Aerosol mass spectrometric analysis of the chemical composition of non- refractory PM1 samples from school environments in Brisbane, Australia

Long term exposure to vehicle emissions has been associated with harmful health effects. Children are amongst the most susceptible group and schools represent an environment where they can experience significant exposure to vehicle emissions. However, there are limited studies on children’s exposure to vehicle emissions in schools. The aim of this study was to quantify the concentration of organic aerosol and in particular, vehicle emissions that children are exposed to during school hours. Therefore an Aerodyne compact time-of-flight aerosol mass spectrometer (TOF-AMS) was deployed at five urban schools in Brisbane, Australia. The TOF-AMS enabled the chemical composition of the non- refractory (NR-PM1) to be analysed with a high temporal resolution to assess the concentration of vehicle emissions and other organic aerosols during school hours. At each school the organic fraction comprised the majority of NR-PM1 with secondary organic aerosols as the main constitute. At two of the schools, a significant source of the organic aerosol (OA) was slightly aged vehicle emissions from nearby highways. More aged and oxidised OA was observed at the other three schools, which also recorded strong biomass burning influences. Primary emissions were found to dominate the OA at only one school which had an O:C ratio of 0.17, due to fuel powered gardening equipment used near the TOF-AMS. The diurnal cycle of OA concentration varied between schools and was found to be at a minimum during school hours. The major organic component that school children were exposed to during school hours was secondary OA. Peak exposure of school children to HOA occurred during school drop off and pick up times. Unless a school is located near major roads, children are exposed predominately to regional secondary OA as opposed to local emissions during schools hours in urban environments.

Error and uncertainty of adult age estimation of the pubic symphysis in an Australian sub-population using computed tomography

After attending this presentation, attendees will gain awareness of: (1) the error and uncertainty associated with the application of the Suchey-Brooks (S-B) method of age estimation of the pubic symphysis to a contemporary Australian population; (2) the implications of sexual dimorphism and bilateral asymmetry of the pubic symphysis through preliminary geometric morphometric assessment; and (3) the value of three-dimensional (3D) autopsy data acquisition for creating forensic anthropological standards. This presentation will impact the forensic science community by demonstrating that, in the absence of demographically sound skeletal collections, post-mortem autopsy data provides an exciting platform for the construction of large contemporary ‘virtual osteological libraries’ for which forensic anthropological research can be conducted on Australian individuals. More specifically, this study assesses the applicability and accuracy of the S-B method to a contemporary adult population in Queensland, Australia, and using a geometric morphometric approach, provides an insight to the age-related degeneration of the pubic symphysis. Despite the prominent use of the Suchey-Brooks (1990) method of age estimation in forensic anthropological practice, it is subject to intrinsic limitations, with reports of differential inter-population error rates between geographical locations1-4. Australian forensic anthropology is constrained by a paucity of population specific standards due to a lack of repositories of documented skeletons. Consequently, in Australian casework proceedings, standards constructed from predominately American reference samples are applied to establish a biological profile. In the global era of terrorism and natural disasters, more specific population standards are required to improve the efficiency of medico-legal death investigation in Queensland. The sample comprises multi-slice computed tomography (MSCT) scans of the pubic symphysis (slice thickness: 0.5mm, overlap: 0.1mm) on 195 individuals of caucasian ethnicity aged 15-70 years. Volume rendering reconstruction of the symphyseal surface was conducted in Amira® (v.4.1) and quantitative analyses in Rapidform® XOS. The sample was divided into ten-year age sub-sets (eg. 15-24) with a final sub-set of 65-70 years. Error with respect to the method’s assigned means were analysed on the basis of bias (directionality of error), inaccuracy (magnitude of error) and percentage correct classification of left and right symphyseal surfaces. Morphometric variables including surface area, circumference, maximum height and width of the symphyseal surface and micro-architectural assessment of cortical and trabecular bone composition were quantified using novel automated engineering software capabilities. The results of this study demonstrated correct age classification utilizing the mean and standard deviations of each phase of the S-B method of 80.02% and 86.18% in Australian males and females, respectively. Application of the S-B method resulted in positive biases and mean inaccuracies of 7.24 (±6.56) years for individuals less than 55 years of age, compared to negative biases and mean inaccuracies of 5.89 (±3.90) years for individuals greater than 55 years of age. Statistically significant differences between chronological and S-B mean age were demonstrated in 83.33% and 50% of the six age subsets in males and females, respectively. Asymmetry of the pubic symphysis was a frequent phenomenon with 53.33% of the Queensland population exhibiting statistically significant (χ2 - p<0.01) differential phase classification of left and right surfaces of the same individual. Directionality was found in bilateral asymmetry, with the right symphyseal faces being slightly older on average and providing more accurate estimates using the S-B method5. Morphometric analysis verified these findings, with the left surface exhibiting significantly greater circumference and surface area than the right (p<0.05). Morphometric analysis demonstrated an increase in maximum height and width of the surface with age, with most significant changes (p<0.05) occurring between the 25-34 and 55-64 year age subsets. These differences may be attributed to hormonal components linked to menopause in females and a reduction in testosterone in males. Micro-architectural analysis demonstrated degradation of cortical composition with age, with differential bone resorption between the medial, ventral and dorsal surfaces of the pubic symphysis. This study recommends that the S-B method be applied with caution in medico-legal death investigations of unknown skeletal remains in Queensland. Age estimation will always be accompanied by error; therefore this study demonstrates the potential for quantitative morphometric modelling of age related changes of the pubic symphysis as a tool for methodological refinement, providing a rigor and robust assessment to remove the subjectivity associated with current pelvic aging methods.

