A-GAME: improving the assembly of pooled functional metagenomics sequence data.

BACKGROUND: Expression screening of environmental DNA (eDNA) libraries is a popular approach for the identification and characterization of novel microbial enzymes with promising biotechnological properties. In such "functional metagenomics" experiments, inserts, selected on the basis of activity assays, are sequenced with high throughput sequencing technologies. Assembly is followed by gene prediction, annotation and identification of candidate genes that are subsequently evaluated for biotechnological applications. RESULTS: Here we present A-GAME (A GAlaxy suite for functional MEtagenomics), a web service incorporating state of the art tools and workflows for the analysis of eDNA sequence data. We illustrate the potential of A-GAME workflows using real functional metagenomics data, showing that they outperform alternative metagenomics assemblers. Dedicated tools available in A-GAME allow efficient analysis of pooled libraries and rapid identification of candidate genes, reducing sequencing costs and saving the need for laborious manual annotation. CONCLUSION: In conclusion, we believe A-GAME will constitute a valuable resource for the functional metagenomics community. A-GAME is publicly available at http://beaconlab.it/agame.

Endometrial Cancer: A Pilot Study of the Tissue Microbiota.

BACKGROUND: The endometrium remains a difficult tissue for the analysis of microbiota, mainly due to the low bacterial presence and the sampling procedures. Among its pathologies, endometrial cancer has not yet been completely investigated for its relationship with microbiota composition. In this work, we report on possible correlations between endometrial microbiota dysbiosis and endometrial cancer. METHODS: Women with endometrial cancer at various stages of tumor progression were enrolled together with women with a benign polymyomatous uterus as the control. Analyses were performed using biopsies collected at two specific endometrial sites during the surgery. This study adopted two approaches: the absolute quantification of the bacterial load, using droplet digital PCR (ddPCR), and the analysis of the bacterial composition, using a deep metabarcoding NGS procedure. RESULTS: ddPCR provided the first-ever assessment of the absolute quantification of bacterial DNA in the endometrium, confirming a generally low microbial abundance. Metabarcoding analysis revealed a different microbiota distribution in the two endometrial sites, regardless of pathology, accompanied by an overall higher prevalence of pathogenic bacterial genera in cancerous tissues. CONCLUSIONS: These results pave the way for future studies aimed at identifying potential biomarkers and gaining a deeper understanding of the role of bacteria associated with tumors.

"Good Wine Makes Good Blood": An Integrated Approach to Characterize Autochthonous Apulian Grapevines as Promising Candidates for Healthy Wines.

Wine production represents an ancient human activity and one of the most economically important markets in Europe. Moreover, the health effects of grapes and related products have been largely demonstrated, and mostly depend on their richness in bioactive molecules such as flavonoid and non-flavonoid phenolic compounds. Italy has the highest global wine production and provides one of the richest grapevine germplasm in the Mediterranean area. In this paper, our attention was focused on the evaluation of the total phenol and anthocyanin content in five autochthonous Apulian grapevine cultivars, in both wines and their non-alcoholic extracts. Moreover, the potential antioxidant effects of the non-alcoholic wine extracts on the cell viability of Caco-2 and HeLa carcinoma cell lines were tested. Finally, for the most promising autochthonous selected cultivars (Negramaro, Nero di Troia and Susumaniello), comparative transcriptomic analysis in berries was performed using high-throughput sequencing technology.

Human Endometrial Microbiota at Term of Normal Pregnancies.

The endometrium is a challenging site for metagenomic analysis due to difficulties in obtaining uncontaminated samples and the limited abundance of the bacterial population. Indeed, solid correlations between endometrial physio-pathologic conditions and bacteria compositions have not yet been firmly established. Nevertheless, the study of the endometrial microbiota is of great interest due to the close correlations between microbiota profiles, women's health, and successful pregnancies. In this study, we decided to tackle the study of the endometrial microbiota through analysis of bacterial population in women subjected to elective caesarean delivery. As a pilot study, a cohort of 19 Caucasian women at full term of normal pregnancy and with a prospection of elective caesarean delivery was enrolled for endometrium sampling at the time of caesarean section. Sampling was carried out by endometrial biopsy soon after the delivery of the newborn and the discharge of the placenta and fetal membranes from the uterus. Bacterial composition was established by a deep metabarcoding next generation sequencing (NGS) procedure addressing the V5-V6 hypervariable region of the 16S rRNA gene. Amplicon sequences were analysed by bioinformatic procedures for denoising and taxonomic classification. The RDP database was used as 16S rRNA reference collection. Metabarcoding analysis showed the presence of a common bacterial composition, including six genera classifiable within the human microbiota (Cutibacterium, Escherichia, Staphylococcus, Acinetobacter, Streptococcus, Corynebacterium), that could be part of the core endometrial microbiota under the specific conditions examined. These results can provide useful information for future studies on the correlations between bacteria and successful pregnancies.

Transcription of two sunflower (Helianthus annuus L.) mitochondrial tRNA genes having different genetic origins.

The divergent transcription of two tRNA genes encoded in sunflower mitochondrial DNA, proposed as genes of different genetic origin, has been studied in detail. The transcription initiation site (TIS) for both transcript precursors has been identified by hybridization with in vitro (32)P-capped total RNAs and primer extension. The location of two TISs and the analysis of distribution of sequence elements (motifs) usually present in higher plant mitochondrial promoters led to the identification of two short regions (about 30-40 bp) which can be proposed as the promoters for the transcription of two genes. This conclusion is supported by the observation that within the short intergenic region included between the 5' termini of two genes (1924 bp) the distribution of those specific motifs is unique around the TISs, although not identical for the two promoters. Based on specific experimental results the trnE promoter shows a higher efficiency in comparison with that of the trnH promoter. This result is in good agreement with its structure which strictly conforms to those described for mitochondrial genes of dicot plants. Instead the other promoter shows some divergences which could be responsible for its lower efficiency. The context in which trnH lies in the sunflower mitochondrial genome and other features described in the paper may suggest that, despite the high similarity with the chloroplast counterpart, the trnH gene could have a native origin.

Mapping of 5' and 3'-ends of sunflower mitochondrial nad6 mRNAs reveals a very complex transcription pattern which includes primary transcripts lacking 5'-UTR.

Nad6 orf, encoded in the sunflower mitochondrial genome in single copy contains a non-conserved 3'-extension. The transcription of the nad6 locus generates a highly complex pattern. Three main transcripts of 1240, 960 and 870 nt have been characterized by different approaches. The two smaller ones are apparently the most abundant of the steady-state RNA population and are generated as primary transcription products as well as by processing of the 1240 nt transcript. Their 5'-UTRs are absent or very short. Whereas the 3'-ends of the 960 nt transcripts contain a TGA codon the shorter one terminates at positions excluding the stop codon. The fate of transcripts to promote the synthesis of NAD6 sunflower protein seems, thus, to rely on the occurrence of mechanisms yet to be identified.