The redox switch/redox coupling hypothesis

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD(+) limitation, under sufficiently reduced cytosolic NAD(+)/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD(+)/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD(+)/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD(+)/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.     

Immediate Effects of Experimental Human Trampling on Mid-Upper Intertidal benthic Invertebrates at the Asinara Island MPA (NW Mediterranean)

To assess potential risks of human visitation to ecological communities, the immediate effects of human trampling were investigated experimentally on small invertebrates inhabiting mid-upper intertidal hard bottoms covered by algae. Two different experimental intensities of trampling (60 and 120 footsteps) and controls (with no trampling) were applied to quadrats 20×20 cm in size (experimental area), within the two ‘no-entry, no-take’ zones of the Asinara Island MPA (Italy, Mediterranean Sea). One day after trampling ended, samples of benthic fauna were collected and the animals attributed to macrofaunal and meiofaunal components. Analyses of variance on the nine most common taxa of macrofauna identified significant higher abundance of bivalves, gammarid amphipods, polychaetes, isopods, oligochaetes in controls than in trampled plots. For nematodes, polychaetes, ostracods, oligochaetes, bivalves, acari, caprellid amphipods and tanaids a significant higher abundance of meiofaunal animals was found in controls than in trampled areas. Although no information on recovery is available, these results suggest that macrofaunal and meiofaunal taxa are vulnerable to this type of disturbance.     

The intrinsic peritrophic matrix protein peritrophin-95 from larvae of Lucilia cuprina is synthesised in the cardia and regurgitated or excreted as a highly immunogenic protein

The intrinsic peritrophic matrix glycoprotein, peritrophin-95, from the midgut of larvae of Lucilia cuprina can only be solubilized from the matrix using strong denaturants. This suggests that the protein has a structural role in the matrix. Consistent with this is the finding that immuno-gold and immuno-fluorescence localizations of the protein showed a uniform distribution within the peritrophic matrix. RT-PCR demonstrated that expression of peritrophin-95 mRNA was restricted to the larval cardia, a small organ located in the anterior midgut from which the type 2 peritrophic matrix originates. Immuno-blots and ELISAs demonstrated that the sera from sheep infested naturally or artificially with these larvae recognised peritrophin-95. This indicates that peritrophin-95 stimulates the ovine immune system during larval infestation even though the protein is firmly attached to the peritrophic matrix in the larval midgut and seemingly "concealed" from the ovine immune surveillance system. Analyses of larval regurgitated or excreted material by immuno-blots, immuno-affinity purification and amino-terminal sequencing demonstrated the presence of soluble monomeric peritrophin-95. These results indicate that peritrophin-95, a candidate vaccine antigen for use in sheep is not a "concealed" antigen as previously thought. The presence of soluble peritrophin-95 in the regurgitated/excreted material from larvae suggests that this protein may be involved in a maturation phase of peritrophic matrix production, a by-product of which is the excretion or regurgitation of soluble peritrophin-95.     

A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056

The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic β-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as β-sheets in the presence of anionic vesicles. Lipid monolayer surface pressure experiments revealed unusual kinetics of monolayer penetration, which suggest lipid-induced aggregation as a membranolytic mechanism. NMR analyses of the linear monomer and the dendrimeric SB056 in water and in 30% trifluoroethanol, on the other hand, yielded essentially unstructured conformations, supporting the excellent solubility and storage properties of this compound. However, simulated annealing showed that many residues lie in the β-region of the Ramachandran plot, and molecular-dynamics simulations confirmed the propensity of this peptide to fold as a β-type conformation. The excellent solubility in water and the lipid-induced oligomerization characteristics of this peptide thus shed light on its mechanism of antimicrobial action, which may also be relevant for systems that can form toxic β-amyloid fibrils when in contact with cellular membranes. Functionally, SB056 showed high activity against Gram-negative bacteria and some limited activity against Gram-positive bacteria. Its potency against Gram-negative strains was comparable (on a molar basis) to that of colistin and polymyxin B, with an even broader spectrum of activity than numerous other reference compounds.     

Overcoming fluconazole resistance in Candida albicans clinical isolates with tetracyclic indoles

Continuing efforts to discover novel means of combating fluconazole resistance in Candida albicans have identified an indole derivative that sensitizes strains demonstrating resistance to fluconazole. This tetracycle (3, ML229) does not appear to act through established Hsp90 or calcineurin pathways to chemosensitize C. albicans, as determined in Saccharomyces cerevisiae models, and may be a useful probe to uncover alternative resistance pathways.     

