Generation of an OMgp allelic series in mice

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin‐derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type‐specific roles of OMgp in CNS injury‐induced axonal plasticity. genesis 47:751–756, 2009. © 2009 Wiley‐Liss, Inc.     

TNF-α-induced exosomal miR-146a mediates mesenchymal stem cell-dependent suppression of urethral stricture

The effective treatment of urethral stricture remains a medical problem. The use of proinflammatory cytokines as stimuli to improve the reparative efficacy of mesenchymal stem cells (MSCs) towards damaged tissues represents an evolving field of investigation. However, the therapeutic benefits of this strategy in the treatment of urethral stricture remain unknown. Here, we enriched exosomes derived from human umbilical cord-derived MSCs pretreated with or without tumor necrosis factor alpha (TNF-α) to evaluate their therapeutic effects in an in vivo model of TGFβ1-induced urethral stricture. Male Sprague-Dawley rats received sham (saline) or TGFβ1 injections to urethral tissues followed by incisions in the urethra. Animals in the TGFβ1 injection (urethral fibrosis) cohort were subsequently injected with vehicle control, or with exosomes derived from MSCs cultured with or without TNF-α. After 4 weeks, rats underwent ultrasound evaluation and, following euthanasia, urethral tissues were harvested for histological and molecular analysis. In vitro, the effects of MSC-derived exosomes on fibroblast secretion of collagen and cytokines were studied by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. Exosomes derived from MSCs pretreated with TNF-α were more effective in suppressing urethral fibrosis and stricture than exosomes from untreated MSCs. We found that miR-146a, an anti-inflammatory miRNA, was strongly upregulated in TNF-α-stimulated MSCs and was selectively packaged into exosomes. Moreover, miR-146a-containing exosomes were taken up by fibroblasts and inhibited fibroblast activation and associated inflammatory responses, a finding that may underlie the therapeutic mechanism for suppression of urethral stricture. Inhibition of miR-146a in TNF-α-treated MSCs partially reduced antifibrotic effects and increased the release of proinflammatory factors of exosomes derived from these cells. Together these findings demonstrate that exosomes derived from TNF-α-treated MSCs are of therapeutic benefit in urethral fibrosis, suggesting that this strategy may have utility as an adjuvant therapy in the treatment of urethral stricture diseases.     

Muon disrupts AKT hydrogen bond network in cancer

A cancer microenvironment generates strong hydrogen bond network system by the positive feedback loops supporting cancer complexity and robustness. Such network functions through the AKT locus generating high entropic energy supporting cancer metastatic robustness. Charged lepton particle muon follows the rule of Bragg effect during a collision with hydrogen network in cancer cells. Muon beam dismantles hydrogen bond network in cancer by the muon-catalyzed fusion, leading to apoptosis of cancer cells. Muon induces cumulative energy appearance on the hydrogen bond network in a cancer cell with its fast decay to an electron and two neutrinos. Thus, muon beam, muonic atom, muon neutrino shower, and electrons simultaneously cause fast neutralization of the AKT hydrogen bond network by the conversion of hydrogen into deuterium or helium, inactivating the hydrogen bond networks and inducing failure of cancer complexity and robustness with the disappearance of a malignant phenotype.     

Amyloid-beta induces chemotaxis and oxidant stress by acting at formylpeptide receptor 2, a G protein-coupled receptor expressed in phagocytes and brain

Amyloid-beta, the pathologic protein in Alzheimer's disease, induces chemotaxis and production of reactive oxygen species in phagocytic cells, but mechanisms have not been fully defined. Here we provide three lines of evidence that the phagocyte G protein-coupled receptor (N-formylpeptide receptor 2 (FPR2)) mediates these amyloid-beta-dependent functions in phagocytic cells. First, transfection of FPR2, but not related receptors, including the other known N-formylpeptide receptor FPR, reconstituted amyloid-beta-dependent chemotaxis and calcium flux in HEK 293 cells. Second, amyloid-beta induced both calcium flux and chemotaxis in mouse neutrophils (which express endogenous FPR2) with similar potency as in FPR2-transfected HEK 293 cells. This activity could be specifically desensitized in both cell types by preincubation with a specific FPR2 agonist, which desensitizes the receptor, or with pertussis toxin, which uncouples it from G(i)-dependent signaling. Third, specific and reciprocal desensitization of superoxide production was observed when N-formylpeptides and amyloid-beta were used to sequentially stimulate neutrophils from FPR -/- mice, which express FPR2 normally. Potential biological relevance of these results to the neuroinflammation associated with Alzheimer's disease was suggested by two additional findings: first, FPR2 mRNA could be detected by PCR in mouse brain; second, induction of FPR2 expression correlated with induction of calcium flux and chemotaxis by amyloid-beta in the mouse microglial cell line N9. Further, in sequential stimulation experiments with N9 cells, N-formylpeptides and amyloid-beta were able to reciprocally cross-desensitize each other. Amyloid-beta was also a specific agonist at the human counterpart of FPR2, the FPR-like 1 receptor. These results suggest a unified signaling mechanism for linking amyloid-beta to phagocyte chemotaxis and oxidant stress in the brain.     

Two bacteria causing farmer's lung: Fine structure ofThermoactinomyces vulgaris andSaccharopolyspora rectivirgula

Larval cuticle ofHelicoverpa (Heliothis)zea and yeast extract added to a minimal medium (MM) induced germination of conidia ofNomuraea rileyi whereas sterile distilled water or MM alone did not. Yeast extract increased mycelial yield, but when cuticle was added, mycelial yield significantly decreased. Proteases and chitinases ofN. rileyi were only expressed when cuticle was added to the MM.This article reports the results of research only. Mention of a proprietary product in this paper does not constitute a recommendation for use by US Department of Agriculture.     

Histopathological studies ofSporothrix schenckii-inoculated mice

Defense mechanisms againstSporothrix schenckii were studied using mouse models. After an intracutaneous injection of the yeast form ofS. schenckii to the dorsal skin of the congenitally athymic nude and normal heterozygote littermate mice, nodules were formed. They regressed and disappeared in 10 weeks in the case of normal mice. On the other hand, nodules and then ulceration developed progressively in nude mice until all animals expired by dissemination of microorganisms at the 11th week of inoculation. Histopathologically the migrated cells were similar in both the normal and the nude mice, particularly during the early phase (within 24 h), with infiltration by PMNs being predominant. Fragmentation ofS. schenckii commenced early during the 12–24 h stage of inoculation in the normal mice, while such fragmentation was scarce in nude mice even though numerous PMNs accumulated. Microscopic observations in the early stages (within 24 h of inoculation) suggested that the lack of killing activity by PMNs in nude mice contributes more to the impaired defense than the lack of macrophage activation by T-cells.     

Identification of opsA,a Gene Involved in Solute Stress Mitigation and Survival in Soil,in the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Novosphingobium sp. Strain LH128

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.     

Total Synthesis of Septocylindrin B and C-Terminus Modified Analogues

The total synthesis is reported of the peptaibol Septocylindrin B which is related to the well documented channel forming peptaibol antibiotic Alamethicin. Several analogues were synthesized with a modified C-terminus, to investigate the SAR of the terminal residue Phaol. All these peptides were tested for their membrane perturbation properties by fluorescent dye leakage assay and for their antibacterial activity.