Vascular remodeling of the mouse yolk sac requires hemodynamic force

The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a^(–/–)). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sa

Xenotransplantation of Mitochondrial Electron Transfer Enzyme, Ndi1, in Myocardial Reperfusion Injury

A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury

Autophagy Induced by Ischemic Preconditioning is Essential for Cardioprotection

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 × 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5K130R, a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5K130R. To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents—UTP, diazoxide, and ranolazine—for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning

Preconditioning Involves Selective Mitophagy Mediated by Parkin and p62/SQSTM1

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia (sI) in vitro and IPC of hearts to investigate the role of Parkin in mediating cardioprotection ex vivo and in vivo. In HL-1 cells, sI induced Parkin translocation to mitochondria and mitochondrial elimination. IPC induced Parkin translocation to mitochondria in Langendorff-perfused rat hearts and in vivo in mice subjected to regional IPC. Mitochondrial depolarization with an uncoupling agent similarly induced Parkin translocation to mitochondria in cells and Langendorff-perfused rat hearts. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports indicating a role for p62/SQSTM1 in mitophagy, we found that depletion of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to sI. While wild type mice showed p62 translocation to mitochondria and an increase in ubiquitination, Parkin knockout mice exhibited attenuated IPC-induced p62 translocation to the mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection

Entomopathogenic Fungi on Hemiberlesia pitysophila

Hemiberlesia pitysophila Takagi is an extremely harmful exotic insect in forest to Pinus species, including Pinus massoniana. Using both morphological taxonomy and molecular phylogenetics, we identified 15 strains of entomogenous fungi, which belong to 9 genera with high diversities. Surprisingly, we found that five strains that were classified as species of Pestalotiopsis, which has been considered plant pathogens and endophytes, were the dominant entomopathogenic fungus of H. pitysophila. Molecular phylogenetic tree established by analyzing sequences of ribosomal DNA internal transcribed spacer showed that entomopathogenic Pestalotiopsis spp. were similar to plant Pestalotiopsis, but not to other pathogens and endophytes of its host plant P. massoniana. We were the first to isolate entomopathogenic Pestalotiopsis spp. from H. pitysophila. Our findings suggest a potential and promising method of H. pitysophila bio-control

A novel strain D5 isolated from Acacia confusa.

We isolated a novel strain D5 from nodules of Acacia confusa. Under strict sterile conditions the strain could successfully nodulate Acacia confusa, A. crassicarpa and A. mangium, with nitrogenase activity ranging from 18.90 to 19.86 nmol·g(-1)·min(-1). In the phylogenetic tree based on a complete 16S rRNA gene sequence, the sequence of strain D5 shared 99% homology with that of four species of genus Pseudomonas. The 685 bp nodA fragment amplified from strain D5 shared 95% homology with the nodA sequence of 9 species of genus Bradyrhizobium, with a genetic distance of 0.01682. The 740 bp nifH gene fragment was amplified from strain D5. This strain D5 nifH gene and Bradyrhizobium spp. formed a branch, showing 98% homology and a genetic distance of 0. The homology between this branch and the Bradyrhizobium spp. DG in another branch was 99%, with a genetic distance of 0.007906. These results indicate that this strain D5 is a new type of nitrogen-fixing bacterium

Phylogenetic dendrogram of strain D5 <i>nifH</i> gene.

<p>Phylogenetic dendrogram of strain D5 <i>nifH</i> gene.</p

Nodulation of strain D5 inoculated plants.

<p>A. <i>Acacia confusa</i>, B. <i>Acacia crassicarpa</i>, C. <i>Acacia mangium</i>, D. <i>Glycine max,</i> and E. Control.</p

The nodulation rates of strain D5 inoculated <i>Acacia confusa</i>, <i>A. crassicarpa, A. mangium</i>, and <i>Glycine max</i>, and the corresponding nitrogenase activities of root nodules.

<p>The nodulation rates of strain D5 inoculated <i>Acacia confusa</i>, <i>A. crassicarpa, A. mangium</i>, and <i>Glycine max</i>, and the corresponding nitrogenase activities of root nodules.</p