A comparison of the histochemical and image-derived proteoglycan content of articular cartilage

There are several methods for determining the proteoglycan content of cartilage in biomechanics experiments. Many of these include assay-based methods and the histochemistry or spectrophotometry protocol where quantification is biochemically determined. More recently a method based on extracting data to quantify proteoglycan content has emerged using the image processing algorithms, e.g., in ImageJ, to process histological micrographs, with advantages including time saving and low cost. However, it is unknown whether or not this image analysis method produces results that are comparable to those obtained from the biochemical methodology. This paper compares the results of a well-established chemical method to those obtained using image analysis to determine the proteoglycan content of visually normal (n=33) and their progressively degraded counterparts with the protocols. The results reveal a strong linear relationship with a regression coefficient (R2) = 0.9928, leading to the conclusion that the image analysis methodology is a viable alternative to the spectrophotometry.

The effect of three different (-135°C) whole body cryotherapy exposure durations on elite rugby league players

Background: Whole body cryotherapy (WBC) is the therapeutic application of extreme cold air for a short duration. Minimal evidence is available for determining optimal exposure time. Purpose: To explore whether the length of WBC exposure induces differential changes in inflammatory markers, tissue oxygenation, skin and core temperature, thermal sensation and comfort. Method: This study was a randomised cross over design with participants acting as their own control. Fourteen male professional first team super league rugby players were exposed to 1, 2, and 3 minutes of WBC at -135°C. Testing took place the day after a competitive league fixture, each exposure separated by seven days. Results: No significant changes were found in the inflammatory cytokine interleukin six. Significant reductions (p<0.05) in deoxyhaemoglobin for gastrocnemius and vastus lateralis were found. In vastus lateralis significant reductions (p<0.05) in oxyhaemoglobin and tissue oxygenation index (p<0.05) were demonstrated. Significant reductions (p<0.05) in skin temperature were recorded. No significant changes were recorded in core temperature. Significant reductions (p<0.05) in thermal sensation and comfort were recorded. Conclusion: Three brief exposures to WBC separated by 1 week are not sufficient to induce physiological changes in IL-6 or core temperature. There are however significant changes in tissue oxyhaemoglobin, deoxyhaemoglobin, tissue oxygenation index, skin temperature and thermal sensation. We conclude that a 2 minute WBC exposure was the optimum exposure length at temperatures of -135°C and could be applied as the basis for future studies.

Alcohol ingestion impairs maximal post-exercise rates of myofibrillar protein synthesis following a single bout of concurrent training

Introduction The culture in many team sports involves consumption of large amounts of alcohol after training/competition. The effect of such a practice on recovery processes underlying protein turnover in human skeletal muscle are unknown. We determined the effect of alcohol intake on rates of myofibrillar protein synthesis (MPS) following strenuous exercise with carbohydrate (CHO) or protein ingestion. Methods In a randomized cross-over design, 8 physically active males completed three experimental trials comprising resistance exercise (8×5 reps leg extension, 80% 1 repetition maximum) followed by continuous (30 min, 63% peak power output (PPO)) and high intensity interval (10×30 s, 110% PPO) cycling. Immediately, and 4 h post-exercise, subjects consumed either 500 mL of whey protein (25 g; PRO), alcohol (1.5 g·kg body mass−1, 12±2 standard drinks) co-ingested with protein (ALC-PRO), or an energy-matched quantity of carbohydrate also with alcohol (25 g maltodextrin; ALC-CHO). Subjects also consumed a CHO meal (1.5 g CHO·kg body mass−1) 2 h post-exercise. Muscle biopsies were taken at rest, 2 and 8 h post-exercise. Results Blood alcohol concentration was elevated above baseline with ALC-CHO and ALC-PRO throughout recovery (P<0.05). Phosphorylation of mTORSer2448 2 h after exercise was higher with PRO compared to ALC-PRO and ALC-CHO (P<0.05), while p70S6K phosphorylation was higher 2 h post-exercise with ALC-PRO and PRO compared to ALC-CHO (P<0.05). Rates of MPS increased above rest for all conditions (~29–109%, P<0.05). However, compared to PRO, there was a hierarchical reduction in MPS with ALC-PRO (24%, P<0.05) and with ALC-CHO (37%, P<0.05). Conclusion We provide novel data demonstrating that alcohol consumption reduces rates of MPS following a bout of concurrent exercise, even when co-ingested with protein. We conclude that alcohol ingestion suppresses the anabolic response in skeletal muscle and may therefore impair recovery and adaptation to training and/or subsequent performance.

