Unveiling the impact of STEC infecting phages on the colon microbiota using an in vitro fermentation model

(Bacterio)phages are considered safe for humans consumption, being regard as excellent biocontrol tools to prevent foodborne pathogens spread. Phages major advantage is their inherent specificity towards a bacterial species, yet some reports have shown phages ability to evolve to infect different hosts when transiting the gastrointestinal tract (GIT). And so, it is of extreme importance to understand the safety outcome of using phages as biocontrol agents in food, with particular interest in the ones that target species from Enterobacteriaceae family, commonly found in the human GIT microbiota. In this study, the impact of a phage infecting Shiga toxin-producing Escherichia coli (STEC), named E. coli phage vB_EcoS_Ace (Ace), towards the colon microbiota was investigated. An in vitro batch fermentation model was used, and the inoculum was the fecal material of three healthy donors. Fermentations metabolome was analyzed through GC and HPLC, and the concentration of both phage Ace and STEC strain were monitored along time (up to 24h). The interference with the gut microbiota composition and functional potential was assessed by shot gun metagenomics. We observed an increase in phage titre only when the host was present, suggesting that there was no other suitable host within the different microbiotas used. Also, the microbiotas composition did not alter when phage Ace was added. Nevertheless, the attenuated version of STEC strain did indeed create some perturbation in the microbiota, which led to different functional potential. This was corroborated by the differences observed for both gas and short chain fatty acid acids dynamics. The microbiotas individuality was an important factor for the observed perturbations. Moreover, phage Ace revealed to be a safe phage when intended to be used as a biocontrol agent for food products. Also, we concluded that the in vitro fermentation model is a reliable, easy, and nonexpensive safety screening methodology for phages.info:eu-repo/semantics/publishedVersio

An in vitro fermentation model to study the impact of bacteriophages targeting shiga toxin-encoding escherichia coli on the colonic microbiota

Lytic bacteriophages are considered safe for human consumption as biocontrol agents against foodborne pathogens, in particular in ready-to-eat foodstuffs. Phages could, however, evolve to infect different hosts when passing through the gastrointestinal tract (GIT). This underlines the importance of understanding the impact of phages towards colonic microbiota, particularly towards bacterial families usually found in the colon such as the Enterobacteriaceae. Here we propose in vitro batch fermentation as model for initial safety screening of lytic phages targeting Shiga toxin-producing Escherichia coli (STEC). As inoculum we used faecal material of three healthy donors. To assess phage safety, we monitored fermentation parameters, including short chain fatty acid production and gas production/intake by colonic microbiota. We performed shotgun metagenomic analysis to evaluate the outcome of phage interference with colonic microbiota composition and functional potential. During the 24h incubation, concentrations of phage and its host were also evaluated. We found the phage used in this study, named E. coli phage vB_EcoS_Ace (Ace), to be safe towards human colonic microbiota, independently of the donors faecal content used. This suggests that individuality of donor faecal microbiota did not interfere with phage effect on the fermentations. However, the model revealed that the attenuated STEC strain used as phage host perturbed the faecal microbiota as based on metagenomic analysis, with potential differences in metabolic output. We conclude that the in vitro batch fermentation model used in this study is a reliable safety screening for lytic phages intended to be used as biocontrol agents.info:eu-repo/semantics/publishedVersio

Three-dimensional self-assembling nanofiber matrix rejuvenates aged/degenerative human tendon stem/progenitor cells

The poor healing capacity of tendons is known to worsen in the elderly. During tendon aging and degeneration, endogenous human tendon stem/progenitor cells (hTSPCs) experience profound pathological changes. Here, we explored a rejuvenation strategy for hTSPCs derived from aged/degenerated Achilles tendons (A-TSPCs) by providing three-dimensional (3D) nanofiber hydrogels and comparing them to young/healthy TSPCs (Y-TSPCs). RADA peptide hydrogel has a self-assembling ability, forms a nanofibrous 3D niche and can be further functionalized by adding RGD motifs. Cell survival, apoptosis, and proliferation assays demonstrated that RADA and RADA/RGD hydrogels support A-TSPCs in a comparable manner to Y-TSPCs. Moreover, they rejuvenated ATSPCs to a phenotype similar to that of Y-TSPCs, as evidenced by restored cell morphology and cytoskeletal architecture. Transmission electron, confocal laser scanning and atomic force microscopies demonstrated comparable ultrastructure, surface roughness and elastic modulus of A- and Y-TSPC-loaded hydrogels. Lastly, quantitative PCR revealed similar expression profiles, as well a significant upregulation of genes related to tenogenesis and multipotency. Taken together, the RADA-based hydrogels exert a rejuvenating effect by recapitulating in vitro specific features of the natural microenvironment of human TSPCs, which strongly indicates their potential to direct cell behaviour and overcome the challenge of cell aging and degeneration in tendon repair.D.D. acknowledges the EU Twinning Grant Achilles (H2020- WIDESPREAD-05-2017-Twinning Grant Nr. 810850). H.Y. thanks for the support of China Scholarship Council (CSC Grant Nr. 201606200072). S.K. and H.C-S. acknowledge the financial support for CANTER by the Bavarian State Ministry for Science and Education. The authors thank Daniela Drenkard for valuable technical assistance and Dr. Girish Pattappa for English proof-readin

