Comparison of genomic and transcriptional microbiome analysis in gastric cancer patients and healthy individuals.

BACKGROUND: Helicobacter pylori and the stomach microbiome play a crucial role in gastric carcinogenesis, and detailed characterization of the microbiome is necessary for a better understanding of the pathophysiology of the disease. There are two common modalities for microbiome analysis: DNA (16S rRNA gene) and RNA (16S rRNA transcript) sequencing. The implications from the use of one or another sequencing approach on the characterization and comparability of the mucosal microbiome in gastric cancer (GC) are poorly studied. AIM: To characterize the microbiota of GC using 16S rRNA gene and its transcript and determine difference in the bacterial composition. METHODS: In this study, 316 DNA and RNA samples extracted from 105 individual stomach biopsies were included. The study cohort consisted of 29 healthy control individuals and 76 patients with GC. Gastric tissue biopsy samples were collected from damaged mucosa and healthy mucosa at least 5 cm from the tumor tissue. From the controls, healthy stomach mucosa biopsies were collected. From all biopsies RNA and DNA were extracted. RNA was reverse transcribed into cDNA. V1-V2 region of bacterial 16S rRNA gene from all samples were amplified and sequenced on an Illumina MiSeq platform. Bray-Curtis algorithm was used to construct sample-similarity matrices abundances of taxonomic ranks in each sample type. For significant differences between groups permutational multivariate analysis of variance and Mann-Whitney test followed by false-discovery rate test were used. RESULTS: Microbial analysis revealed that only a portion of phylotypes (18%-30%) overlapped between microbial profiles obtained from DNA and RNA samples. Detailed analysis revealed differences between GC and controls depending on the chosen modality, identifying 17 genera at the DNA level and 27 genera at the RNA level. Ten of those bacteria were found to be different from the control group at both levels. The key taxa showed congruent results in various tests used; however, differences in 7 bacteria taxa were found uniquely only at the DNA level, and 17 uniquely only at the RNA level. Furthermore, RNA sequencing was more sensitive for detecting differences in bacterial richness, as well as differences in the relative abundance of Reyranella and Sediminibacterium according to the type of GC. In each study group (control, tumor, and tumor adjacent) were found differences between DNA and RNA bacterial profiles. CONCLUSION: Comprehensive microbial study provides evidence for the effect of choice of sequencing modality on the microbiota profile, as well as on the identified differences between case and control.

Microbial composition of tumorous and adjacent gastric tissue is associated with prognosis of gastric cancer.

Helicobacter pylori (H. pylori) infection has been considered as the main causal factor in gastric carcinogenesis, but other bacterial species may also play an important role in pathophysiology of gastric cancer. The aim of the study was to explore the link between gastric cancer prognosis and the mucosal microbial community in tumorous and adjacent gastric tissue. The bacterial profile was analysed using 16S sequencing (V1-V2 region). Microbial differences were mostly characterized by lower relative abundances of H. pylori in tumorous gastric tissues. Bacterial community and outcome data analysis revealed the genus Fusobacterium and Prevotella significantly associated with worse overall survival in gastric cancer patients. In particular, Fusobacterium was associated with significant increase in hazard ratio in both univariable and multivariable analysis and independently validated using TCMA data. Phylogenetic biodiversity of Fusobacterium species in the stomach revealed F. periodonticum as the most prevalent in healthy subjects, while F. nucleatum was most abundant in patients with gastric cancer. Bacterial community network analysis in gastric cancer suggests substantial complexity and a strong interplay between F. nucleatum and Prevotella. In summary, mucosal microbial community in the stomach was associated with worse overall survival in gastric cancer patients. Strongest negative impact on prognosis was linked to the abundance of F. nucleatum in tumorous specimens, suggesting its translational relevance in management of gastric cancer patients.

Characterization of Maladaptive Processes in Acute, Chronic and Remission Phases of Experimental Colitis in C57BL/6 Mice.

Inflammatory bowel disease (IBD) is a chronic recurrent inflammatory disease with unknown etiology. Dextran sulfate sodium (DSS) induced colitis is a widely used mouse model in IBD research. DSS colitis involves activation of the submucosal immune system and can be used to study IBD-like disease characteristics in acute, chronic, remission and transition phases. Insight into colon inflammatory parameters is needed to understand potentially irreversible adaptations to the chronification of colitis, determining the baseline and impact of further inflammatory episodes. We performed analyses of non-invasive and invasive colitis parameters in acute, chronic and remission phases of the DSS colitis in C57BL/6 mice. Non-invasive colitis parameters poorly reflected inflammatory aspects of colitis in chronic remission phase. We found invasive inflammatory parameters, positively linked to repeated DSS-episodes, such as specific colon weight, inflamed colon area, spleen weight, absolute cell numbers of CD4+ and CD8+ T cells as well as B cells, blood IFN-γ level, colonic chemokines BLC and MDC as well as the prevalence of Turicibacter species in feces. Moreover, microbial Lactobacillus species decreased with chronification of disease. Our data point out indicative parameters of recurrent gut inflammation in context of DSS colitis.

Primers matter: Influence of the primer selection on human fungal detection using high throughput sequencing.

Microbiota research has received an increasing attention for its role in disease development and fungi are considered as one of the key players in the microbial niche. Various sequencing approaches have been applied to uncover the role of fungal community in health and disease; however, little is known on the performance of various primers and comparability between the studies. Motivated by the recent publications, we performed a systematic comparison of the 18S and ITS regions to identify the impact of various primers on the sequencing results. Using four pairs of primers extensively used in literature, fungal community was retrieve from 25 fecal samples, and applying high throughput sequencing; and the results were compared in order to select the most suitable primers for fungal detection in human fecal samples. Considering the high variability between samples, primers described in the Earth microbiome project detected the broadest fungal spectrum suggesting its superior performance in mycobiome research.

