Modelling of amino acid turnover in the horse during training and racing: A basis for developing a novel supplementation strategy.

Horses in heavy training in preparation for racing and competition have increased metabolic demands to support the more intensive levels of exercise and recovery. However, little is known at the metabolic level about amino acid turnover and the specific alterations of demand caused by high intensity exercise. During exercise, certain amino acids are required in greater quantities due to disproportionate losses via excretory systems and usage in biosynthetic pathways. This investigation has built a theoretical computer model in an attempt to bring together the published rates of protein intake and utilisation to try to understand how some amino acids might be in higher demand than others. The model indicated that after evaluation of the daily amino acid turnover, glutamine/glutamic acid (Glx), serine and ornithine were in negative nitrogen balance which identified these amino acids as critical limiting factors for anabolism. Adjustment of the modelling conditions to cater for high intensity training indicated that an additional demand was placed on eight amino acids, including GLx, valine, lysine, histidine and phenylalanine which could thus become limiting under these conditions. The modelling results indicated that an amino acid supplement with the correct amino acids to match demand could theoretically be beneficial to a 500Kg horse in quantities of 20-80g/day. These results open new avenues of research for specifically tailoring amino acid supplementation to meet demands for sports horses in heavy training and improving general well-being, especially in hotter climates.

Relationships between electrolyte and amino acid compositions in sweat during exercise suggest a role for amino acids and K+ in reabsorption of Na+ and Cl- from sweat.

Concentrations of free amino acids and [K+] in human sweat can be many times higher than in plasma. Conversely, [Na+] and [Cl-] in sweat are hypotonic to plasma. It was hypothesised that the amino acids and K+ were directly or indirectly associated with the resorption of Na+ and Cl- in the sweat duct. The implication would be that, as resources of these components became limiting during prolonged exercise then the capacity to resorb [Na+] and [Cl-] would diminish, resulting in progressively higher levels in sweat. If this were the case, then [Na+] and [Cl-] in sweat would have inverse relationships with [K+] and the amino acids during exercise. Forearm sweat was collected from 11 recreational athletes at regular intervals during a prolonged period of cycling exercise after 15, 25, 35, 45, 55 and 65 minutes. The subjects also provided passive sweat samples via 15 minutes of thermal stimulation. The sweat samples were analysed for concentrations of amino acids, Na+, Cl-, K+, Mg2+ and Ca2+. The exercise sweat had a total amino acid concentration of 6.4 ± 1.2mM after 15 minutes which was lower than the passive sweat concentration at 11.6 ± 0.8mM (p<0.05) and showed an altered array of electrolytes, indicating that exercise stimulated a change in sweat composition. During the exercise period, [Na+] in sweat increased from 23.3 ± 3.0mM to 34.6 ± 2.4mM (p<0.01) over 65 minutes whilst the total concentrations of amino acids in sweat decreased from 6.4 ± 1.2mM to 3.6 ± 0.5mM. [Na+] showed significant negative correlations with the concentrations of total amino acids (r = -0.97, p<0.05), K+ (r = -0.93, p<0.05) and Ca2+ (r = -0.83, p<0.05) in sweat. The results supported the hypothesis that amino acids and K+, as well as Ca2+, were associated with resorption of Na+ and Cl-.

An overview on biodegradation of polystyrene and modified polystyrene: the microbial approach.

Polystyrene is a widely used plastic in many aspects of human life and in industries due to its useful characteristics of low cost, light weight, ease of manufacture, versatility, thermal efficiency, durability, and moisture resistance. However, polystyrene is very stable and extremely hard to degrade in the environment after disposal. Polystyrene can be used as a carbon source for microorganisms similar to many other hydrocarbons. The ability of microorganisms to use polystyrene as a carbon source has been recently established. However, the high molecular weight of polystyrene limits its use as a substrate for enzymatic reactions to take place. In this paper, we review studies on biodegradation of polystyrene to give an overview and direction for future studies.

