The effect of high-load vs. high-repetition training on endurance performance

The purpose of this study was to compare the effects of high-load (H-load) periodized resistance training and high-repetition (H-rep) reverse step loading periodized resistance training on endurance performance. Twenty-six female university rowers (age = 20 +/- 1 year) were randomly assigned to H-load (5 novice, 8 varsity) or H-rep (7 novice, 6 varsity) groups. Subjects were pre- and posttested using a 2,000-m rowing ergometer test. Outcome variables included VO2 peak, time to test completion, total power, average power per stroke, total number of strokes, stroke rate, and body mass. Subjects trained for 8 weeks using identical exercises. Varsity rowers who performed H-load training demonstrated greater improvement compared with those who performed H-rep training. Novice rowers who performed H-rep training demonstrated greater improvement compared with those who performed H-load training. High-load periodized training appears to be more effective for athletes with advanced training status, and H-rep reverse step loading periodized training is more effective for those who are relatively untrained.     

Characterization of Six Bacteriophages of Serratia liquefaciens CP6 Isolated from the Sugar Beet Phytosphere

Six phages (ΦCP6-1 to ΦCP6-6) that are commonly found in the phytosphere of sugar beet (Beta vulgaris var. Amethyst) were investigated, and their relative impacts on their host (Serratia liquefaciens CP6) were compared. There were fundamental differences between the two most abundant predators of CP6 (ΦCP6-1 and ΦCP6-4). Like ΦCP6-2 and ΦCP6-5, ΦCP6-1 belonged to the family Siphoviridae, while ΦCP6-4 exhibited the morphology of the family Podoviridae. The other phages were members of the family Myoviridae. DNA-DNA cross-hybridization revealed that ΦCP6-1 and ΦCP6-4 had little common DNA, although all of the other phages exhibited some genetic similarity. Like ΦCP6-2, ΦCP6-3, and ΦCP6-5, ΦCP6-1 was capable of forming a lysogenic association with its host, while ΦCP6-4 and ΦCP6-6 appeared to be entirely virulent. Single-step growth curve experiments revealed that ΦCP6-4 had a much shorter latent period and a smaller burst size than ΦCP6-1. Also, ΦCP6-1 could transduce a number of host chromosomal markers with transfer frequencies of 2.9 × 10⁻⁹ to 3.9 × 10⁻⁷, whereas ΦCP6-4 could not transduce S. liquefaciens CP6 genes. When viewed in the context of the strikingly different temporal niches of these phages, our data provide an insight into how bacteriophage interactions with their hosts might reflect the natural ecology of bacteriophages. Our data also illustrate how the potential for gene transfer changes over time in an environment that supports several different phages.     

Enhanced control of pathogenic Simian immunodeficiency virus SIVmac239 replication in macaques immunized with an interleukin-12 plasmid and a DNA prime-viral vector boost vaccine regimen

DNA priming has previously been shown to elicit augmented immune responses when administered by electroporation (EP) or codelivered with a plasmid encoding interleukin-12 (pIL-12). We hypothesized that the efficacy of a DNA prime and recombinant adenovirus 5 boost vaccination regimen (DNA/rAd5) would be improved when incorporating these vaccination strategies into the DNA priming phase, as determined by pathogenic simian immunodeficiency virus SIVmac239 challenge outcome. The whole SIVmac239 proteome was delivered in 5 separate DNA plasmids (pDNA-SIV) by EP with or without pIL-12, followed by boosting 4 months later with corresponding rAd5-SIV vaccine vectors. Remarkably, after repeated low-dose SIVmac239 mucosal challenge, we demonstrate 2.6 and 4.4 log reductions of the median SIV peak and set point viral loads in rhesus macaques (RMs) that received pDNA-SIV by EP with pIL-12 compared to the median peak and set point viral loads in mock-immunized controls (P < 0.01). In 5 out of 6 infected RMs, strong suppression of viremia was observed, with intermittent "blips" in virus replication. In 2 RMs, we could not detect the presence of SIV RNA in tissue and lymph nodes, even after 13 viral challenges. RMs immunized without pIL-12 demonstrated a typical maximum of 1.5 log reduction in virus load. There was no significant difference in the overall magnitude of SIV-specific antibodies or CD8 T-cell responses between groups; however, pDNA delivery by EP with pIL-12 induced a greater magnitude of SIV-specific CD4 T cells that produced multiple cytokines. This vaccine strategy is relevant for existing vaccine candidates entering clinical evaluation, and this model may provide insights into control of retrovirus replication.     

Increasing Anaerobic Acetate Consumption and Ethanol Yields in Saccharomyces cerevisiae with NADPH-Specific Alcohol Dehydrogenase

Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic redox engineering of S. cerevisiae. Introduction of an NADPH-specific alcohol dehydrogenase (NADPH-ADH) not only reduces the NADH demand of the acetate-to-ethanol pathway but also allows the cell to effectively exchange NADPH for NADH during sugar fermentation. Unlike NADH, NADPH can be freely generated under anoxic conditions, via the oxidative pentose phosphate pathway. We show that an industrial bioethanol strain engineered with the original pathway (expressing acetylating acetaldehyde dehydrogenase from Bifidobacterium adolescentis and with deletions of glycerol-3-phosphate dehydrogenase genes GPD1 and GPD2) consumed 1.9 g liter⁻¹ acetate during fermentation of 114 g liter⁻¹ glucose. Combined with a decrease in glycerol production from 4.0 to 0.1 g liter⁻¹, this increased the ethanol yield by 4% over that for the wild type. We provide evidence that acetate consumption in this strain is indeed limited by NADH availability. By introducing an NADPH-ADH from Entamoeba histolytica and with overexpression of ACS2 and ZWF1, we increased acetate consumption to 5.3 g liter⁻¹ and raised the ethanol yield to 7% above the wild-type level.     

A splice variant of the Drosophila vesicular monoamine transporter contains a conserved trafficking domain and functions in the storage of dopamine, serotonin, and octopamine

Vesicular monoamine transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related vesicular acetylcholine transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of vesicular transporter function and regulation, we have cloned the Drosophila homolog of the vesicular monoamine transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila vesicular glutamate transporter. Our data suggest that DVMAT-A functions as a vesicular transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of transporter trafficking and regulation.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.     

Disentangling environmental and host sources of fungal endophyte communities in an experimental beachgrass study

Disentangling the ecological factors that contribute to the assembly of the microbial symbiont communities within eukaryotic hosts is an ongoing challenge. Broadly speaking, symbiont propagules arrive either from external sources in the environment or from internal sources within the same host individual. To understand the relative importance of these propagule sources to symbiont community assembly, we characterized symbiotic fungal endophyte communities within the roots of three species of beachgrass in a field experiment. We manipulated two aspects of the external environment, successional habitat and physical disturbance. To determine the role of internal sources of propagules for endophyte community assembly, we used beachgrass individuals with different pre‐existing endophyte communities. Endophyte species richness and community composition were characterized using culture‐based and next‐generation sequencing approaches. Our results showed that external propagule sources associated with successional habitat, but not disturbance, were particularly important for colonization of most endophytic taxa. In contrast, internal propagule sources played a minor role for most endophytic taxa but were important for colonization by the dominant taxon Microdochium bolleyi. Our findings highlight the power of manipulative field experiments to link symbiont community assembly to its underlying ecological processes, and to ultimately improve predictions of symbiont community assembly across environments.     

Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel,CNS penetrant pan-muscarinic antagonists

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M₄ (hM₄ IC₅₀s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K_p = 2.1, K_p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M_1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.