Evaluation of the Suchey-Brooks method for aging Australian Caucasian Populations using multislice Computed Tomography reconstructions of the Pubic Symphyseal Surface

Establishing age-at-death for skeletal remains is a vital component of forensic anthropology. The Suchey-Brooks (S-B) method of age estimation has been widely utilised since 1986 and relies on a visual assessment of the pubic symphyseal surface in comparison to a series of casts. Inter-population studies (Kimmerle et al., 2005; Djuric et al., 2007; Sakaue, 2006) demonstrate limitations of the S-B method, however, no assessment of this technique specific to Australian populations has been published. Aim: This investigation assessed the accuracy and applicability of the S-B method to an adult Australian Caucasian population by highlighting error rates associated with this technique. Methods: Computed tomography (CT) and contact scans of the S-B casts were performed; each geometrically modelled surface was extracted and quantified for reference purposes. A Queensland skeletal database for Caucasian remains aged 15 – 70 years was initiated at the Queensland Health Forensic and Scientific Services – Forensic Pathology Mortuary (n=350). Three-dimensional reconstruction of the bone surface using innovative volume visualisation protocols in Amira® and Rapidform® platforms was performed. Samples were allocated into 11 sub-sets of 5-year age intervals and changes associated with the surface geometry were quantified in relation to age, gender and asymmetry. Results: Preliminary results indicate that computational analysis was successfully applied to model morphological surface changes. Significant differences in observed versus actual ages were noted. Furthermore, initial morphological assessment demonstrates significant bilateral asymmetry of the pubic symphysis, which is unaccounted for in the S-B method. These results propose refinements to the S-B method, when applied to Australian casework. Conclusion: This investigation promises to transform anthropological analysis to be more quantitative and less invasive using CT imaging. The overarching goal contributes to improving skeletal identification and medico-legal death investigation in the coronial process by narrowing the range of age-at-death estimation in a biological profile.

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samples

Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N-acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock.

Comparison of genomic DNA extraction techniques from whole blood samples : a time, cost and quality evaluation study

Genomic DNA obtained from patient whole blood samples is a key element for genomic research. Advantages and disadvantages, in terms of time-efficiency, cost-effectiveness and laboratory requirements, of procedures available to isolate nucleic acids need to be considered before choosing any particular method. These characteristics have not been fully evaluated for some laboratory techniques, such as the salting out method for DNA extraction, which has been excluded from comparison in different studies published to date. We compared three different protocols (a traditional salting out method, a modified salting out method and a commercially available kit method) to determine the most cost-effective and time-efficient method to extract DNA. We extracted genomic DNA from whole blood samples obtained from breast cancer patient volunteers and compared the results of the product obtained in terms of quantity (concentration of DNA extracted and DNA obtained per ml of blood used) and quality (260/280 ratio and polymerase chain reaction product amplification) of the obtained yield. On average, all three methods showed no statistically significant differences between the final result, but when we accounted for time and cost derived for each method, they showed very significant differences. The modified salting out method resulted in a seven- and twofold reduction in cost compared to the commercial kit and traditional salting out method, respectively and reduced time from 3 days to 1 hour compared to the traditional salting out method. This highlights a modified salting out method as a suitable choice to be used in laboratories and research centres, particularly when dealing with a large number of samples.