Physiological tolerance times while wearing explosive ordnance disposal protective clothing in simulated environmental extremes

Explosive ordnance disposal (EOD) technicians are required to wear protective clothing to protect themselves from the threat of overpressure, fragmentation, impact and heat. The engineering requirements to minimise these threats results in an extremely heavy and cumbersome clothing ensemble that increases the internal heat generation of the wearer, while the clothing’s thermal properties reduce heat dissipation. This study aimed to evaluate the heat strain encountered wearing EOD protective clothing in simulated environmental extremes across a range of differing work intensities. Eight healthy males [age 25±6 years (mean ± sd), height 180±7 cm, body mass 79±9 kg, V˙O2max 57±6 ml.kg−1.min−1] undertook nine trials while wearing an EOD9 suit (weighing 33.4 kg). The trials involved walking on a treadmill at 2.5, 4 and 5.5 km⋅h−1 at each of the following environmental conditions, 21, 30 and 37°C wet bulb globe temperature (WBGT) in a randomised controlled crossover design. The trials were ceased if the participants’ core temperature reached 39°C, if heart rate exceeded 90% of maximum, if walking time reached 60 minutes or due to fatigue/nausea. Tolerance times ranged from 10–60 minutes and were significantly reduced in the higher walking speeds and environmental conditions. In a total of 15 trials (21%) participants completed 60 minutes of walking; however, this was predominantly at the slower walking speeds in the 21°C WBGT environment. Of the remaining 57 trials, 50 were ceased, due to attainment of 90% maximal heart rate. These near maximal heart rates resulted in moderate-high levels of physiological strain in all trials, despite core temperature only reaching 39°C in one of the 72 trials.

Expression, regulation and putative nutrient-sensing function of taste GPCRs in the heart

G protein-coupled receptors (GPCRs) are critical for cardiovascular physiology. Cardiac cells express >100 nonchemosensory GPCRs, indicating that important physiological and potential therapeutic targets remain to be discovered. Moreover, there is a growing appreciation that members of the large, distinct taste and odorant GPCR families have specific functions in tissues beyond the oronasal cavity, including in the brain, gastrointestinal tract and respiratory system. To date, these chemosensory GPCRs have not been systematically studied in the heart. We performed RT-qPCR taste receptor screens in rodent and human heart tissues that revealed discrete subsets of type 2 taste receptors (TAS2/Tas2) as well as Tas1r1 and Tas1r3 (comprising the umami receptor) are expressed. These taste GPCRs are present in cultured cardiac myocytes and fibroblasts, and are enriched in myocytes, which we corroborated using in situ hybridization. Tas1r1 gene-targeted mice (Tas1r1Cre/Rosa26tdRFP) strikingly recapitulated these data. In vivo taste receptor expression levels were developmentally regulated in the postnatal period. Intriguingly, several Tas2rs were upregulated in cultured rat myocytes and in mouse heart in vivo following starvation. The discovery of taste GPCRs in the heart opens an exciting new field of cardiac research. We predict that these taste receptors may function as nutrient sensors in the heart.

