The effect of 8-day oral taurine supplementation on thermoregulation during low-intensity exercise at fixed heat production in hot conditions of incremental humidity.

PURPOSE: To determine the effect of taurine supplementation on sweating and core temperature responses, including the transition from compensable to uncompensable heat stress, during prolonged low-intensity exercise of a fixed-heat production (~ 200W/m2) in hot conditions (37.5 °C), at both fixed and incremental vapour-pressure. METHODS: Fifteen females (n = 3) and males (n = 12; 27 ± 5 years, 78 ± 9 kg, V ˙ O2max 50.3 ± 7.8 mL/kg/min), completed a treadmill walking protocol (~ 200W/m2 heat production [Hprod]) in the heat (37.5 ± 0.1 °C) at fixed-(16-mmHg) and ramped-humidity (∆1.5-mmHg/5-min) following 1 week of oral taurine supplementation (50 mg/kg/bm) or placebo, in a double-blind, randomised, cross-over design. Participants were assessed for whole-body sweat loss (WBSL), local sweat rate (LSR), sweat gland activation (SGA), core temperature (Tcore), breakpoint of compensability (Pcrit) and calorimetric heat transfer components. Plasma volume and plasma taurine concentrations were established through pre- and post-trial blood samples. RESULTS: Taurine supplementation increased WBSL by 26.6% and 5.1% (p = 0.035), LSR by 15.5% and 7.8% (p = 0.013), SGA (1 × 1 cm) by 32.2% and 29.9% (p < 0.001) and SGA (3 × 3 cm) by 22.1% and 17.1% (p = 0.015) during the fixed- and ramped-humidity exercise periods, respectively. Evaporative heat loss was enhanced by 27% (p = 0.010), heat-storage reduced by 72% (p = 0.024) and Pcrit was greater in taurine vs placebo (25.0-mmHg vs 21.7-mmHg; p = 0.002). CONCLUSION: Taurine supplementation increased sweating responses during fixed Hprod in hot conditions, prior to substantial heat strain and before the breakpoint of compensability, demonstrating improved thermoregulatory capacity. The enhanced evaporative cooling and reduced heat-storage delayed the subsequent upward inflection in Tcore-represented by a greater Pcrit-and offers a potential dietary supplementation strategy to support thermoregulation.

Embedding retrieval practice in undergraduate biochemistry teaching using PeerWise.

Retrieval practice is an evidence-based approach to teaching; here, we evaluate the use of PeerWise for embedding retrieval practice into summative assessment. PeerWise allows anonymous authoring, sharing, answering, rating, and feedback on peer-authored multiple choice questions. PeerWise was embedded as a summative assessment in a large first-year introductory biochemistry module. Engagement with five aspects of the tool was evaluated against student performance in coursework, exam, and overall module outcome. Results indicated a weak-to-moderate positive but significant correlation between engagement with PeerWise and assessment performance. Student feedback showed PeerWise had a polarizing effect; the majority recognized the benefits as a learning and revision tool, but a minority strongly disliked it, complaining of a lack of academic moderation and irrelevant questions unrelated to the module. PeerWise can be considered a helpful learning tool for some students and a means of embedding retrieval practice into summative assessment.

Antimicrobial Volatiles of the Insect Pathogen Metarhizium brunneum.