SHOX gene is expressed in vertebral body growth plates in idiopathic and congenital scoliosis : implications for the etiology of scoliosis in Turner Syndrome

Reduced SHOX gene expression has been demonstrated to be associated with all skeletal abnormalities in Turner syndrome, other than scoliosis (and kyphosis). There is evidence to suggest that Turner syndrome scoliosis is clinically and radiologically similar to idiopathic scoliosis, although the phenotypes are dissimilar. This pilot gene expression study used relative quantitative real-time PCR (qRT-PCR) of the SHOX (short stature on X) gene to determine whether it is expressed in vertebral body growth plates in idiopathic and congenital scoliosis. After vertebral growth plate dissection, tissue was examined histologically and RNA was extracted and its integrity was assessed using a Bio-Spec Mini, NanoDrop ND-1000 spectrophotometer and standard denaturing gel electrophoresis. Following cDNA synthesis, gene-specific optimization in a Corbett RotorGene 6000 real-time cycler was followed by qRT-PCR of vertebral tissue. Histological examination of vertebral samples confirmed that only growth plate was analyzed for gene expression. Cycling and melt curves were resolved in triplicate for all samples. SHOX abundance was demonstrated in congenital and idiopathic scoliosis vertebral body growth plates. SHOX expression was 11-fold greater in idiopathic compared to congenital (n = 3) scoliosis (p = 0.027). This study confirmed that SHOX was expressed in vertebral body growth plates, which implies that its expression may also be associated with the scoliosis (and kyphosis) of Turner syndrome. SHOX expression is reduced in Turner syndrome (short stature). In this study, increased SHOX expression was demonstrated in idiopathic scoliosis (tall stature) and congenital scoliosis.

A sensitive assay for creatine kinase in serum samples dried on paper : enhanced thermal stability of the dried enzyme

A new bioluminescent creatine kinase (CK) assay using purified luciferase was used to analyse CK activity in serum samples dried on filter paper. Enzyme activity was preserved for over 1 wk on paper stored at room temperature. At 60°C, CK activity in liquid serum samples was rapidly inactivated, but the activity of enzyme stored on paper was preserved for at least 2 days.

Nutrient provision increases signalling and protein synthesis in human skeletal muscle after repeated sprints

The effect of nutrient availability on the acute molecular responses following repeated sprint exercise is unknown. The aim of this study was to determine skeletal muscle cellular and protein synthetic responses following repeated sprint exercise with nutrient provision. Eight healthy young male subjects undertook two sprint cycling sessions (10 × 6 s, 0.75 N m torque kg -1, 54 s recovery) with either pre-exercise nutrient (24 g whey, 4.8 g leucine, 50 g maltodextrin) or non-caloric placebo ingestion. Muscle biopsies were taken from vastus lateralis at rest, and after 15 and 240 min post-exercise recovery to determine muscle cell signalling responses and protein synthesis by primed constant infusion of L-[ring- 13C 6] phenylalanine. Peak and mean power outputs were similar between nutrient and placebo trials. Post-exercise myofibrillar protein synthetic rate was greater with nutrient ingestion compared with placebo ( ? 48%, P<0.05) but the rate of mitochondrial protein synthesis was similar between treatments. The increased myofibrillar protein synthesis following sprints with nutrient ingestion was associated with coordinated increases in Akt-mTOR-S6KrpS6 phosphorylation 15 min post-exercise (?200-600%, P<0.05), while there was no effect on these signalling molecules when exercise was undertaken in the fasted state. For the first time we report a beneficial effect of nutrient provision on anabolic signalling and muscle myofibrillar protein synthesis following repeated sprint exercise. Ingestion of protein/carbohydrate in close proximity to high-intensity sprint exercise provides an environment that increases cell signalling and protein synthesis.

