Gastric microbial community profiling reveals a dysbiotic cancer-associated microbiota

Objective Gastric carcinoma development is triggered by Helicobacter pylori. Chronic H. pylori infection leads to reduced acid secretion, which may allow the growth of a different gastric bacterial community. This change in the microbiome may increase aggression to the gastric mucosa and contribute to malignancy. Our aim was to evaluate the composition of the gastric microbiota in chronic gastritis and in gastric carcinoma. Design The gastric microbiota was retrospectively investigated in 54 patients with gastric carcinoma and 81 patients with chronic gastritis by 16S rRNA gene profiling, using next-generation sequencing. Differences in microbial composition of the two patient groups were assessed using linear discriminant analysis effect size. Associations between the most relevant taxa and clinical diagnosis were validated by real-time quantitative PCR. Predictive functional profiling of microbial communities was obtained with PICRUSt. Results The gastric carcinoma microbiota was characterised by reduced microbial diversity, by decreased abundance of Helicobacter and by the enrichment of other bacterial genera, mostly represented by intestinal commensals. The combination of these taxa into a microbial dysbiosis index revealed that dysbiosis has excellent capacity to discriminate between gastritis and gastric carcinoma. Analysis of the functional features of the microbiota was compatible with the presence of a nitrosating microbial community in carcinoma. The major observations were confirmed in validation cohorts from different geographic origins. Conclusions Detailed analysis of the gastric microbiota revealed for the first time that patients with gastric carcinoma exhibit a dysbiotic microbial community with genotoxic potential, which is distinct from that of patients with chronic gastritis.This research was supported by a Worldwide Cancer Research grant to CF and JCM (Reference 16-1352). RMF, JPM and IPR have fellowships from Fundacao para a Ciencia e a Tecnologia (FCT; SFRH/BPD/84084/2012, PD/BD/114014/2015 and SFRH/BD/110803/2015, respectively) through Programa Operacional Capital Humano (POCH) and the European Union. JPM's fellowship is in the framework of FCT's PhD Programme BiotechHealth (Ref PD/0016/2012). i3S-Instituto de Investigacao e Inovacao em Saude is funded by Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through Fundacao para a Ciencia e a Tecnologia (FCT)/Ministerio da Ciencia, Tecnologia e Inovacao (POCI-01-0145-FEDER-007274)

Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of helicobacter pylori clarithromycin resistance in gastric biopsy specimens

Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin- resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach

The lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes

Bergantin LB, Figueiredo LB, Godinho RO. the lumbrical muscle: a novel in situ system to evaluate adult skeletal muscle proteolysis and anticatabolic drugs for therapeutic purposes. J Appl Physiol 111: 1710-1718, 2011. First published September 15, 2011; doi:10.1152/japplphysiol.00586.2011.-The molecular regulation of skeletal muscle proteolysis and the pharmacological screening of anticatabolic drugs have been addressed by measuring tyrosine release from prepubertal rat skeletal muscles, which are thin enough to allow adequate in vitro diffusion of oxygen and substrates. However, the use of muscle at accelerated prepubertal growth has limited the analysis of adult muscle proteolysis or that associated with aging and neurodegenerative diseases. Here we established the adult rat lumbrical muscle (4/hindpaw; 8/rat) as a new in situ experimental model for dynamic measurement of skeletal muscle proteolysis. By incubating lumbrical muscles attached to their individual metatarsal bones in Tyrode solution, we showed that the muscle proteolysis rate of adult and aged rats (3-4 to 24 mo old) is 45-25% of that in prepubertal animals (1 mo old), which makes questionable the usual extrapolation of proteolysis from prepubertal to adult/senile muscles. While acute mechanical injury or 1- to 7-day denervation increased tyrosine release from adult lumbrical muscle by up to 60%, it was reduced by 20-28% after 2-h incubation with beta-adrenoceptor agonists, forskolin or phosphodiesterase inhibitor IBMX. Using inhibitors of 26S-proteasome (MG132), lysosome (methylamine), or calpain (E64/leupeptin) systems, we showed that ubiquitin-proteasome is accountable for 40-50% of total lumbrical proteolysis of adult, middle-aged, and aged rats. in conclusion, the lumbrical model allows the analysis of muscle proteolysis rate from prepubertal to senile rats. By permitting eight simultaneous matched measurements per rat, the new model improves similar protocols performed in paired extensor digitorum longus (EDL) muscles from prepubertal rats, optimizing the pharmacological screening of drugs for anticatabolic purposes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Div Cellular Pharmacol, Dept Pharmacol, Escola Paulista Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Div Cellular Pharmacol, Dept Pharmacol, Escola Paulista Med, BR-04044020 São Paulo, BrazilFAPESP: 05/59006-1FAPESP: 08/55988-2CNPq: 304602/2008-6Web of Scienc

Impact of host DNA and sequencing depth on the taxonomic resolution of whole metagenome sequencing for microbiome analysis