Fungal volatile organic compounds (VOCs) represent promising candidates for biopesticide fumigants to control crop pests and pathogens. Herein, VOCs produced using three strains of the entomopathogenic fungus Metarhizium brunneum were identified via GC-MS and screened for antimicrobial activity. The VOC profiles varied with fungal strain, development state (mycelium, spores) and culture conditions. Selected VOCs were screened against a range of rhizosphere and non-rhizosphere microbes, including three Gram-negative bacteria (Escherichia coli, Pantoea agglomerans, Pseudomonas aeruginosa), five Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, B. megaterium, B. thuringiensis), two yeasts (Candida albicans, Candida glabrata) and three plant pathogenic fungi (Pythium ultimum, Botrytis cinerea, Fusarium graminearum). Microbes differed in their sensitivity to the test compounds, with 1-octen-3-ol and isovaleric acid showing broad-spectrum antimicrobial activity. Yeasts and bacteria were inhibited by the same VOCs. Cryo-SEM showed that both yeasts and bacteria underwent some form of "autolysis", where all components of the cell, including the cell wall, disintegrated with little evidence of their presence in the clear, inhibition zone. The oomycete (P. ultimum) and ascomycete fungi (F. graminearum, B. cinerea) were sensitive to a wider range of VOCs than the bacteria, suggesting that eukaryotic microbes are the main competitors to M. brunneum in the rhizosphere. The ability to alter the VOC profile in response to nutritional cues may assist M. brunneum to survive among the roots of a wide range of plant species. Our VOC studies provided new insights as to how M. brunneum may protect plants from pathogenic microbes and correspondingly promote healthy growth.

Response of Key Metabolites during a UV-A Exposure Time-Series in the Cyanobacterium Chlorogloeopsis fritschii PCC 6912.

Ultraviolet A (UV-A) is the major component of UV radiation reaching the Earth's surface, causing indirect damage to photosynthetic organisms via the production of reactive oxygen species (ROS). In comparison, UV-B causes both direct damage to biomolecules and indirect damage. UV-B is well studied in cyanobacterial research due to their long evolutionary history and adaptation to high levels of UV, with less work on the effects of UV-A. In this study, the response of key metabolites in Chlorogloeopsis fritschii (C. fritschii) during 48 h of photosynthetically active radiation (PAR, 15 µmol·m-2·s-1) supplemented with UV-A (11 µmol·m-2·s-1) was investigated using gas chromatography- mass spectrometry (GC-MS). Results showed an overall significant increase in metabolite levels up to 24 h of UV-A exposure. Compared with previously reported UV-B (PAR + UV-B) and PAR only results, UV-A showed more similarity compared to PAR only exposure as opposed to supplemented UV-B. The amino acids glutamate, phenylalanine and leucine showed differences in levels between UV (both supplemented UV-A and supplemented UV-B) and PAR only (non-supplemented PAR), hinting to their relevance in UV stress response. The fatty acids, palmitic and stearic acid, showed positive log2 fold-change (FC) in supplemented UV-A and PAR only experiments but negative log2 FC in UV-B, indicating the more harmful effect of UV-B on primary metabolism. Less research has been conducted on UV-A exposure and cyanobacteria, a potential environmental stimuli for the optimisation of metabolites for industrial biotechnology. This study will add to the literature and knowledge on UV-A stress response at the metabolite level in cyanobacteria, especially within the less well-known species C. fritschii.

Volatile organic compounds of Metarhizium brunneum influence the efficacy of entomopathogenic nematodes in insect control.

The entomopathogenic fungus (EPF) Metarhizium brunneum occupies the same ecological niche as entomopathogenic nematodes (EPN), with both competing for insects as a food source in the rhizosphere. Interactions between these biocontrol agents can be antagonistic or synergistic. To better understand these interactions, this study focussed on investigating the effect of M. brunneum volatile organic compounds (VOCs), 1-octen-3-ol and 3-octanone, on EPN survival and behaviour. These VOCs proved to be highly toxic to the infective juveniles (IJs) of the EPN Steinernema carpocapsae, Steinernema feltiae and Heterorhabditis bacteriophora with mortality being dose dependent. Chemotaxis studies of H. bacteriophora IJs in Pluronic F127 gel revealed significant preference for the VOCs compared with controls for all tested concentrations. The VOCs also impacted on the test insects in a dose-dependent manner with 3-octanone being more toxic to Galleria mellonella, Cydia splendana and Curculio elephas larvae than 1-octen-3-ol. Mortality of C. splendana and G. mellonella larvae was significantly higher when exposed to relatively high doses (>25%) of 3-octanone. Lower doses of 3-octanone and 1-octen-3-ol immobilised test insects, which recovered after exposure to fresh air for 2 hrs. In depth studies on H. bacteriophora showed that exposure of IJs to > 10% concentration of 3-octanone or 1-octen-3-ol negatively affected infectivity whereas exposure to lower doses (0.1%, 0.01%) had no effect. The VOCs affected IJs, reducing penetration efficacy and the number of generations inside G. mellonella but they failed to inhibit the bacterial symbiont, Photorhabdus kayaii. The ecological significance of VOCs and how they could influence EPF-EPN insect interactions is discussed.

