High resolution analysis of meiotic chromosome structure and behaviour in barley (Hordeum vulgare L.)

Reciprocal crossing over and independent assortment of chromosomes during meiosis generate most of the genetic variation in sexually reproducing organisms. In barley, crossovers are confined primarily to distal regions of the chromosomes, which means that a substantial proportion of the genes of this crop rarely, if ever, engage in recombination events. There is potentially much to be gained by redistributing crossovers to more proximal regions, but our ability to achieve this is dependent upon a far better understanding of meiosis in this species. This study explores the meiotic process by describing with unprecedented resolution the early behaviour of chromosomal domains, the progression of synapsis and the structure of the synaptonemal complex (SC). Using a combination of molecular cytogenetics and advanced fluorescence imaging, we show for the first time in this species that non-homologous centromeres are coupled prior to synapsis. We demonstrate that at early meiotic prophase the loading of the SC-associated structural protein ASY1, the cluster of telomeres, and distal synaptic initiation sites occupy the same polarised region of the nucleus. Through the use of advanced 3D image analysis, we show that synapsis is driven predominantly from the telomeres, and that new synaptic initiation sites arise during zygotene. In addition, we identified two different SC configurations through the use of super-resolution 3D structured illumination microscopy (3D-SIM).     

A versatile method for preparation of hydrated microbial–latex biocatalytic coatings for gas absorption and gas evolution

We describe a latex wet coalescence method for gas-phase immobilization of microorganisms on paper which does not require drying for adhesion. This method reduces drying stresses to the microbes. It is applicable for microorganisms that do not tolerate desiccation stress during latex drying even in the presence of carbohydrates. Small surface area, 10-65?μm thick coatings were generated on chromatography paper strips and placed in the head-space of vertical sealed tubes containing liquid to hydrate the paper. These gas-phase microbial coatings hydrated by liquid in the paper pore space demonstrated absorption or evolution of H(2), CO, CO(2) or O(2). The microbial products produced, ethanol and acetate, diffuse into the hydrated paper pores and accumulate in the liquid at the bottom of the tube. The paper provides hydration to the back side of the coating and also separates the biocatalyst from the products. Coating reactivity was demonstrated for Chlamydomonas reinhardtii CC124, which consumed CO(2) and produced 10.2?±?0.2?mmol?O(2)?m(-2)?h(-1), Rhodopseudomonas palustris CGA009, which consumed acetate and produced 0.47?±?0.04?mmol?H(2)?m(-2)?h(-1), Clostridium ljungdahlii OTA1, which consumed 6?mmol CO?m(-2)?h(-1), and Synechococcus sp. PCC7002, which consumed CO(2) and produced 5.00?±?0.25?mmol O(2)?m(-2)?h(-1). Coating thickness and microstructure were related to microbe size as determined by digital micrometry, profilometry, and confocal microscopy. The immobilization of different microorganisms in thin adhesive films in the gas phase demonstrates the utility of this method for evaluating genetically optimized microorganisms for gas absorption and gas evolution.     

A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase (LpxD) catalyze the first and third steps of lipid A biosynthesis, respectively. Both enzymes have been found to be essential for survival among gram-negative bacteria that synthesize lipopolysaccharide and are viable targets for antimicrobial development. Catalytically, both acyltransferases catalyze an acyl-acyl carrier protein (ACP)-dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring. Here, we exploited the single free thiol unveiled on holo-ACP after transfer of the fatty acyl group to the glucosamine ring using the thiol-specific labeling reagent, ThioGlo. The assay was continuously monitored as a change in fluorescence at λ(ex)=379 nm and λ(em)=513 nm using a microtiter plate reader. This assay marks the first continuous and nonradioactive assay for either acyltransferase.     