Sweat Facilitated Amino Acid Losses in Male Athletes during Exercise at 32-34°C.

Sweat contains amino acids and electrolytes derived from plasma and athletes can lose 1-2L of sweat per hour during exercise. Sweat may also contain contributions of amino acids as well as urea, sodium and potassium from the natural moisturizing factors (NMF) produced in the stratum corneum. In preliminary experiments, one participant was tested on three separate occasions to compare sweat composition with surface water washings from the same area of skin to assess contributions from NMF. Two participants performed a 40 minute self-paced cycle session with sweat collected from cleansed skin at regular intervals to assess the contributions to the sweat load from NMF over the period of exercise. The main study investigated sweat amino acid composition collected from nineteen male athletes following standardised endurance exercise regimes at 32-34°C and 20-30% RH. Plasma was also collected from ten of the athletes to compare sweat and plasma composition of amino acids. The amino acid profiles of the skin washings were similar to the sweat, suggesting that the NMF could contribute certain amino acids into sweat. Since the sweat collected from athletes contained some amino acid contributions from the skin, this fluid was subsequently referred to as "faux" sweat. Samples taken over 40 minutes of exercise showed that these contributions diminished over time and were minimal at 35 minutes. In the main study, the faux sweat samples collected from the athletes with minimal NMF contributions, were characterised by relatively high levels of serine, histidine, ornithine, glycine and alanine compared with the corresponding levels measured in the plasma. Aspartic acid was detected in faux sweat but not in the plasma. Glutamine and proline were lower in the faux sweat than plasma in all the athletes. Three phenotypic groups of athletes were defined based on faux sweat volumes and composition profiles of amino acids with varying relative abundances of histidine, serine, glycine and ornithine. It was concluded that for some individuals, faux sweat resulting from exercise at 32-34°C and 20-30% RH posed a potentially significant source of amino acid loss.

Small changes in environmental parameters lead to alterations in antibiotic resistance, cell morphology and membrane fatty acid composition in Staphylococcus lugdunensis.

Staphylococcus lugdunensis has emerged as a major cause of community-acquired and nosocomial infections. This bacterium can rapidly adapt to changing environmental conditions to survive and capitalize on opportunities to colonize and infect through wound surfaces. It was proposed that S. lugdunensis would have underlying alterations in metabolic homeostasis to provide the necessary levels of adaptive protection. The aims of this project were to examine the impacts of subtle variations in environmental conditions on growth characteristics, cell size and membrane fatty acid composition in S. lugdunensis. Liquid broth cultures of S. lugdunensis were grown under varying combinations of pH (6-8), temperature (35-39°C) and osmotic pressure (0-5% sodium chloride w/w) to reflect potential ranges of conditions encountered during transition from skin surfaces to invasion of wound sites. The cells were harvested at the mid-exponential phase of growth and assessed for antibiotic minimal inhibitory concentration (MIC), generation time, formation of small colony variants, cell size (by scanning electron microscopy) and membrane fatty acid composition. Stress regimes with elevated NaCl concentrations resulted in significantly higher antibiotic resistance (MIC) and three of the combinations with 5% NaCl had increased generation times (P<0.05). It was found that all ten experimental growth regimes, including the control and centroid cultures, yielded significantly different profiles of plasma membrane fatty acid composition (P<0.0001). Alterations in cell size (P<0.01) were also observed under the range of conditions with the most substantial reduction occurring when cells were grown at 39°C, pH 8 (514±52 nm, mean ± Standard Deviation) compared with cells grown under control conditions at 37°C with pH 7 (702±76 nm, P<0.01). It was concluded that S. lugdunensis responded to slight changes in environmental conditions by altering plasma membrane fatty acid composition, growth rates and morphology to achieve optimal adaptations for survival in changing environments.

Phenotypic variants of staphylococci and their underlying population distributions following exposure to stress.