Adopting a flipped classroom approach for teaching molar calculations to biochemistry and genetics students.

The flipped classroom is a relatively new active learning pedagogical intervention, gaining popularity as a blended learning methodology. The flipped classroom comprises two distinct parts, directed learning carried out at the student's own pace away from the classroom and an interactive, class-based activity encouraging problem-solving and experiential learning. This research presents a 1-year study to measure student performance and perception toward a flipped classroom approach to teaching core biochemical calculations to first-year undergraduate biochemistry and genetics students. A post-task questionnaire showed an overall positive student perception with an associated significant improvement in the end of module summative assessment. These results suggest that this teaching approach offers some advantages over more traditional teaching pedagogies.

MALDI Profiling and Applications in Medicine.

Matrix assisted laser desorption ionization (MALDI) mass spectrometry allows for the rapid profiling of different biomolecular species from both biofluids and tissues. Whilst originally focused upon the analysis of intact proteins and peptides, MALDI mass spectrometry has found further applications in lipidomic analysis, genotyping, microorganism identification, biomarker discovery and metabolomics. The combining of multiple profiles data from differing locations across a sample furthermore, allows for spatial distribution of biomolecules to be established utilizing imaging MALDI analysis. This chapter discusses the MALDI process, its usual applications in the field of protein identification via peptide mass fingerprinting before focusing upon advances in the application of the profiling potential of MALDI mass spectrometry and its' various applications in biomedicine.

Metabolomics and Biomarker Discovery.

Recently, metabolomics-the study of metabolite profiles within biological samples-has found a wide range of applications. This chapter describes the different techniques available for metabolomic analysis, the various samples that can be utilised for analysis and applications of both global and targeted metabolomic analysis to biomarker discovery in medicine.

Intracellular and Extracellular Metabolites from the Cyanobacterium Chlorogloeopsis fritschii, PCC 6912, During 48 Hours of UV-B Exposure.

Cyanobacteria have many defence strategies to overcome harmful ultraviolet (UV) stress including the production of secondary metabolites. Metabolomics can be used to investigate this altered metabolism via targeted and untargeted techniques. In this study we assessed the changes in the intra- and extracellular low molecular weight metabolite levels of Chlorogloeopsis fritschii (C. fritschii) during 48 h of photosynthetically active radiation (PAR) supplemented with UV-B (15 µmol m-2 s-1 of PAR plus 3 µmol m-2 s-1 of UV-B) and intracellular levels during 48 h of PAR only (15 µmol m-2 s-1) with sampling points at 0, 2, 6, 12, 24 and 48 h. Gas chromatography-mass spectrometry (GC-MS) was used as a metabolite profiling tool to investigate the global changes in metabolite levels. The UV-B time series experiment showed an overall significant reduction in intracellular metabolites involved with carbon and nitrogen metabolism such as the amino acids tyrosine and phenylalanine which have a role in secondary metabolite production. Significant accumulation of proline was observed with a potential role in stress mitigation as seen in other photosynthetic organisms. 12 commonly identified metabolites were measured in both UV-B exposed (PAR + UV-B) and PAR only experiments with differences in significance observed. Extracellular metabolites (PAR + UV-B) showed accumulation of sugars as seen in other cyanobacterial species as a stress response to UV-B. In conclusion, a snapshot of the metabolome of C. fritschii was measured. Little work has been undertaken on C. fritschii, a novel candidate for use in industrial biotechnology, with, to our knowledge, no previous literature on combined intra- and extracellular analysis during a UV-B treatment time-series. This study is important to build on experimental data already available for cyanobacteria and other photosynthetic organisms exposed to UV-B.