The amount of host DNA poses a major challenge to metagenome analysis. However, there is no guidance on the levels of host DNA, nor on the depth of sequencing needed to acquire meaningful information from whole metagenome sequencing (WMS). Here, we evaluated the impact of a wide range of amounts of host DNA and sequencing depths on microbiome taxonomic profiling using WMS. Synthetic samples with increasing levels of host DNA were created by spiking DNA of a mock bacterial community, with DNA from a mouse-derived cell line. Taxonomic analysis revealed that increasing proportions of host DNA led to decreased sensitivity in detecting very low and low abundant species. Reduction of sequencing depth had major impact on the sensitivity of WMS for profiling samples with 90% host DNA, increasing the number of undetected species. Finally, analysis of simulated datasets with fixed depth of 10 million reads confirmed that microbiome profiling becomes more inaccurate as the level of host DNA increases in a sample. In conclusion, samples with high amounts of host DNA coupled with reduced sequencing depths, decrease WMS coverage for characterization of the microbiome. This study highlights the importance of carefully considering these aspects in the design of WMS experiments to maximize microbiome analyses.This work was supported by European Regional Development Funds (ERDF) funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by FCT – Fundação para a Ciência e a Tecnologia (POCI-01-0145-FEDER-032532). JP-M and IP-R have fellowships from FCT (PD/BD/114014/2015 and SFRH/BD/110803/2015, respectively) through Programa Operacional Capital Humano (POCH) and the European Social Fund. JP-M’s have fellowship from the framework of FCT’s Ph.D. Program Biotech Health (Ref. PD/0016/2012)

DNA-Based Asymmetric Inverse Electron-Demand Hetero-Diels-Alder

International audienceWhile artificial cyclases hold great promise in chemical synthesis, this work presents the first example of a DNA-catalyzed inverse electron-demand hetero-Diels-Alder (IEDHDA) between dihydrofuran and various α,β-unsaturated acyl imidazoles. The resulting fused bicyclic O,O-acetals containing three contiguous stereogenic centers are obtained in high yields (up to 99 %) and excellent diastereo- (up to >99:1 dr) and enantioselectivities (up to 95 % ee) using a low catalyst loading. Most importantly, these results show that the concept of DNA-based asymmetric catalysis can be expanded to new synthetic transformations offering an efficient, sustainable, and highly selective tool for the construction of chiral building blocks

Effect of maternal Schistosoma mansoni infection and praziquantel treatment during pregnancy on Schistosoma mansoni infection and immune responsiveness among offspring at age five years.

INTRODUCTION: Offspring of Schistosoma mansoni-infected women in schistosomiasis-endemic areas may be sensitised in-utero. This may influence their immune responsiveness to schistosome infection and schistosomiasis-associated morbidity. Effects of praziquantel treatment of S. mansoni during pregnancy on risk of S. mansoni infection among offspring, and on their immune responsiveness when they become exposed to S. mansoni, are unknown. Here we examined effects of praziquantel treatment of S. mansoni during pregnancy on prevalence of S. mansoni and immune responsiveness among offspring at age five years. METHODS: In a trial in Uganda (ISRCTN32849447, http://www.controlled-trials.com/ISRCTN32849447/elliott), offspring of women treated with praziquantel or placebo during pregnancy were examined for S. mansoni infection and for cytokine and antibody responses to SWA and SEA, as well as for T cell expression of FoxP3, at age five years. RESULTS: Of the 1343 children examined, 32 (2.4%) had S. mansoni infection at age five years based on a single stool sample. Infection prevalence did not differ between children of treated or untreated mothers. Cytokine (IFNγ, IL-5, IL-10 and IL-13) and antibody (IgG1, Ig4 and IgE) responses to SWA and SEA, and FoxP3 expression, were higher among infected than uninfected children. Praziquantel treatment of S. mansoni during pregnancy had no effect on immune responses, with the exception of IL-10 responses to SWA, which was higher in offspring of women that received praziquantel during pregnancy than those who did not. CONCLUSION: We found no evidence that maternal S. mansoni infection and its treatment during pregnancy influence prevalence and intensity of S. mansoni infection or effector immune response to S. mansoni infection among offspring at age five years, but the observed effects on IL-10 responses to SWA suggest that maternal S. mansoni and its treatment during pregnancy may affect immunoregulatory responsiveness in childhood schistosomiasis. This might have implications for pathogenesis of the disease

Analytical and numerical analyses of the micromechanics of soft fibrous connective tissues

State of the art research and treatment of biological tissues require accurate and efficient methods for describing their mechanical properties. Indeed, micromechanics motivated approaches provide a systematic method for elevating relevant data from the microscopic level to the macroscopic one. In this work the mechanical responses of hyperelastic tissues with one and two families of collagen fibers are analyzed by application of a new variational estimate accounting for their histology and the behaviors of their constituents. The resulting, close form expressions, are used to determine the overall response of the wall of a healthy human coronary artery. To demonstrate the accuracy of the proposed method these predictions are compared with corresponding 3-D finite element simulations of a periodic unit cell of the tissue with two families of fibers. Throughout, the analytical predictions for the highly nonlinear and anisotropic tissue are in agreement with the numerical simulations