This study investigated whether alterations in environmental conditions would induce the formation of small colony variant phenotypes (SCV) with associated changes in cell morphology and ultra-structure in S. aureus, s. epidermidis, and S. lugdunensis. Wild-type clinical isolates were exposed to low temperature (4 °C), antibiotic stress (penicillin G and vancomycin; 0-10,000 µg mL(-1)), pH stress (pH 3-9) and osmotic challenge (NaCl concentrations of 0-20%). Changes in cell diameter, cell-wall thickness, and population distribution changes (n ≥ 300) were assessed via scanning and transmission electron microscopy (SEM and TEM), and compared to control populations. Our analyses found that prolonged exposure to all treatments resulted in the subsequent formation of SCV phenotypes. Observed SCVs manifested as minute colonies with reduced haemolysis and pigmentation (NaCl, pH and 4°C treatments), or complete lack thereof (antibiotic treatments). SEM comparison analyses revealed significantly smaller cell sizes for SCV populations except in S. aureus and S. epidermidis 10% NaCl, and S. epidermidis 4 °C (p<0.05). Shifts in population distribution patterns were also observed with distinct sub-populations of smaller cells appearing for S. epidermidis, and S. lugdunensis. TEM analyses revealed significantly thicker cell-walls in all treatments and species except S. lugdunensis exposed to 4 °C. These findings suggest that staphylococci adapted to environmental stresses by altering their cell size and wall thickness which could represent the formation of altered phenotypes which facilitate survival under harsh conditions. The phenotypic response was governed by the type of prevailing environmental stress regime leading to appropriate alterations in ultra-structure and size, suggesting downstream changes in gene expression, the proteome, and metabolome.

Effect of low temperature on growth and ultra-structure of Staphylococcus spp.

The effect of temperature fluctuation is an important factor in bacterial growth especially for pathogens such as the staphylococci that have to remain viable during potentially harsh and prolonged transfer conditions between hosts. The aim of this study was to investigate the response of S. aureus, S. epidermidis, and S. lugdunensis when exposed to low temperature (4°C) for prolonged periods, and how this factor affected their subsequent growth, colony morphology, cellular ultra-structure, and amino acid composition in the non-cytoplasmic hydrolysate fraction. Clinical isolates were grown under optimal conditions and then subjected to 4°C conditions for a period of 8 wks. Cold-stressed and reference control samples were assessed under transmission electron microscopy (TEM) to identify potential ultra-structural changes. To determine changes in amino acid composition, cells were fractured to remove the lipid and cytoplasmic components and the remaining structural components were hydrolysed. Amino acid profiles for the hydrolysis fraction were then analysed for changes by using principal component analysis (PCA). Exposure of the three staphylococci to prolonged low temperature stress resulted in the formation of increasing proportions of small colony variant (SCV) phenotypes. TEM revealed that SCV cells had significantly thicker and more diffuse cell-walls than their corresponding WT samples for both S. aureus and S. epidermidis, but the changes were not significant for S. lugdunensis. Substantial species-specific alterations in the amino acid composition of the structural hydrolysate fraction were also observed in the cold-treated cells. The data indicated that the staphylococci responded over prolonged periods of cold-stress treatment by transforming into SCV populations. The observed ultra-structural and amino acid changes were proposed to represent response mechanisms for staphylococcal survival amidst hostile conditions, thus maintaining the viability of the species until favourable conditions arise again.

Filterability of staphylococcal species through membrane filters following application of stressors.

BACKGROUND: Passage of bacterial cells through filter pores has been reported for a number of bacterial species. In this investigation, we tested the filterability of staphylococcal cultures that were exposed to several environmental stress conditions by passing them through 0.22 and 0.45 mum sterile filters, which are industry standards. FINDINGS: Results showed repeated passage of viable staphylococcal cells through both pore sizes, although more passage was seen through the 0.45 mum pore size. Of the three staphylococcal species, S. lugdunensis showed the best passage at relatively higher numbers regardless of the treatment, while both S. aureus and S. epidermidis showed limited passage or complete inhibition. CONCLUSION: The data showed that staphylococcal bacteria were capable of passing through sterile filters in a viable state. There was better passage through 0.45 mum sterile filters than through the 0.22 mum sterile filters. Application of a stress condition did not appear to enhance filterability of these bacterial cultures.