Distinct mechanisms by which two forms of miR-140 suppress the malignant properties of lung cancer cells.

In this study we attempted to determine the molecular mechanisms underlying the two mature products of pre-miR-140 (3p and 5p) in malignant properties of lung cancer cells. The differential expression of the two forms of miR-140 in both NSCLC tissues and cell lines was determined by quantitative real-time PCR (qRT-PCR). The effects of the miR-140 mimics on the malignant properties of lung cancer cells were evaluated using invasion assay, adhesion assay, tubule formation assay and metabolite profiling. Biotin-miRNA pulldown and transcriptome profiling by RNA-seq were utilized to distinguish their mRNA targets of the miR-140 strands. Their downstream signalling pathways were unveiled using a high-throughput antibody array. Although both strands of the miR-140 are downregulated in the NSCLC, miR-140-3p is more predominant compared to miR-140-5p in lung cancer cell lines. Both miR-140 mimics suppress the invasion of lung cancer cells and the inhibitory effect of the miR-140 on adhesion is cell-dependent. Tumor conditioned media from A549 cells after treatment with miR-140-3p mimic reduce the tubule formation ability of the endothelial cells. Metabolite profiling indicates the alteration of glycine in both lung cancer cells following treatment with miR-140 mimics. The data from the RNA-sequencing and antibody array indicate that two miR-140 strands present different targeting and signalling profiles despite the existence of mutual targets such as IGF1R and FOS. In conclusion, two forms of miR-140 both suppress the malignant properties of lung cancer cells but through distinct and multiple mechanisms.

Limitations in the use of PSMγ, agr, RNAIII, and biofilm formation as biomarkers to define invasive Staphylococcus epidermidis from chronic biomedical device-associated infections.

Staphylococcus epidermidis is a common cause of biomedical device-associated infections. Agr is the major quorum sensing system in staphylococci and regulates virulence factors. Four agr-specificity groups exist in S. epidermidis, and chronic S. epidermidis infections are hypothesised to select for agr-negative phenotypes. Therefore, we investigated S. epidermidis strains from prosthetic joint- and catheter-associated infections to establish i) whether an infection selects for an agr-negative phenotype; ii) the importance of PSMγ and iii) if the agr-specificity group is infection dependent. S. epidermidis nasal isolates from healthy volunteers were used as controls. The distribution of agr-specificity groups was significantly different between infection and control episodes, but did not distinguish between the infection types. PSMγ secretion was used to determine agr-activity and HPLC analysis showed that 44% of prosthetic and 32% of catheter-associated episodes produced no PSMγ in comparison to 8% of the control strains. However, PSMγ expression did not always correlate with RNAIII up-regulation, indicating that PSMγ synthesis is likely influenced by additional post-transcriptional control. The data suggests chronic S. epidermidis infections favour agr-specificity group 1 but the results suggest that they do not select for an agr-negative phenotype. Further studies are required to explore the mechanisms underlying the selection and survival of these S. epidermidis phenotypes isolated from biomedical device-associated infections.

Detection and partial characterization of antifungal bioactivity from the secretions of the medicinal maggot, Lucilia sericata.

The antibacterial properties of the excretions/secretions (ES) of the medicinal maggot, Lucilia sericata have long been known and the effectiveness of maggot debridement therapy in relation to the clearance of bacteria from the surface of wounds has been the source of much research over recent years. Less well known, however, are the antifungal properties of L. sericata ES. Here, we show by means of the colony forming unit assay and optical density assays, that L. sericata native ES possess significant antifungal properties and appears to possess a highly heat stable, freeze/thaw, and lyophilization resistant antifungal component. We also show that the antifungal activity present in the native ES consists of a number of antifungal components present in three fraction masses consisting of >10, 10-0.5, and <0.5 kDa, with the greatest level of activity being seen in the <0.5 kDa fraction.

