivation.

Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation.

Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to prevent reactivation of latent tuberculosis.

Accelerated pentose utilization by Corynebacterium glutamicum for accelerated production of lysine, glutamate, ornithine and putrescine.

Because of their abundance in hemicellulosic wastes arabinose and xylose are an interesting source of carbon for biotechnological production processes. Previous studies have engineered several Corynebacterium glutamicum strains for the utilization of arabinose and xylose, however, with inefficient xylose utilization capabilities. To improve xylose utilization, different xylose isomerase genes were tested in C. glutamicum. The gene originating from Xanthomonas campestris was shown to have the highest effect, resulting in growth rates of 0.14 h(-1), followed by genes from Bacillus subtilis, Mycobacterium smegmatis and Escherichia coli. To further increase xylose utilization different xylulokinase genes were expressed combined with X. campestris xylose isomerase gene. All combinations further increased growth rates of the recombinant strains up to 0.20 h(-1) and moreover increased biomass yields. The gene combination of X. campestris xylose isomerase and C. glutamicum xylulokinase was the fastest growing on xylose and compared with the previously described strain solely expressing E. coli xylose isomerase gene delivered a doubled growth rate. Productivity of the amino acids glutamate, lysine and ornithine, as well as the diamine putrescine was increased as well as final titres except for lysine where titres remained unchanged. Also productivity in medium containing rice straw hydrolysate as carbon source was increased.

Corynebacterium glutamicum as a potent biocatalyst for the bioconversion of pentose sugars to value-added products.

Corynebacterium glutamicum, the industrial microbe traditionally used for the production of amino acids, proved its value for the fermentative production of diverse products through genetic/metabolic engineering. A successful demonstration of the heterologous expression of arabinose and xylose utilization genes made them interesting biocatalysts for pentose fermentation, which are the main components in lignocellulosic hydrolysates. Its ability to withstand substantial amount of general growth inhibitors like furfurals, hydroxyl methyl furfurals and organic acids generated from the acid/alkali hydrolysis of lignocellulosics in growth arrested conditions and its ability to produce amino acids like glutamate and lysine in acid hydrolysates of rice straw and wheat bran, indicate the future prospective of this bacterium as a potent biocatalyst in fermentation biotechnology. However, the efforts so far on these lines have not yet been reviewed, and hence an attempt is made to look into the efficacy and prospects of C. glutamicum to utilize the normally non-fermentable pentose sugars from lignocellulosic biomass for the production of commodity chemicals.

Amino acid production from rice straw and wheat bran hydrolysates by recombinant pentose-utilizing Corynebacterium glutamicum.

Corynebacterium glutamicum wild type lacks the ability to utilize the pentose fractions of lignocellulosic hydrolysates, but it is known that recombinants expressing the araBAD operon and/or the xylA gene from Escherichia coli are able to grow with the pentoses xylose and arabinose as sole carbon sources. Recombinant pentose-utilizing strains derived from C. glutamicum wild type or from the L-lysine-producing C. glutamicum strain DM1729 utilized arabinose and/or xylose when these were added as pure chemicals to glucose-based minimal medium or when they were present in acid hydrolysates of rice straw or wheat bran. The recombinants grew to higher biomass concentrations and produced more L-glutamate and L-lysine, respectively, than the empty vector control strains, which utilized the glucose fraction. Typically, arabinose and xylose were co-utilized by the recombinant strains along with glucose either when acid rice straw and wheat bran hydrolysates were used or when blends of pure arabinose, xylose, and glucose were used. With acid hydrolysates growth, amino acid production and sugar consumption were delayed and slower as compared to media with blends of pure arabinose, xylose, and glucose. The ethambutol-triggered production of up to 93 ± 4 mM L-glutamate by the wild type-derived pentose-utilizing recombinant and the production of up to 42 ± 2 mM L-lysine by the recombinant pentose-utilizing lysine producer on media containing acid rice straw or wheat bran hydrolysate as carbon and energy source revealed that acid hydrolysates of agricultural waste materials may provide an alternative feedstock for large-scale amino acid production.