Hematologic and urinary excretion anomalies in patients with chronic fatigue syndrome.

Patients with chronic fatigue syndrome (CFS) have a broad and variable spectrum of signs and symptoms with variable onsets. This report outlines the results of a single-blind, cross-sectional research project that extensively investigated a large cohort of 100 CFS patients and 82 non fatigued control subjects with the aim of performing a case-control evaluation of alterations in standard blood parameters and urinary amino and organic acid excretion profiles. Blood biochemistry and full blood counts were unremarkable and fell within normal laboratory ranges. However, the case-control comparison of the blood cell data revealed that CFS patients had a significant decrease in red cell distribution width and increases in mean platelet volume, neutrophil counts, and the neutrophil-lymphocyte ratio. Evaluation of the urine excretion parameters also revealed a number of anomalies. The overnight urine output and rate of amino acid excretion were both reduced in the CFS group (P < 0.01). Significant decreases in the urinary excretion of asparagine (P < 0.0001), phenylalanine (P < 0.003), the branch chain amino acids (P < 0.005), and succinic acid (P < 0.0001), as well as increases in 3-methylhistidine (P < 0.05) and tyrosine (P < 0.05) were observed. It was concluded that the urinary excretion and blood parameters data supported the hypothesis that alterations in physiologic homeostasis exist in CFS patients.

Evaluating potential applications of faecal sterols in distinguishing sources of faecal contamination from mixed faecal samples.

Faecal samples from humans, herbivores, carnivores and birds as well as samples from septic tanks and effluents from a sewage treatment plant (STP) were extracted and analyzed by gas chromatography-mass spectrometry for faecal sterols including coprostanol, epicoprostanol, cholestanol, cholesterol, stigmasterol, campesterol, 24-ethylcoprostanol and beta-sitosterol. Coprostanol was observed in the highest concentrations from the human derived samples, but it was also present in substantial quantities in a range of herbivores. There was no unique marker of human faecal contamination. Multivariate analyses revealed that the faecal sterol profiles were significantly different between the four groups of animals (Wilks' lambda=0.007, P<0.002), and coprostanol and 24-ethylcoprostanol were the major discriminant factors. However, when faecal samples were mixed, the confounding of faecal sterol levels prevented accurate identification of contributing species. Conversely, faecal sterol ratios were highly efficient at identifying which mixtures contained human contribution, but could not appropriately determine percentage contributions of sources.

Comparisons of water quality parameters from diverse catchments during dry periods and following rain events.

In this study, 12 catchments sites located along the north coast of New South Wales in Australia were grouped into the four categories of septic, cattle, sewage treatment plant (STP) and forested sites via cluster analysis based on their land use patterns. Water samples from all these sites were collected between October 2004 and June 2006 at a regular monthly interval and within 48 h of rain events. The samples were analyzed for bacterial counts including faecal coliform and total coliform; faecal sterols including coprostanol, epicoprostanol, cholesterol, cholestanol, 24-ethylcoprostanol, campesterol, stigmasterol and beta-sitosterol; and the elements including Na, Rb, Sr, Ag, Cd, Sn, Cs, Ba, Hg, Tl, Pb, Bi, U, Mg, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, K, As, Se, P and Mo. Over the course of the sampling period, the STP site had the highest average coprostanol level of 1693+/-567 ng/L which was significantly higher (p<0.05) than the septic sites (190+/-71 ng/L), the cattle sites (163+/-94 ng/L) and forested sites (14+/-4 ng/L). As expected, the forested sites had significantly lower average level of faecal coliforms (373+/-87 cfu/100 mL) compared with the STP (1395+/-574 cfu/100 mL), septic (1243+/-494 cfu/100 mL) and cattle sites (535+/-112 cfu/100 mL). The concentrations of coprostanol were not correlated with the numbers of faecal coliform bacteria when the entire data set was evaluated. The forested sites generally had the lowest average levels of elemental compositions, with significantly lower levels noted for Na, U, Mg, V, Cu, Sr, K, As, P and Mo, whereas Fe was the only element notably higher in the forested sites. Temporal and rain events analyses of the data set revealed that elevated levels of both coprostanol and faecal coliforms were not exclusive to rain events. The average coprostanol levels in rain event samples at each site were not significantly different compared with the corresponding dry event samples. Conversely, faecal coliform numbers increased by 2-4 times in rain events samples from septic, cattle and forested sites, but did not alter in the STP site. Multivariate analyses identified coprostanol and Sr as major contributing factors for the discrimination of septic, cattle, STP and forested sites for both rain and dry events samples. It was clear that each land use type of catchment could be characterized by biochemical, bacteriological and elemental parameters.