Reproduction potentiated in nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) by adding a synthetic peptide to their aqueous environment.

Ambient exposure to a short synthetic peptide has enhanced fecundity (number of offspring) in invertebrates and vertebrates, ostensibly by disinhibiting reproduction. In separate experiments, nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) were exposed via their aqueous environment to a dissolved synthetic hexamer (6mer) peptide, IEPVFT (EPL036), at a concentration of 1 µmol l(-1). In the case of the worms, peptide was added to their aqueous buffer daily throughout the experiment (14 days); for the guppies, peptide administration was on the first 15 alternate days in a 50 week experiment. Fecundity rose by 79% among the worms. The number of descendants of the treated guppies was more than four times that of controls by week 26 (103 versus 25, including 72 juveniles versus 6), with 15.4% more estimated biomass in the test tank in total (i.e. including founders). It was deduced that treated females bred earlier, at a smaller size, and had larger brood sizes. The total number of fish in the control tank had caught up by termination, but biomass continued to lag the test tank. There were no overt signs of toxicity among either the worms or the fish. Bioinformatics has been unilluminating in explaining these results in terms, for example, of mimicry of an endogenous regulator. A mass spectrometric campaign to identify a receptor, using murine brain for expediency, proved inconclusive. Molecular modelling in silico indicated unexpectedly that the hexamer EPL036 might be acting as an antagonist, to pro-fecundity effect; that is, as a blocker of an inhibitor. This suggests that there awaits discovery an evolutionarily conserved reproductive inhibitor and its (anti-fecundity) receptor.

Multiplatform metabolome and proteome profiling identifies serum metabolite and protein signatures as prospective biomarkers for schizophrenia.

Schizophrenia is a severe mental illness with a biological basis. However, the search for reliable biomarkers suitable for clinical routine has been futile so far. Accordingly, there is a need for innovative approaches such as genomics and proteomics to achieve this goal. In the present study, we compared metabolomic and proteomic data from 26 schizophrenia patients as well as from unaffected controls carefully matched for age and gender in a multi-platform approach. The combined analysis identified many signatures with initially good biomarker characteristics. After statistical analysis and comparison of these identified serum metabolites (analysed by Gas Chromatography Mass Spectrometry) and hydrophobic serum proteins (analysed by matrix-assisted laser desorption ionisation mass spectrometry), several markers (e.g., 2-piperidinec carboxylic acid, 6-deoxy-mannofuranose, galactoseoxime and a serum peptide of m/z 3177) were determined as having the best discriminating value between the groups. Our findings represent a proof of principle indicating that metabolomic and proteomic approaches can be successfully used in psychiatric biomarker research, even though the results should be regarded as preliminary with a need for replication in larger samples.

Phosphoproteomic techniques and applications.

Phosphoproteomic analysis seeks to determine the overall level of protein phosphorylation, as a result of kinase and phosphatase activity, and determine the identity of proteins which are phosphorylated and the amino acid residues which hold the phosphate group. The methodologies available have improved with increased research efforts; however, the most commonly followed procedure is to enrich for phosphoproteins or peptides and undertake tandem mass spectrometric analysis focusing on specific signature losses which represent phosphopeptides. There have been many advances in this area and these are detailed both in relation to available protocols for phosphoproteomic analysis and to the widening range of biomedical fields in which such approaches are being commonly applied.

MALDI profiling and applications in medicine.

Matrix-assisted laser desorption ionisation (MALDI) mass spectrometry allows for the rapid profiling of different biomolecular species from both biofluids and tissues. Whilst originally focussed upon the analysis of intact proteins and peptides, MALDI mass spectrometry has found further applications in lipidomic analysis, genotyping, micro-organism identification, biomarker discovery and metabolomics. The combining of multiple profiles data from differing locations across a sample, furthermore, allows for spatial distribution of biomolecules to be established utilising imaging MALDI analysis. This chapter discusses the MALDI process, its usual applications in the field of protein identification via peptide mass fingerprinting before focusing upon advances in the application of the profiling potential of MALDI mass spectrometry and its various applications in biomedicine.

