The integrity of transplanted mesenchymal stem cells (MSCs) for cardiac regeneration is dependent on cell-cell or cell-matrix adhesion, which is inhibited by reactive oxygen species (ROS) generated in ischemic surroundings after myocardial infarction. Intracellular ROS play a key role in the regulation of cell adhesion, migration, and proliferation. This study was designed to investigate the role of ROS on MSC adhesion. In H 2 O 2 treated MSCs, adhesion and spreading were inhibited and detachment was increased in a dose-dependent manner, and these effects were significantly rescued by co-treatment with the free radical scavenger, N-acetyl-L-cysteine (NAC, 1 mM). A similar pattern was observed on plates coated with different matrices such as fibronectin and cardiogel. Hydrogen peroxide treatment resulted in a marked decrease in the level of focal adhesion-related molecules, such as phospho-FAK and p-Src in MSCs. We also observed a significant decrease in the integrin-related adhesion molecules, aV and b1, in H 2 O 2 treated MSCs. When injected into infarcted hearts, the adhesion of MSCs co-injected with NAC to the border region was significantly improved. Consequently, we observed that fibrosis and infarct size were reduced in MSC and NAC-injected rat hearts compared to in MSC-only injected hearts. These results indicate that ROS inhibit cellular adhesion of engrafted MSCs and provide evidence that the elimination of ROS might be a novel strategy for improving the survival of engrafted MSCs.

Melanoma is one of the deadliest cancers, yet the cells of origin and mechanisms of tumor initiation remain unclear. The majority of melanomas emerge from clear skin without a precursor lesion, but it is unknown whether these melanomas can arise from melanocyte stem cells (MCSCs). Here we employ mouse models to define the role of MCSCs as melanoma cells of origin, demonstrate that MCSC quiescence acts as a tumor suppressor, and identify the extrinsic environmental and molecular factors required for the critical early steps of melanoma initiation. Specifically, melanomas originate from melanoma-competent MCSCs upon stimulation by UVB, which induces MCSC activation and translocation via an inflammation-dependent process. Moreover, the chromatin-remodeling factor Hmga2 in the skin plays a critical role in UVB-mediated melanomagenesis. These findings delineate melanoma formation from melanoma-competent MCSCs following extrinsic stimuli, and they suggest that abrogation of Hmga2 function in the microenvironment can suppress MCSC-originating cutaneous melanomas.

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BackgroundTumour-derived extracellular vesicles (EVs) play a role in tumour progression; however, the spectrum of molecular mechanisms regulating EV secretion and cargo selection remain to be fully elucidated. We have reported that cavin-1 expression in prostate cancer PC3 cells reduced the abundance of a subset of EV proteins, concomitant with reduced xenograft tumour growth and metastasis.MethodsWe examined the functional outcomes and mechanisms of cavin-1 expression on PC3-derived EVs (PC3-EVs).ResultsPC3-EVs were internalized by osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs) in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. Strikin gly, EVs derived from cavin-1-expressing PC3 cells (cavin-1-PC3-EVs) failed to induce multinucleate osteoblasts or hOB proliferation. Cavin-1 was not detected in EVs, indicating an indirect mechanism of action. EV morphology, size and quantity were also not affected by cavin-1 expression, suggesting that cavin-1 modulated EV cargo recruitment rather than release. While cavin-1-EVs had no osteoclastogenic function, they were internalized by RAW264.7 cells but at a reduced efficiency compared to control EVs. EV surface proteins are required for internalization of PC3-EVs by RAW264.7 cells, as proteinase K treatment abolished uptake of both control and cavin-1-PC3-EVs. Removal of sialic acid modifications by neuraminidase treatment increased the amount of control PC3-EVs internalized by RAW264.7 cells, without affecting cavin-1-PC3-EVs. This suggests that cavin-1 expression altered the glycosylation modifications on PC3-EV surface. Finally, cavin-1 expression did not affect EV in vivo tissue targeting as both control and cavin-1-PC3-EVs were predominantly retained in the lung and bone 24 hours after injection into mice.DiscussionTaken together, our results reveal a novel pathway for EV cargo sorting, and highlight the potential of utilizing cavin-1-mediated pathways to attenuate metastatic prostate cancer.

is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in -mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.

ObjectivesThe purpose of this study was to evaluate the antiarrhythmic potential of mesenchymal stem cells (MSC) under a different environment.

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Low-nutrient media and long incubation times facilitated the cultivation of 20 taxonomically diverse Gram-negative marine bacteria within the phyla Bacteroidetes and Proteobacteria. These strains comprise as many as three new families and include members of clades that had only been observed using culture-independent techniques. Chemical studies of the type strains representing two new families within the order Cytophagales led to the isolation of nine new alkaloid secondary metabolites that can be grouped into four distinct structure classes, including azepinones, aziridines, quinolones, and pyrazinones. Several of these compounds possess antibacterial properties and appear, on structural grounds, to be produced by amino acid-based biosynthetic pathways. Our results demonstrate that relatively simple cultivation techniques can lead to the isolation of new bacterial taxa that are capable of the production of alkaloid secondary metabolites with antibacterial activities. These findings support continued investment in cultivation techniques as a method for natural product discovery.