Bacterial source tracking from diverse land use catchments by sterol ratios.

Water samples from sites potentially impacted by septic tanks, cattle, sewage treatment plant (STP) and natural forests were collected at regular monthly intervals and within 48 h of rainfall events between October 2004 and June 2006. All samples (n=296) were analysed for faecal coliforms and faecal sterols including coprostanol, epicoprostanol, cholestanol, cholesterol and 24-ethylcoprostanol. Faecal sterol ratios were used to assign human and/or herbivore contamination sources and to estimate their percentage relative contributions in water samples. The catchments had significantly different profiles of designated contamination origins (p<0.05), which were consistent with land use patterns. The STP impacted site had the highest incidence of human contamination assignations and the highest mean levels of coprostanol, whilst the forested site had the highest incidence of uncontaminated samples and the lowest mean concentration of coprostanol. Coprostanol concentrations were not always correlated with faecal coliform counts.

Babesia canis vogeli: a novel PCR for its detection in dogs in Australia.

Babesia canis vogeli is known to cause disease in dogs in Australia, and the rapid detection of various subspecies would enable effective treatment and management. A 21 bp oligonucleotide, "Bab-f" was proposed for the production of larger PCR products with high species specificity that would enable effective sequence analyses to yield subspecies identification. The new forward primer when paired with a previously reported "Babesia common" reverse primer generated a 394 bp product which was successfully amplified and provided subspecies differentiation by sequence analyses. Specificity and sensitivity were reported at 100% on a cohort of 55 dogs.

Anaplasma platys: an improved PCR for its detection in dogs.

This study compares two PCR assays for the detection of Anaplasma platys in dog blood using primers based on the A. platys 16S rRNA gene. The first approach utilized a "standard" PCR protocol composed of a "single-step" direct amplification using an Ehrlichia genus-specific primer set. The second assay being a "nested" PCR screen that first involved a universal bacterial primer set that amplified the majority of the 16S rRNA gene, followed by the nested round of PCR using an A. platys-specific primer set. Of the 22 dogs sampled, 10 were found to contain A. platys DNA using both protocols, and an additional two dogs were found positive using the nested technique. An extract of A. platys positive genomic DNA was serially diluted and comparison of sensitivities determined between the nested PCR, and a direct assay using A. platys-specific primers. The nested protocol demonstrated an increased sensitivity by at least 2 orders of magnitude when compared to the direct assay alone. Our results indicated that the nested PCR assay with its increased sensitivity would be useful for experimental research investigations as well as offer the potential for use as a routine test in diagnostic pathology.

Intravenous immunoglobulin (IVIG): modes of action in the clinical management of recurrent pregnancy loss (RPL) and selected autoimmune disorders.

Recurrent pregnancy loss has been associated with autoimmune responses to membrane phospholipids and alloimmune reactions against paternally derived molecules on the trophoblast. The problem is psychologically and economically stressful as it undermines the capacity of some couples to reproduce and participate effectively in the day-to-day economic activities. This article reviews the adoption of intravenous immunoglobulin as a form of therapy for the clinical management of recurrent pregnancy loss and of selected autoimmune disorders. Side effects, contraindications and safety of use are discussed.