A Broad Range of Conformations Contribute to the Solution Ensemble of the Essential Splicing Factor U2AF(65)

U2AF(65) is essential for pre-mRNA splicing in most eukaryotes. Two consecutive RNA recognition motifs (RRM) of U2AF(65) recognize a polypyrimidine tract at the 3' splice site. Here, we use small-angle X-ray scattering to demonstrate that the tandem U2AF(65) RRMs exhibit a broad range of conformations in the solution ensemble. The majority of U2AF(65) conformations exhibit few contacts between the RRMs, such as observed in the crystal structure. A subpopulation adopts tight inter-RRM contacts, such as independently reported based on paramagnetic relaxation enhancements. These complementary structural methods demonstrate that diverse splice sites have the opportunity to select compact or extended inter-RRM proximities from the U2AF(65) conformational pool.     

A mediated glucose/oxygen enzymatic fuel cell based on printed carbon inks containing aldose dehydrogenase and laccase as anode and cathode

Enzyme electrodes show great potential for many applications, as biosensors and more recently as anodes and cathodes in biocatalytic fuel cells for power generation. Enzymes have advantages over metal catalysts, as they provide high specificity and reaction rates, while operating under mild conditions. Here we report on studies related to development of mass-producible, completely enzymatic printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks containing mediators and laccase, for reduction of oxygen, or aldose dehydrogenase, for oxidation of glucose. Mediator performance in these printed formats is compared to relative rate constants for the enzyme-mediator reaction in solution, for a range of anode and cathode mediators. The power output and stability of fuels cells using an acidophilic laccase isolated from Trametes hirsuta is greater, at pH 5, than that for cells based on Melanocarpus albomyces laccase, that shows optimal activity closer to neutral pH, at pH 6. Highest power output, although of limited stability, was observed for ThL/ABTS cathodes, providing a maximum power density of 3.5 μWcm(-2) at 0.34 V, when coupled to an ALDH glucose anode mediated by an osmium complex. The stability of cell voltage above a threshold of 200 mV under a moderate 75 kΩ load is used to benchmark printed fuel cell performance. Highest stability was obtained for a printed fuel cell using osmium complexes as mediators of glucose oxidation by aldose dehydrogenase, and oxygen reduction by T. hirsuta laccase, maintaining cell voltage above 200 mV for 137 h at pH 5. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells.     

Two independent activities define Ccm1p as a moonlighting protein in Saccharomyces cerevisiae

Ccm1p is a nuclear-encoded PPR (pentatricopeptide repeat) protein that localizes into mitochondria of Saccharomyces cerevisiae. It was first defined as an essential factor to remove the bI4 [COB (cytochrome b) fourth intron)] and aI4 [COX1 (cytochrome c oxidase subunit 1) fourth intron] of pre-mRNAs, along with bI4 maturase, a protein encoded by part of bI4 and preceding exons that removes the intronic RNA sequence that codes for it. Later on, Ccm1p was described as key to maintain the steady-state levels of the mitoribosome small subunit RNA (15S rRNA). bI4 maturase is produced inside the mitochondria and therefore its activity depends on the functionality of mitochondrial translation. This report addresses the dilemma of whether Ccm1p supports bI4 maturase activity by keeping steady-state levels of 15S rRNA or separately and directly supports bI4 maturase activity per se. Experiments involving loss of Ccm1p, SMDC (sudden mitochondrial deprivation of Ccm1p) and mutations in one of the PPR (pentatricopeptide repeat) motifs revealed that the failure of bI4 maturase activity in CCM1 deletion mutants was not due to a malfunction of the translational machinery. Both functions were found to be independent, defining Ccm1p as a moonlighting protein. bI4 maturase activity was significantly more dependent on Ccm1p levels than the maintenance of 15S rRNA. The novel strategy of SMDC described here allowed the study of immediate short-term effects, before the mutant phenotype was definitively established. This approach can be also applied for further studies on 15S rRNA stability and mitoribosome assembly.     