Mass spectrometry analysis of nucleosides and nucleotides.

Mass spectrometry has been widely utilised in the study of nucleobases, nucleosides and nucleotides as components of nucleic acids and as bioactive metabolites in their own right. In this review, the application of mass spectrometry to such analysis is overviewed in relation to various aspects regarding the analytical mass spectrometric and chromatographic techniques applied and also the various applications of such analysis.

Conidia of the insect pathogenic fungus, Metarhizium anisopliae, fail to adhere to mosquito larval cuticle.

Adhesion of conidia of the insect pathogenic fungus, Metarhizium anisopliae, to the arthropod host cuticle initially involves hydrophobic forces followed by consolidation facilitated by the action of extracellular enzymes and secretion of mucilage. Gene expression analysis and atomic force microscopy were used to directly quantify recognition and adhesion between single conidia of M. anisopliae and the cuticle of the aquatic larval stage of Aedes aegypti and a representative terrestrial host, Tenebrio molitor. Gene expression data indicated recognition by the pathogen of both hosts; however, the forces for adhesion to the mosquito were approximately five times lower than those observed for Tenebrio. Although weak forces were recorded in response to Aedes, Metarhizium was unable to consolidate firm attachment. An analysis of the cuticular composition revealed an absence of long-chain hydrocarbons in Aedes larvae which are thought to be required for fungal development on host cuticle. This study provides, to our knowledge, the first evidence that Metarhizium does not form firm attachment to Ae. aegypti larvae in situ, therefore preventing the normal route of invasion and pathogenesis from occuring.

Metarhizium anisopliae pathogenesis of mosquito larvae: a verdict of accidental death.

Metarhizium anisopliae, a fungal pathogen of terrestrial arthropods, kills the aquatic larvae of Aedes aegypti, the vector of dengue and yellow fever. The fungus kills without adhering to the host cuticle. Ingested conidia also fail to germinate and are expelled in fecal pellets. This study investigates the mechanism by which this fungus adapted to terrestrial hosts kills aquatic mosquito larvae. Genes associated with the M. anisopliae early pathogenic response (proteinases Pr1 and Pr2, and adhesins, Mad1 and Mad2) are upregulated in the presence of larvae, but the established infection process observed in terrestrial hosts does not progress and insecticidal destruxins were not detected. Protease inhibitors reduce larval mortality indicating the importance of proteases in the host interaction. The Ae. aegypti immune response to M. anisopliae appears limited, whilst the oxidative stress response gene encoding for thiol peroxidase is upregulated. Cecropin and Hsp70 genes are downregulated as larval death occurs, and insect mortality appears to be linked to autolysis through caspase activity regulated by Hsp70 and inhibited, in infected larvae, by protease inhibitors. Evidence is presented that a traditional host-pathogen response does not occur as the species have not evolved to interact. M. anisopliae retains pre-formed pathogenic determinants which mediate host mortality, but unlike true aquatic fungal pathogens, does not recognise and colonise the larval host.

Protein and peptide profiling as a tool for biomarker discovery in depression.

This study sought to determine whether protein and/or peptide profiles from serum were able to distinguish patients suffering from depression from healthy control subjects and thereby act as biomarker candidates with potential diagnostic value. Serum samples were collected from patients (n = 39) and controls (n = 30). A C8 magnetic bead protocol was used to prepare serum proteins, while a microextraction C18 packed tip was used to isolate serum peptides. Both protein and peptide profiles were recorded by MALDI-MS and the data were exported for further analysis. No protein signals differentiated patients from controls and principle component analysis of the entire peptide profile did not allow for distinct clustering of the two groups. Further analysis of individual peptides however identified three peptide signals whose intensities were significantly different between patients and control subjects. The efficacy of these potential biomarker candidates to identify the patients was therefore studied using a receiver operating characteristic (ROC) curve and the combined use of all three candidates together offered the most specific and sensitive identification of true positive cases of depression.