Osteoidosis leads to altered differentiation and function of osteoclasts

In patients with osteomalacia, a defect in bone mineralization leads to changed characteristics of the bone surface. Considering that the properties of the surrounding matrix influence function and differentiation of cells, we aimed to investigate the effect of osteoidosis on differentiation and function of osteoclasts. Based on osteomalacic bone biopsies, a model for osteoidosis in vitro (OIV) was established. Peripheral blood mononuclear cells were differentiated to osteoclasts on mineralized surfaces (MS) as internal control and on OIV. We observed a significantly reduced number of osteoclasts and surface resorption on OIV. Atomic force microscopy revealed a significant effect of the altered degree of mineralization on surface mechanics and an unmasking of collagen fibres on the surface. Indeed, coating of MS with RGD peptides mimicked the resorption phenotype observed in OIV, suggesting that the altered differentiation of osteoclasts on OIV might be associated with an interaction of the cells with amino acid sequences of unmasked extracellular matrix proteins containing RGD sequences. Transcriptome analysis uncovered a strong significant up-regulation of transmembrane glycoprotein TROP2 in osteoclastic cultures on OIV. TROP2 expression on OIV was also confirmed on the protein level and found on the bone surface of patients with osteomalacia. Taken together, our results show a direct influence of the mineralization state of the extracellular matrix surface on differentiation and function of osteoclasts on this surface which may be important for the pathophysiology of osteomalacia and other bone disorders with changed ratio of osteoid to bone

The microRNA processing subunit DGCR8 is required for a T cell-dependent germinal center response

We have previously shown that the microRNA (miRNA) processor complex consisting of the RNAse Drosha and the DiGeorge Critical Region (DGCR) 8 protein is essential for B cell maturation. To determine whether miRNA processing is required to initiate T cell-mediated antibody responses, we deleted DGCR8 in maturing B2 cells by crossing a mouse with loxP-flanked DGCR8 alleles with a CD23-Cre mouse. As expected, non-immunized mice showed reduced numbers of mature B2 cells and IgG-secreting cells and diminished serum IgG titers. In accordance, germinal centers and antigen-specific IgG-secreting cells were absent in mice immunized with T-dependent antigens. Therefore, DGCR8 is required to mount an efficient T-dependent antibody response. However, DGCR8 deletion in B1 cells was incomplete, resulting in unaltered B1 cell numbers and normal IgM and IgA titers in DGCR8-knock-out mice. Therefore, this mouse model could be used to analyze B1 responses in the absence of functional B2 cells

Nationale Forschungsdateninfrastruktur für die Ingenieurwissenschaften (NFDI4Ing)

NFDI4Ing ist ein 2017 gegründetes Konsortium mit dem Ziel, Wissenschaftler:innen aller Disziplinen zu ermöglichen, ingenieurwissenschaftliche Forschungsprozesse in ihrer Gesamtheit nachvollziehen oder reproduzieren zu können. Die Besonderheit an NFDI4Ing ist der Aufbau, welcher sich in drei Bereiche aufteilt. Die Archetypen, die an den methodischen Bedarfen ausgerichtet sind, die Community Cluster und die Base Services. NFDI4Ing erarbeitet technologische Methoden und Lösungen, bietet Aus- und Weiterbildungsprogramme und trägt zur Verbreitung des Forschungsdatenmanagements (FDM) in den Ingenieurwissenschaften bei

Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element

A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30-to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74-0.81, p = 3.1 x 10(-31)).Peer reviewe

Genome-wide association study of classical Hodgkin lymphoma identifies key regulators of disease susceptibility

Several susceptibility loci for classical Hodgkin lymphoma (cHL) have been reported, however much of the heritable risk is unknown. Here, we perform a meta-analysis of two existing genome-wide association studies (GWAS), a new GWAS, and replication totalling 5,314 cases and 16,749 controls. We identify risk loci for all cHL at 6q22.33 (rs9482849, P=1.52 × 10-8) and for nodular sclerosis HL (NSHL) at 3q28 (rs4459895, P=9.43 × 10-17), 6q23.3 (rs6928977, P=4.62 × 10-55 11), 10p14 (rs3781093, P=9.49 × 10-13), 13q34 (rs112998813, P=4.58 × 10-8) and 16p13.13 (rs34972832, P=2.12 × 10-8). Additionally, independent loci within the HLA region are observed for NSHL (rs9269081, HLA-DPB1*03:01, Val86 in HLA-DRB1) and mixed cellularity HL (rs1633096, rs13196329, Val86 in HLA-DRB1). The new and established risk loci localise to areas of active chromatin and show an over-representation of transcription factor binding for determinants of B-cell development and immune response.In the United Kingdom, Bloodwise (LLR; 10021) provided principal funding for the study. Support from Cancer Research UK (C1298/A8362 supported by the Bobby Moore Fund) and the Lymphoma Research Trust is also acknowledged. A.S. is supported by a clinical fellowship from Cancer Research UK. For the UK-GWAS, sample and data acquisition were supported by Breast Cancer Now, the European Union and the Lymphoma Research Trust. The UK-GWAS made use of control genotyping data generated by the WTCCC. For further information, please visit the publishr's website

Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma

Genome-wide association studies (GWAS) have transformed our understanding of susceptibility to multiple myeloma (MM), but much of the heritability remains unexplained. We report a new GWAS, a meta-analysis with previous GWAS and a replication series, totalling 9974 MM cases and 247,556 controls of European ancestry. Collectively, these data provide evidence for six new MM risk loci, bringing the total number to 23. Integration of information from gene expression, epigenetic profiling and in situ Hi-C data for the 23 risk loci implicate disruption of developmental transcriptional regulators as a basis of MM susceptibility, compatible with altered B-cell differentiation as a key mechanism. Dysregulation of autophagy/apoptosis and cell cycle signalling feature as recurrently perturbed pathways. Our findings provide further insight