Pain intensity, illness duration, and protein catabolism in temporomandibular disorder patients with chronic muscle pain.

AIMS: To investigate whether the duration of chronic pain in temporomandibular disorder (TMD) patients is associated with a net depletion of amino acids, and a distinct process from pain intensity. METHODS: Twenty-nine patients defined by the research diagnostic criteria/TMD as having Type 1a muscle pain (TMD1A group), and 34 age- and sex-matched control subjects, were assessed for variation in urinary organic and amino acid excretion by gas chromatography-mass spectrometry. RESULTS: The TMD1A patients' mean pain intensity, assessed on a visual analog scale (VAS), was 5.4 (95% confidence limits: 4.5 to 6.3), TMD1A illness duration was 5.0 +/- 1.2 (SD) years, number of body areas with pain/subject was 6.3 +/- 2.4 (range 0 to 10), and symptom prevalence from the Symptom Check List-90-Revised (SCL-90-R) was 25.5 +/- 11.3 symptoms/subject, which was higher than the controls (5.2 +/- 5.0 symptoms/subject, P < .001). TMD1A patient illness duration was positively correlated with symptom prevalence and body pain distribution, and all were independent of pain intensity. The TMD1A patients had: (1) and increased tyrosine:leucine ratio; and (2) reduced leucine concentrations (both P < .001), which suggests deregulated catabolism. Pain intensity was associated with: (1) changes in the multivariate urinary metabolite excretion patterns (P < .001); (2) reduced leucine concentrations (P < .001); and (3) increases in total urinary metabolites (P < .04), and in 2 unidentified molecules, UM28 (P < .001) and CFSUM1 (P < .002). TMD1A illness duration was associated with lower (1) urinary metabolite concentrations and (2) succinic acid and combined glutamine + glutamic acid levels, suggesting a progressive depletion of metabolite reserves. CONCLUSION: In TMD1A patients, total amino acid excretion was positively correlated with pain intensity and negatively correlated with illness duration, which indicated that illness duration was associated with a different set of metabolic anomalies compared with those identified for pain intensity.

Coagulase-negative staphylococcal membrane-damaging toxins, pain intensity, and metabolic changes in temporomandibular disorder patients with chronic muscle pain.

AIMS: To investigate the association between toxin-producing staphylococci, symptom expression, and changes in urinary excretion of metabolites in temporomandibular disorder (TMD) patients and age- and sex-matched control subjects. METHODS: Twenty-nine patients defined by the research diagnostic criteria/TMD as having Type 1a muscle pain (TMD1A), and 34 age- and sex-matched control subjects were assessed for the carriage of staphylococcal species, staphylococcal toxin production, expression of symptoms, and changes in urinary excretion of amino and organic acids. RESULTS: TMD1A patients had an increased incidence of carriage of toxin-producing coagulase-negative staphylococcus (MDT-CoNS, P < .004), which produced increased levels of delta-like membrane-damaging toxins. The TMD1A patients also had a reduction in the incidence of carriage of Staphylococcus aureus (P < .02). Increased incidence of MDT-CoNS was positively associated with increased pain intensity as assessed by a visual analog scale (P < .001). Odds ratio analysis revealed a 9.2-fold increase in MDT-CoNS recovery from the nose of TMD1A patients compared with the control subjects (odds ratio = 9.2, > 95% confidence limits: 2.3 to 37.5, P < .001). Increases in the carriage incidence of MDT-CoNS were also associated with increases in the urinary tyrosine:leucine ratio (P < .004), which represents a change in the balance of proteolysis and protein synthesis. The toxin production by these CoNS species was also associated with an increased urinary excretion of glutamic acid (P < .03). CONCLUSION: These data suggest that an increased colonization of MDT-CoNS on skin and mucosal membranes was associated with changed proteolysis, increased pain intensity, and an increase in excitatory amino acids consistent with events associated with the development of chronic orofacial muscle pain in TMD patients.