Influence of wrestling on the physiological and skill demands of small-sided games

This study investigated the influence of wrestling on the physiological and skill demands of small-sided games. Twenty-eight elite rugby league players ([mean ± SE] age, 21.6 ± 0.5 years) participated in this within-subject crossover study. On day 1, 14 players played 2, 8-minute small-sided games, whereas the remaining 14 players played identical games with intermittent wrestling throughout. Each game was separated by 90 seconds. On day 2, the groups were crossed over. Movement was recorded by a global positioning system unit (miniMaxX, Catapult Innovations, Melbourne, Australia), sampling at 5 Hz. Each small-sided game was filmed to track the number of possessions and the number and quality of disposals. The games without wrestling resulted in a greater (p < 0.05) total distance covered (2,475 ± 31 vs. 1,964 ± 27 m) and greater distance covered in low (930 ± 19 vs. 842 ± 19 m), moderate (1,120 ± 28 vs. 752 ± 26 m), high (332 ± 16 vs. 240 ± 12 m), and very-high (24 ± 4 vs. 15 ± 3 m) velocity movement intensities. Conversely, the games with wrestling resulted in a significantly greater (p < 0.05) distance covered in mild, moderate, and maximal accelerations and a greater number of repeated high-intensity effort bouts (2.1 ± 0.2 bouts vs. 0.2 ± 0.1 bouts). No significant differences (p > 0.05) were detected between games with and without wrestling for the total number of involvements, receives, passes, effective passes, ineffective passes, and disposal efficiency. The results of this study demonstrate that intermittent wrestling reduces the running demands but increases the repeated high-intensity effort demands of small-side games. Furthermore, these physiological changes occur without compromising the volume of skill executions, the number of errors, or disposal efficiency. From a practical perspective, these results suggest that intermittent wrestling may be a useful supplement to small-sided games to concurrently train repeated-effort ability and skills under game-specific fatigue.     

Human electromuscular incapacitation devices characterization: a comparative study on stress and the physiological effects on swine

Human electromuscular incapacitation devices or electromuscular disruption (EMD) devices are increasingly used in police and military applications. Most individuals who experience electromuscular incapacitation are in a stress-filled state, and the effects of prolonged or repeated exposures are not well understood. Three different commercially available EMD devices were tested randomly on 6 anesthetized pigs each for a total of 18 pigs. Each animal was exposed to an initial 60-second application of the EMD device as an initial stressor. The animals were then allowed to rest under anesthesia for 60 minutes followed immediately by a 180-second application of the same device. Arterial blood gases and serum samples were collected throughout the experiment to measure catecholamines (epinephrine, norepinephrine, and dopamine) and cortisol. All the devices produced some level of muscle tetany as a result of the electrical delivery to the animal. All the pigs showed a mixed metabolic and respiratory acidosis. Cortisol tended to decrease after the initial exposure and slightly increased over the rest period. The extreme muscular work caused by the electrical stimulation resulting in muscle contractions did not result in a strong stress response but did result in an immediate sympathetic response during both applications of the device leading to the conclusion that initial stressor followed by rest and prolonged EMD device application did not exhaust the sympathetic system. For healthy adult animals, despite the prolonged muscular exertion and physiological stress caused by EMD devices, the body should be able to mount an appropriate sympathetic response and recover normally.     

Impact of a one-week intensive ‘training of trainers’ workshop for community health workers in south-west Nigeria

Background There is a huge unmet need for mental health services in low- and middle-in-come countries such as Nigeria. It has been suggested that one way of bridging the service gap is to plan for the effective integration of mental health services into primary care. We present the impact of a one-week training workshop on attitudes to and knowledge of mental health issues among the tutors of community health workers.Method An intensive one-week training workshop was organised for 24 trainers of community health officers from eight Nigerian states. The package was designed for the training of primary care workers in low-income countries by one of the authors (RJ). Participants completed a questionnaire designed to assess knowledge of and attitudes to mental health issues before and on completion of the training.Results There were 24 participants with a mean age of 47 years (SD ± 4.89). Eighteen (75%) of the participants were female. The overall assessment of knowledge of mental health issues increased from a mean score of 60.4% before training to a mean score of 73.7% after the training (t-test = 4.48, P = 0.001).Conclusion We reported a significant improvement in the knowledge and attitudes of tutors of community health workers following an intensive one-week training workshop. This, we believe, should improve the quality of pre-service mental health training for community health workers and hopefully impact on mental health service delivery at the primary healthcare level.