Genetic diversity of wild grapevine populations in Spain and their genetic relationships with cultivated grapevines

The wild grapevine, Vitis vinifera L. ssp. sylvestris (Gmelin) Hegi, considered as the ancestor of the cultivated grapevine, is native from Eurasia. In Spain, natural populations of V. vinifera ssp. sylvestris can still be found along river banks. In this work, we have performed a wide search of wild grapevine populations in Spain and characterized the amount and distribution of their genetic diversity using 25 nuclear SSR loci. We have also analysed the possible coexistence in the natural habitat of wild grapevines with naturalized grapevine cultivars and rootstocks. In this way, phenotypic and genetic analyses identified 19% of the collected samples as derived from cultivated genotypes, being either naturalized cultivars or hybrid genotypes derived from spontaneous crosses between wild and cultivated grapevines. The genetic diversity of wild grapevine populations was similar than that observed in the cultivated group. The molecular analysis showed that cultivated germplasm and wild germplasm are genetically divergent with low level of introgression. Using a model‐based approach implemented in the software structure , we identified four genetic groups, with two of them fundamentally represented among cultivated genotypes and two among wild accessions. The analyses of genetic relationships between wild and cultivated grapevines could suggest a genetic contribution of wild accessions from Spain to current Western cultivars.     

Production of engineered long-life and male sterile Pelargonium plants

ABSTRACT: BACKGROUND: Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. RESULTS: The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3--4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. CONCLUSION: The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.     

Longitudinal changes in response to a cycle-run field test of young male national "talent identification" and senior elite triathlon squads

This study investigated the changes in cardiorespiratory response and running performance of 9 male "Talent Identification" (TID) and 6 male Senior Elite (SE) Spanish National Squad triathletes during a specific cycle-run (C-R) test. The TID and SE triathletes (initial age 15.2 ± 0.7 vs. 23.8 ± 5.6 years, p = 0.03; V(O2)max 77.0 ± 5.6 vs. 77.8 ± 3.6 ml · kg(-1) · min(-1), nonsignificant) underwent 3 tests through the competitive period and the preparatory period, respectively, of 2 consecutive seasons: test 1 was an incremental cycle test to determine the ventilatory threshold (Th(vent)); test 2 (C-R) was 30-minute constant load cycling at the Th(vent) power output followed by a 3-km time-trial run; and test 3 (isolated control run [R]) was an isolated 3-km time-trial control run, in randomized counterbalanced order. In both seasons, the time required to complete the C-R 3-km run was greater than for R in TID (11:09 ± 00:24 vs. 10:45 ± 00:16 min:ss, p < 0.01 and 10:24 ± 00:22 vs. 10:04 ± 00:14, p = 0.006, for season 2005-2006 and 2006-2007, respectively) and SE (10:15 ± 00:19 vs. 09:45 ± 00:30, p < 0.001 and 09:51 ± 00:26 vs. 09:46 ± 00:06, p = 0.02 for season 2005-2006 and 2006-2007, respectively). Compared with the first season, the completion of the time-trial run was faster in the second season (6.6%, p < 0.01 and 6.4%, p < 0.01, for C-R and R tests, respectively) only in TID. Changes in post cycling run performance were accompanied by changes in pacing strategy, but there were only slight or nonsignificant changes in the cardiorespiratory response. Thus, the negative effect of cycling on performance may persist, independently of the period, over 2 consecutive seasons in TID and SE triathletes; however, improvements over time suggests that monitoring running pacing strategy after cycling may be a useful tool to control performance and training adaptations in TID.     

Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry

The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p?=?0.048; CI 95%: 1.01-10.24; p?=?0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.     

Muscle damage and its relationship with muscle fatigue during a half-iron triathlon

Background

To investigate the cause/s of muscle fatigue experienced during a half-iron distance triathlon.

Methodology/Principal Findings

We recruited 25 trained triathletes (36±7 yr; 75.1±9.8 kg) for the study. Before and just after the race, jump height and leg muscle power output were measured during a countermovement jump on a force platform to determine leg muscle fatigue. Body weight, handgrip maximal force and blood and urine samples were also obtained before and after the race. Blood myoglobin and creatine kinase concentrations were determined as markers of muscle damage.

Results

Jump height (from 30.3±5.0 to 23.4±6.4 cm; P<0.05) and leg power output (from 25.6±2.9 to 20.7±4.6 W · kg⁻¹; P<0.05) were significantly reduced after the race. However, handgrip maximal force was unaffected by the race (430±59 to 430±62 N). Mean dehydration after the race was 2.3±1.2% with high inter-individual variability in the responses. Blood myoglobin and creatine kinase concentration increased to 516±248 µg · L⁻¹ and 442±204 U · L⁻¹, respectively (P<0.05) after the race. Pre- to post-race jump change did not correlate with dehydration (r = 0.16; P>0.05) but significantly correlated with myoglobin concentration (r = 0.65; P<0.001) and creatine kinase concentration (r = 0.54; P<0.001).

Conclusions/significance

During a half-iron distance triathlon, the capacity of leg muscles to produce force was notably diminished while arm muscle force output remained unaffected. Leg muscle fatigue was correlated with blood markers of muscle damage suggesting that muscle breakdown is one of the most relevant sources of muscle fatigue during a triathlon.