Acute hypoxia reduces plasma myostatin independent of hypoxic dose

Background: Muscle atrophy is seen ~ 25 % of patients with cardiopulmonary disorders, such as chronic obstructive pulmonary disorder and chronic heart failure. Multiple hypotheses exist for this loss, including inactivity, inflammation, malnutrition and hypoxia. Healthy individuals exposed to chronic hypobaric hypoxia also show wasting, suggesting hypoxia alone is sufficient to induce atrophy. Myostatin regulates muscle mass and may underlie hypoxic-induced atrophy. Our previous work suggests a decrease in plasma myostatin and increase in muscle myostatin following 10 hours of exposure to 12 % O2. Aims: To establish the effect of hypoxic dose on plasma myostatin concentration. Concentration of plasma myostatin following two doses of normobaric hypoxia (10.7 % and 12.3 % O2) in a randomised, single-blinded crossover design (n = 8 lowlanders, n = 1 Sherpa), with plasma collected pre (0 hours), post (2 hours) and 2 hours following (4 hours) exposure. Results: An effect of time was noted, plasma myostatin decreased at 4 hours but not 2 hours relative to 0 hours (p = 0.01; 0 hours = 3.26 [0.408] ng.mL-1, 2 hours = 3.33, [0.426] ng.mL-1, 4 hours = 2.92, [0.342] ng.mL-1). No difference in plasma myostatin response was seen between hypoxic conditions (10.7 % vs. 12.3 % O2). Myostatin reduction in the Sherpa case study was similar to the lowlander cohort. Conclusions: Decreased myostatin peptide expression suggests hypoxia in isolation is sufficient to challenge muscle homeostasis, independent of confounding factors seen in chronic cardiopulmonary disorders, in a manner consistent with our previous work. Decreased myostatin peptide may represent flux towards peripheral muscle, or a reduction to protect muscle mass. Chronic adaption to hypoxia does not appear to protect against this response, however larger cohorts are needed to confirm this. Future work will examine tissue changes in parallel with systemic effects.

Genomics and virulence factors of Fusobacterium necrophorum

Fusobacterium necrophorum, a Gram negative, anaerobic bacterium, is a common cause of acute pharyngitis and tonsillitis and a rare cause of more severe infections of the head and neck. At the beginning of the project, there was no available genome sequence for F. necrophorum. The aim of this project was to sequence the F. necrophorum genome and identify and study its putative virulence factors contained using in silico and in vitro analysis. Type strains JCM 3718 and JCM 3724,F. necrophorum subspecies necrophorum (Fnn) and funduliforme (Fnf), respectively, and strain ARU 01 (Fnf), isolated from a patient with LS, were commercially sequenced by Roche 454 GS-FLX+ next generation sequencing and assembled into contigs using Roche GS Assembler. Sequence data was annotated semi-automatically, using the xBASE pipeline, BLASTp and Pfam. The F. necrophorum genome was determined to be approximately 2.1 – 2.3 Mb in size, with an estimated 1,950 ORFs and includes genes for a leukotoxin, ecotin, haemolysin, haemagglutinin, haemin receptor, adhesin and type Vb and Vc secretion systems. The prevalence of the leukotoxin gene was investigated in strains JCM 3718, JCM 3724 and ARU 01, as well as a clinical collection of 25 Fnf strains, identified using biochemical and molecular tests. The leukotoxin operon was found to be universal within the strain collection by PCR. HL-60 cells subjected to aliquots of concentrated high molecular weight culture supernatant, predicted to contain the secreted leukotoxins of strains JCM 3718, JCM 3724 and ARU 01, were killed in a dose-dependent manner. The cytotoxic effect of the leukotoxin against human donor white blood cells was also tested to validate the HL-60 assay. The differences in the results between the two assays were not statistically significant. Ecotin, a serine protease inhibitor, was found to be present in 100 % of the strain collection and had a highly conserved sequence with primary and secondary binding sites exposed on opposing sides of the protein. During enzyme inhibition studies, a purified recombinant F. necrophorum ecotin protein inhibited human neutrophil elastase, a protease that degrades bacteria at inflammation sites, and human plasma kallikrein, a component of the host clotting cascade. The recombinant ecotin also prolonged human plasma clotting times by up to 7-fold for the extrinsic pathway, and up to 40-fold for the intrinsic pathway. The genome sequence data provides important information about F. necrophorum type strains and enables comparative study between strains and subspecies. Results from the leukotoxin and ecotin assays can be used to build up an understanding of how the organism behaves during infection.