Contraction-induced changes in TNFα and Akt-mediated signalling are associated with increased myofibrillar protein in rat skeletal muscle

Resistance training results in skeletal muscle hypertrophy, but the molecular signalling mechanisms responsible for this altered phenotype are incompletely understood. We used a resistance training (RT) protocol consisting of three sessions [day 1 (d1), day 3 (d3), day 5 (d5)] separated by 48 h recovery (squat exercise, 4 sets × 10 repetitions, 3 min recovery) to determine early signalling responses to RT in rodent skeletal muscle. Six animals per group were killed 3 h after each resistance training session and 24 and 48 h after the last training session (d5). There was a robust increase in TNF? protein expression, and IKKSer180/181 and p38MAPK Thr180/Tyr182 phosphorylation on d1 (P < 0.05), which abated with subsequent RT, returning to control levels by d5 for TNF? and IKK Ser180/181. There was a trend for a decrease in MuRF-1 protein expression, 48 h following d5 of training (P = 0.08). Notably, muscle myofibrillar protein concentration was elevated compared to control 24 and 48 h following RT (P < 0.05). AktSer473 and mTORSer2448 phosphorylation were unchanged throughout RT. Phosphorylation of p70S6k Thr389 increased 3 h post-exercise on d1, d3 and d5 (P < 0.05), whilst phosphorylation of S6Ser235/236 increased on d1 and d3 (P < 0.05). Our results show a rapid attenuation of inflammatory signalling with repeated bouts of resistance exercise, concomitant with summation in translation initiation signalling in skeletal muscle. Indeed, the cumulative effect of these signalling events was associated with myofibrillar protein accretion, which likely contributes to the early adaptations in response to resistance training overload in the skeletal muscle.

Effect of consecutive repeated sprint and resistance exercise bouts on acute adaptive responses in human skeletal muscle

We examined acute molecular responses in skeletal muscle to repeated sprint and resistance exercise bouts. Six men [age, 24.7 ± 6.3 yr; body mass, 81.6 ± 7.3 kg; peak oxygen uptake, 47 ± 9.9 ml·kg -1 ·min -1; one repetition maximum (1-RM) leg extension 92.2 ± 12.5 kg; means ± SD] were randomly assigned to trials consisting of either resistance exercise (8 × 5 leg extension, 80% 1-RM) followed by repeated sprints (10 × 6 s, 0.75 N·m torque·kg -1) or vice-versa. Muscle biopsies from vastus lateralis were obtained at rest, 15 min after each exercise bout, and following 3-h recovery to determine early signaling and mRNA responses. There was divergent exercise order-dependent phosphorylation of p70 S6K (S6K). Specifically, initial resistance exercise increased S6K phosphorylation (?75% P < 0.05), but there was no effect when resistance exercise was undertaken after sprints. Exercise decreased IGF-I mRNA following 3-h recovery (?50%, P = 0.06) independent of order, while muscle RING finger mRNA was elevated with a moderate exercise order effect (P < 0.01). When resistance exercise was followed by repeated sprints PGC-1? mRNA was increased (REX1-SPR2; P = 0.02) with a modest distinction between exercise orders. Repeated sprints may promote acute interference on resistance exercise responses by attenuating translation initiation signaling and exacerbating ubiquitin ligase expression. Indeed, repeated sprints appear to generate the overriding acute exercise-induced response when undertaking concurrent repeated sprint and resistance exercise. Accordingly, we suggest that sprint-activities are isolated from resistance training and that adequate recovery time is considered within periodized training plans that incorporate these divergent exercise modes.

mTOR function in skeletal muscle : a focal point for overnutrition and exercise

The mammalian target of rapamycin (mTOR) is a highly conserved atypical serine-threonine kinase that controls numerous functions essential for cell homeostasis and adaptation in mammalian cells via 2 distinct protein complex formations. Moreover, mTOR is a key regulatory protein in the insulin signalling cascade and has also been characterized as an insulin-independent nutrient sensor that may represent a critical mediator in obesity-related impairments of insulin action in skeletal muscle. Exercise characterizes a remedial modality that enhances mTOR activity and subsequently promotes beneficial metabolic adaptation in skeletal muscle. Thus, the metabolic effects of nutrients and exercise have the capacity to converge at the mTOR protein complexes and subsequently modify mTOR function. Accordingly, the aim of the present review is to highlight the role of mTOR in the regulation of insulin action in response to overnutrition and the capacity for exercise to enhance mTOR activity in skeletal muscle.

