Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.
Interleukin-8 appears to be associated with development of radicular pain by back extension and to be activated on acute or subacute disc herniations. IL-8 seems to participate in the pathomechanism of nerve root inflammation in lumbar disc herniations, which implies that it may be considered a target for therapeutic intervention.
The ability of Listeria monocytogenes to invade non-phagocytic cells is important for development of a systemic listeriosis infection. The authors previously reported that a L. monocytogenes DsigB strain is defective in invasion into human intestinal epithelial cells, in part, due to decreased expression of a major invasion gene, inlA. To characterize additional invasion mechanisms under the control of s B , mutants were generated carrying combinations of in-frame deletions in inlA, inlB and sigB. Quantitative assessment of bacterial invasion into the human enterocyte Caco-2 and hepatocyte HepG-2 cell lines demonstrated that s B contributes to both InlA and InlB-mediated invasion of L. monocytogenes. Previous identification of the s B -dependent P2 prfA promoter upstream of the major virulence gene regulator, positive regulatory factor A (PrfA), suggested that the contributions of s B to expression of various virulence genes, including inlA, could be at least partially mediated through PrfA. To test this hypothesis, relative invasion capabilities of DsigB and DprfA strains were compared. Exponential-phase cells of the DsigB and DprfA strains were similarly defective at invasion; however, stationary-phase DsigB cells were significantly less invasive than stationary-phase DprfA cells, suggesting that the contributions of s B to invasion extend beyond those mediated through PrfA in stationary-phase L. monocytogenes. TaqMan quantitative reverse-transcriptase PCRs further demonstrated that expression of inlA and inlB was greatly increased in a s B -dependent manner in stationary-phase L. monocytogenes. Together, results from this study provide strong biological evidence of a critical role for s B in L. monocytogenes invasion into non-phagocytic cells, primarily mediated through control of inlA and inlB expression.
Recently, matrix metalloproteinases (MMPs) are emerging as important molecules in neuroinflammation as well as neuronal cell death. However, the role of MMPs in activated microglia remains unclear. In the present study, we found that expressions of MMP‐1, ‐3, ‐8 and ‐9 were significantly induced by single or combined treatment of immunostimulants lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) in primary cultured microglia and BV2 microglial cells. Inhibition of MMP‐3 or ‐9 significantly suppressed the expression of iNOS and pro‐inflammatory cytokines and the activities of NF‐κB, AP‐1, and MAPK in LPS‐stimulated microglia. The results suggest that MMP‐3 and ‐9 both mediate LPS‐induced inflammatory reactions. Inhibition of reactive oxygen species (ROS) by N‐acetyl‐cysteine or diphenylene iodonium significantly suppressed the expression of MMP‐3, MMP‐9, NO and TNF‐α in LPS‐stimulated microglia, suggesting that ROS is an early signaling inducer in LPS‐stimulated microglial cells. MMP inhibitors also suppressed ROS production, suggesting a cross‐talk between ROS and MMPs. Collectively, the present study demonstrates that MMP‐3 and MMP‐9 play a role as inflammatory mediators in activated microglia. Pharmacological intervention of MMPs especially MMP‐3 and ‐9 would be a therapeutic strategy for the treatment of inflammatory diseases in the CNS caused by over‐activation of microglial cells.
Dopamine beta-hydroxylase (DBH) catalyzes the conversion of dopamine to noradrenaline and is selectively expressed in noradrenergic and adrenergic neurons and neuroendocrine cells. Recent data from this laboratory showed that a paired-like homeodomain (HD) protein, Phox2a, interacts with the HD-binding site residing within a composite promoter of the human DBH gene, designated domain IV, in a cell-specific manner and directly controls noradrenergic-specific DBH promoter activity. In this report, we demonstrate that three additional protein-binding sites (i.e., domains I, II, and III) between domain IV and the TATA box are critical for intact DBH promoter activity in noradrenergic cells and that they activate DBH transcription in a highly concerted manner. Transient transfection assays of mutant DBH reporter constructs indicated that domain I was active in every cell line tested, whereas domain III was preferentially active in DBH-positive cells. Remarkably, mutation of domain II was associated with inactivation of DBH promoter activity exclusively in DBH-positive cell lines, defining it as another noradrenergic-specific promoter element. The cell-specific profile of the promoter function of these sequence motifs was further supported by in vitro DNA-binding studies and Southwestern analysis. Furthermore, competition and antibody supershift assays show that transcription factors Sp1 and AP2 are the cognate nuclear factors interacting with domains I and III, respectively. Parallel evidence indicates that domain II is another Phox2a-binding site, demonstrating at least two binding sites for this factor in the upstream DBH promoter. Strikingly, four tandem copies of domain II increased the promoter activity of a minimal DBH promoter by 100- to 200-fold in DBH-positive cell lines without compromising cell specificity. Cotransfection of Phox2a-expression vector dramatically increased the activity of the multiple domain II promoter only in DBH-negative cell lines, confirming that domain II is responsive to Phox2a. Collectively, this study emphasizes a critical role of Phox2a as well as its functional synergism with other transcription factors (e.g., CREB, AP2, and Sp1) in transcriptional activation of the DBH gene.
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