Gut microbial similarity in twins is driven by shared environment and aging.

BACKGROUND: Human gut microbiome composition is influenced by genetics, diet and environmental factors. We investigated the microbial composition in several gastrointestinal (GI) compartments to evaluate the impact of genetics, delivery mode, diet, household sharing and aging on microbial similarity in monozygotic and dizygotic twins. METHODS: Fecal, biopsy and saliva samples were obtained from total 108 twins. DNA and/or RNA was extracted and the region V1-V2 of the 16S rRNA gene was amplified and sequenced. Bray-Curtis similarity was used for further microbiome comparisons, Mann-Whitney test was applied to evaluate the significant differences between groups and Spearman test was applied to reveal potential correlations between data. FINDINGS: The global bacterial profiles were grouped into two clusters separating the upper and lower GI. The upper GI microbiome composition was strictly dependent on the Helicobacter pylori status. With a positivity rate of 55%, H. pylori completely colonized the stomach and separated infected twins from non-infected twins irrespective of zygosity status. Lower GI microbiome similarity between the twins was defined mainly by household-sharing and aging; whereas delivery mode and host genetics had no influence. There was a progredient decrease in the bacterial similarity with aging. Shared vs. non-shared phylotypes analysis showed that in both siblings the shared phylotypes progressively diminished with aging, while the non-shared phylotypes increased. INTERPRETATION: Our findings strongly highlight the aging and shared household as they key determinants in gut microbial similarity and drift in twins irrespective of their zygotic state. FUNDING: This work was supported by the grant of the Research Council of Lithuania (Project no. APP-2/2016) and also partially supported by the funds of European Commission through the "European funds for regional development" (EFRE) as well as by the regional Ministry of Economy, Science and Digitalization as part of the "LiLife" Project as part of the "Autonomy in old Age" research group (Project ID: ZS/2018/11/95324).

A new method for the preparation of non-terminal alkynes: application to the total syntheses of tulearin†A and C.

Lactones are known to react with the reagent generated in situ from CCl4 and PPh3 in a Wittig-type fashion to give gem-dichloro-olefin derivatives. Such compounds are now shown to undergo reductive alkylation on treatment with organolithium reagents RLi to furnish acetylene derivatives bearing the substituent R at their termini (R=Me, n-, sec-, tert-alkyl, silyl); the reaction can be catalyzed with either Cu(acac)2 or Fe(acac)3 /1,2-diaminobenzene. Two alkynol derivatives prepared in this way from readily accessible lactone precursors served as the key building blocks for the total syntheses of the cytotoxic marine macrolides tulearin A (1) and C (2). The assembly of these fragile targets hinged upon ring closing alkyne metathesis (RCAM) followed by a formal trans-reduction of the resulting cycloalkynes via trans-hydrosilylation/protodesilylation.

Assignment of relative configuration of desoxypropionates by 1H NMR spectroscopy: method development, proof of principle by asymmetric total synthesis of xylarinic acid A and applications.

The determination of the relative configuration of 1,3-dimethyl-substituted alkyl chains is possible by interpretation of (1)H NMR shift differences. Additionally, assignments are feasible in a variety of deuterated solvents, because the corresponding shift differences are not significantly influenced by the solvent. The trends for Δδ values depending on functional groups adjacent to the stereogenic centers are shown. Based on a thorough comparison with literature data, the relative configuration of natural products can be predicted. For this purpose, we derived an empirical rule for the ranges in which Δδ values usually occur. Furthermore, we were able to proof the validity of our method by the successful prediction of the relative configuration for the polyketide natural product xylarinic acid A, which was confirmed by the asymmetric total synthesis of its enantiomer. Based on the proposed simple analysis of published (1)H NMR data and the determination of the relevant chemical-shift differences, we predicted the relative configurations of several previously unassigned natural products.

Enantioselective total synthesis of the unnatural and the natural stereoisomers of vittatalactone.

The striped cucumber beetle, Acalymma vittatum, is a cause of major damage to cucurbit crops in North America. To develop an environmentally benign plant protection strategy, recent research has focused on identifying sex pheromones of the cucumber beetle. In this context, we developed the asymmetric total synthesis of the aggregation pheromone of A. vittatum, Vittatalactone, to determine its absolute configuration and to further examine the pheromone response in field studies. The synthesis features an enzyme-catalyzed approach toward the deoxypropionate structural motif. A preformed organocopper reagent could then be coupled in an enantioselective o-DPPB-directed allylic substitution with a functionalized o-DPPB-ester. By means of this efficient transformation, Vittatalactone could be obtained following a highly convergent synthetic process.

Palladium-catalyzed asymmetric allylic alkylation of 2-acylimidazoles as ester enolate equivalents.

A broad range of highly enantioenriched 2-acylimidazoles are synthesized by palladium-catalyzed decarboxylative asymmetric allylic alkylation (DAAA) of 2-imidazolo-substituted enol carbonates. The enantioenriched 2-acylimidazole products can easily be converted to the corresponding carboxylic acid, ester, amide, and ketone derivatives with complete retention of the enantiopurity. The synthetic utility of this new method is demonstrated in the short, efficient synthesis of cetiedil.