To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved method has been named as the THSTI method to depict the Institute where the method was developed.

The gastric microbiome is suspected to have a role in the causation of diseases by Helicobacter pylori. Reports on their relative abundance vis-à-vis H. pylori are available from various ethnic and geographic groups, but little is known about their interaction patterns. Endoscopic mucosal biopsy samples from the gastric antrum and corpus of 39 patients with suspected H. pylori infection were collected and microbiomes were analyzed by 16S rDNA profiling. Four groups of samples were identified, which harbored Helicobacter as well as a diverse group of bacteria including Lactobacillus, Halomonas and Prevotella. There was a negative association between the microbiome diversity and Helicobacter abundance. Network analyses showed that Helicobacter had negative interactions with members of the gastric microbiome, while other microbes interacted positively with each other, showing a higher tendency towards intra-cluster co-occurrence/co-operation. Cross-geographic comparisons suggested the presence of region-specific microbial abundance profiles. We report the microbial diversity, abundance variation and interaction patterns of the gastric microbiota of Indian patients with H. pylori infection and present a comparison of the same with the gastric microbial ecology in samples from different geographic regions. Such microbial abundance profiles and microbial interactions can help in understanding the pathophysiology of gastric ailments and can thus help in development of new strategies to curb it.The acidic pH in the stomach lumen impedes bacterial growth 1 . However, it is now known that the human stomach is not sterile but is rather colonized by diverse microbiota 2 . High-throughput sequencing of gastric biopsy samples suggests that the human stomach may harbor 128 phyla, although mainly dominated by five phyla, namely, Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Fusobacteria 2-5 . Among these, several species of Helicobacter are known to be natural inhabitants of the human stomach. Helicobacter pylori (H. pylori), a Gram-negative bacterium and frequent isolate of stomach specimens, with reported links to the causation of chronic/atrophic gastritis, duodenal ulcers, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and adenocarcinoma 2,6,7 , has coevolved with its human host.While H. pylori has been incriminated in disease, an aspect that has only been recently investigated is the role of the stomach microbiome in the causation of these diseases, especially adenocarcinoma. Studies focusing on the microbial composition of the stomach in healthy individuals 4,[8][9][10] have indicated the presence of genera like Streptococcus, Prevotella, Veillonella, Fusobacterium, Haemophilus and Clostridium. H. pylori is known to utilize specific molecular mechanisms to modulate host immune response and create a local micro-environment that aids its colonization 5,11 . The influence of Helicobacter abundance on the populations of other genera and vice versa, and any interactions between them in the causation ...

BackgroundThe gastric microbiota has recently been implicated in the causation of organic/structural gastroduodenal diseases (gastric and duodenal ulcers, gastric cancer) in patients with Helicobacter pylori (H. pylori) infection. We aimed to ascertain, in patients harbouring H. pylori, the role of the gastric microbiota in the causation of symptoms (chronic dyspepsia) in the absence of organic disease.MethodsSeventy-four gastric biopsy samples obtained at endoscopy from patients with (n = 21) or without (n = 53) chronic dyspepsia, and that tested positive by the bedside rapid urease test for H. pylori infection, were cultured for detection of H. pylori and non-H. pylori organisms. The cultured organisms were identified by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS).ResultsA total of 106 non-H. pylori isolates were obtained from 74 patients’ samples. This included 33 isolates (median 2, range 1–2 per patient) from dyspeptic and 73 (median 2, range 1–2 per patient) from non-dyspeptic patients. These were identified from the Bruker Biotyper 2 database as Staphylococcus spp., Streptococcus spp., Lactobacillus spp., Micrococcus spp., Enterococcus spp., Pseudomonas spp., Escherichia spp., Klebsiella spp. and Bacillus spp., Staphylococcus and Lactobacillus were identified significantly more commonly in dyspeptics and Streptococcus, Pseudomonas, Escherichia coli and Klebsiella pneumoniae in non-dyspeptics. All identified organisms belonged to the phyla Firmicutes and Proteobacteria.ConclusionsThere is a qualitative difference in the gastric microbial spectrum between patients harbouring H. pylori with and without chronic dyspepsia. Whether these organisms have an independent role in the development or prevention of dyspepsia or act in concurrence with H. pylori needs study.

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Background: Chuvash polycythemia is a hematological disorder present worldwide but endemic to the Chuvash population, a Turkish ethnic group in Russia. The disorder is caused by a homozygous germline mutation (R200W) in the von Hippel Lindau gene (1,2). This mutation impairs binding of pVHL to hypoxia-inducible factor 1-alpha (HIF-1a); lack of this interaction prevents degradation of HIF-1a (2,3). The resultant upregulation of HIF-1a, even in a normal oxygen state, increases the activity of erythropoietin, thereby causing polycythemia (2,3). Affected individuals experience increased rates of arterial and venous thrombosis unrelated to the increased concentration of hemoglobin (4,5). Aims: To determine whether upregulation of HIF-1a in Chuvash polycythemia individuals causes a decreased level of the antithrombotic Protein S. A decreased level of Protein S may explain the increased risks of thrombotic events and vascular abnormalities in the Chuvash population. Methods: Enzyme-linked immunosorbent assays (ELISA) were performed to measure total Protein S concentration in Chuvash and control plasma. Immunoblotting was performed to confirm the ELISA measurements. Additional assays to measure free Protein S and thrombin generation assays with and without Protein S will be performed to determine whether Chuvash plasma has low free Protein S and whether Protein S supplementation will improve the thrombotic phenotypes. Results: Total Protein S concentration measured by ELISA was lower in Chuvash individuals compared with controls. In addition, we have completed immunoblotting of seven Chuvash samples and three control samples. Our results indicated that Chuvash plasma had lower amounts of Protein S compared with the controls. Immunoblotting of additional Chuvash samples is in progress, and we are planning to measure the free Protein S in Chuvash samples. Conclusion: Our preliminary results suggest a decreased level of total Protein S in individuals with Chuvash polycythemia. Because an increased hemoglobin concentration does not increase the rates of thromboembolic events in this population, our finding of a reduced amount of anticoagulant Protein S may explain the hypercoagulability that Chuvash individuals experience. References  Gordeuk, V.R., et al., Chuvash polycythemia VHLR200W mutation is associated with down-regulation of hepcidin expression. Blood, 2011. 118(19): p. 5278-82. Gordeuk, V.R., et al., Congenital disorder of oxygen sensing: association of the homozygous Chuvash polycythemia VHL mutation with thrombosis and vascular abnormalities but not tumors. Blood, 2004. 103(10): p. 3924-32. Gordeuk, V.R. and J.T. Prchal, Vascular complications in Chuvash polycythemia. Semin Thromb Hemost, 2006. 32(3): p. 289-94. Sergueeva, A., et al., Prospective study of thrombosis and thrombospondin-1 expression in Chuvash polycythemia. Haematologica, 2017. 102(5): p. e166-e169. Gordeuk, V.R., N.S. Key, and J.T. Prchal, Re-evaluation of hematocrit as a determinant of thrombotic risk in erythrocytosis. Haematologica, 2019. 104(4): p. 653-658. Disclosures Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy.