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RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S. epidermidis protein- and PIA-biofilm matrix producers differ with respect to RsaE and metabolic gene expression. RsaE is spatiotemporally expressed within S. epidermidis PIA-mediated biofilms, and its overexpression triggers a PIA biofilm phenotype as well as eDNA release in an S. epidermidis protein biofilm matrix-producing strain background. dRNA-seq and Northern blot analyses revealed RsaE to exist as a major full-length 100-nt transcript and a minor processed species lacking approximately 20 nucleotides at the 5'-end. RsaE processing results in expansion of the mRNA target spectrum. Thus, full-length RsaE interacts with S. epidermidis antiholin-encoding lrgA mRNA, facilitating bacterial lysis and eDNA release. Processed RsaE, however, interacts with the 5'-UTR of icaR and sucCD mRNAs, encoding the icaADBC biofilm operon repressor IcaR and succinyl-CoA synthetase of the tricarboxylic acid (TCA) cycle, respectively. RsaE augments PIA-mediated biofilm matrix production, most likely through activation of icaADBC operon expression via repression of icaR as well as by TCA cycle inhibition and re-progamming of staphylococcal central carbon metabolism towards PIA precursor synthesis. Additionally, RsaE supports biofilm formation by mediating the release of eDNA as stabilizing biofilm matrix component. As RsaE itself is heterogeneously expressed within biofilms, we consider this sRNA to function as a factor favoring phenotypic heterogeneity and supporting division of labor in S. epidermidis biofilm communities. [ABSTRACT FROM AUTHOR]

The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced Arabidopsis thaliana-DNA (A.t.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of A.t.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified A.t.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of A.t.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development. [ABSTRACT FROM AUTHOR]

Introduction: Obliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast differentiation in response to SSc-microenvironment defining growth factors and determined responsible mechanisms. Methods: We studied responses of multipotent MSCs from SSc-patients (SSc-MSCs) and healthy controls (H-MSCs) to long-term exposure to CTGF, b-FGF, PDGF-BB or TGF-β1. Differentiation towards VSMC and myofibroblast lineages was analyzed on phenotypic, biochemical, and functional levels. Intracellular signaling studies included analysis of TGF-β receptor regulation, SMAD, AKT, ERK1/2 and autocrine loops. Results: VSMC differentiation towards both, contractile and synthetic VSMC phenotypes in response to CTGF and b-FGF was disturbed in SSc-MSCs. H-MSCs and SSc-MSCs responded equally to PDGF-BB with prototypic fibroblastic differentiation. TGF-β1 initiated myofibroblast differentiation in both cell types, yet with striking phenotypic and functional differences: In relation to H-MSC-derived myofibroblasts induced by TGF-β1, those obtained from SSc-MSCs expressed more contractile proteins, migrated towards TGF-β1, had low proliferative capacity, and secreted higher amounts of collagen paralleled by reduced MMP expression. Higher levels of TGF-β receptor 1 and enhanced canonical and noncanonical TGF-β signaling in SSc-MSCs accompanied aberrant differentiation response of SSc-MSCs in comparison to H-MSCs. Conclusions: Deregulated VSMC differentiation with a shift towards myofibroblast differentiation expands the concept of disturbed endogenous regenerative capacity of MSCs from SSc patients. Disease related intrinsic hyperresponsiveness to TGF-β1 with increased collagen production may represent one responsible mechanism. Better understanding of repair barriers and harnessing beneficial differentiation processes in MSCs could widen options of autologous MSC application in SSc patients. [ABSTRACT FROM AUTHOR]

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions. [ABSTRACT FROM AUTHOR]

Background: Successful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals. Methods and Findings: Mouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals. Conclusions: Our data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure. [ABSTRACT FROM AUTHOR]