Consecutive bouts of diverse contractile activity alter acute responses in human skeletal muscle

We examined acute molecular responses in skeletal muscle to divergent exercise stimuli by combining consecutive bouts of resistance and endurance exercise. Eight men [22.9 ± 6.3 yr, body mass of 73.2 ± 4.5 kg, peak O2 uptake (V?O2peak) of 54.0 ± 5.7 ml·kg-1·min-1] were randomly assigned to complete trials consisting of either resistance exercise (8 x 5 leg extension, 80% 1 repetition maximum) followed by a bout of endurance exercise (30 min cycling, 70% V?O2peak) or vice versa. Muscle biopsies were obtained from the vastus lateralis at rest, 15 min after each exercise bout, and after 3 h of passive recovery to determine early signaling and mRNA responses. Phosphorylation of Akt and Akt1Ser473 were elevated 15 min after resistance exercise compared with cycling, with the greatest increase observed when resistance exercise followed cycling (?55%; P < 0.01). TSC2-mTOR-S6 kinase phosphorylation 15 min after each bout of exercise was similar regardless of the exercise mode. The cumulative effect of combined exercise resulted in disparate mRNA responses. IGF-I mRNA content was reduced when cycling preceded resistance exercise (-42%), whereas muscle ring finger mRNA was elevated when cycling was undertaken after resistance exercise (?52%; P < 0.05). The hexokinase II mRNA level was higher after resistance cycling (?45%; P < 0.05) than after cycling-resistance exercise, whereas modest increases in peroxisome proliferator-activated receptor gamma coactivator-1? mRNA did not reveal an order effect. We conclude that acute responses to diverse bouts of contractile activity are modified by the exercise order. Moreover, undertaking divergent exercise in close proximity influences the acute molecular profile and likely exacerbates acute "interference".

Effect of high-frequency resistance exercise on adaptive responses in skeletal muscle

PURPOSE: Regulation of skeletal muscle mass is highly dependent on contractile loading. The purpose of this study was to examine changes in growth factor and inflammatory pathways following high-frequency resistance training. METHODS: Using a novel design in which male Sprague-Dawley rats undertook a "stacked" resistance training protocol designed to generate a summation of transient exercise-induced signaling responses (four bouts of three sets × 10 repetitions of squat exercise, separated by 3 h of recovery), we determined the effects of high training frequency on signaling pathways and transcriptional activity regulating muscle mass. RESULTS: The stacked training regimen resulted in acute suppression of insulin-like growth factor 1 mRNA abundance (P < 0.05) and Akt phosphorylation (P < 0.05), an effect that persisted 48 h after the final training bout. Conversely, stacked training elicited a coordinated increase in the expression of tumor necrosis factor alpha, inhibitor kappa B kinase alpha/beta activity (P < 0.05), and p38 mitogen-activated protein kinase phosphorylation (P < 0.05) at 3 h after each training bout. In addition, the stacked series of resistance exercise bouts induced an increase in p70 S6 kinase phosphorylation 3 h after bouts ×3 and ×4, independent of the phosphorylation state of Akt. CONCLUSIONS: Our results indicate that high resistance training frequency extends the transient activation of inflammatory signaling cascades, concomitant with persistent suppression of key mediators of anabolic responses. We provide novel insights into the effects of the timing of exercise-induced overload and recovery on signal transduction pathways and transcriptional activity regulating skeletal muscle mass in vivo.

Exercise-induced phosphorylation of the novel Akt substrates AS160 and filamin A in human skeletal muscle

Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metabolic and transcriptional events. Akt/protein kinase B regulates metabolism and growth/muscle hypertrophy, but contraction effects on this target and its substrates are varied and may depend on the mode of the contractile stimulus. Accordingly, we determined the effects of endurance or resistance exercise on phosphorylation of Akt and downstream substrates in six trained cyclists who performed a single bout of endurance or resistance exercise separated by ?7 days. Muscle biopsies were taken from the vastus lateralis at rest and immediately after exercise. Akt Ser 473 phosphorylation was increased (1.8-fold; P = 0.011) after endurance but was unchanged after resistance exercise. Conversely, Akt Thr 308 phosphorylation was unaltered after either bout of exercise. Several exercise-responsive phosphoproteins were detected by immunoblot analysis with a phospho-Akt substrate antibody. pp160 and pp300 were identified as AS160 and filamin A, respectively, with increased phosphorylation (2.0- and 4.9-fold, respectively; P < 0.05) after endurance but not resistance exercise. In conclusion, AS160 and filamin A may provide an important link to mediate endurance exercise-induced bioeffects in skeletal muscle.