Mechanical tension as a driver of connective tissue growth in vitro

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Bug reproduction : a collaborative practice within software maintenance activities

Software development settings provide a great opportunity for CSCW researchers to study collaborative work. In this paper, we explore a specific work practice called bug reproduction that is a part of the software bug-fixing process. Bug re-production is a highly collaborative process by which software developers attempt to locally replicate the ‘environment’ within which a bug was originally encountered. Customers, who encounter bugs in their everyday use of systems, play an important role in bug reproduction as they provide useful information to developers, in the form of steps for reproduction, software screenshots, trace logs, and other ways to describe a problem. Bug reproduction, however, poses major hurdles in software maintenance as it is often challenging to replicate the contextual aspects that are at play at the customers’ end. To study the bug reproduction process from a human-centered perspective, we carried out an ethnographic study at a multinational engineering company. Using semi-structured interviews, a questionnaire and half-a-day observation of sixteen software developers working on different software maintenance projects, we studied bug reproduction. In this pa-per, we present a holistic view of bug reproduction practices from a real-world set-ting and discuss implications for designing tools to address the challenges developers face during bug reproduction.

Characterization of ENU-induced mutations in red blood cell structural proteins

Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia (kiwifruit) hybrid population

Background Red colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour. Results Four inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals. A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia. The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays. Conclusions The transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR βgeo/+ mice were generated from embryonic stem (ES) cells with a gene trap integration of a β-galactosidase-neomycin phosphotransferase (βgeo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GRβgeo/+ mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin- aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GRβgeo/+ mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GRβgeo/+ mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet.

The effects of metabolic work rate and ambient environment on physiological tolerance times while wearing explosive and chemical personal protective equipment

This study evaluated the physiological tolerance times when wearing explosive and chemical (>35kg) personal protective equipment (PPE) in simulated environmental extremes across a range of differing work intensities. Twelve healthy males undertook nine trials which involved walking on a treadmill at 2.5, 4 and 5.5 km.h-1 in the following environmental conditions, 21, 30 and 37 °C wet bulb globe temperature (WBGT). Participants exercised for 60 min or until volitional fatigue, core temperature reached 39 °C, or heart rate exceeded 90% of maximum. Tolerance time, core temperature, skin temperature, mean body temperature, heart rate and body mass loss were measured. Exercise time was reduced in the higher WBGT environments (WBGT37

Ibuprofen supplementation and its effects on NF-KB activation in skeletal muscle following resistance exercise

Resistance exercise triggers a subclinical inflammatory response that plays a pivotal role in skeletal muscle regeneration. Nuclear factor‐κB (NF‐κB) is a stress signalling transcription factor that regulates acute and chronic states of inflammation. The classical NF‐κB pathway regulates the early activation of post‐exercise inflammation; however there remains scope for this complex transcription factor to play a more detailed role in post‐exercise muscle recovery. Sixteen volunteers completed a bout of lower body resistance exercise with the ingestion of three 400 mg doses of ibuprofen or a placebo control. Muscle biopsy samples were obtained prior to exercise and at 0, 3 and 24 h post‐exercise and analysed for key markers of NF‐κB activity. Phosphorylated p65 protein expression and p65 inflammatory target genes were elevated immediately post‐exercise independent of the two treatments. These changes did not translate to an increase in p65 DNA binding activity. NF‐κB p50 protein expression and NF‐κB p50 binding activity were lower than pre‐exercise at 0 and 3 h post‐exercise, but were elevated at 24 h post‐exercise. These findings provide novel evidence that two distinct NF‐κB pathways are active in skeletal muscle after resistance exercise. The initial wave of activity involving p65 resembles the classical pathway and is associated with the onset of an acute inflammatory response. The second wave of NF‐κB activity comprises the p50 subunit, which has been previously shown to resolve an acute inflammatory program. The current study showed no effect of the ibuprofen treatment on markers of the NF‐κB pathway, however examination of the within group effects of the exercise protocol suggests that this pathway warrants further research.

Multilevel competing risk models to evaluate the risk of nosocomial infection

Introduction Risk factor analyses for nosocomial infections (NIs) are complex. First, due to competing events for NI, the association between risk factors of NI as measured using hazard rates may not coincide with the association using cumulative probability (risk). Second, patients from the same intensive care unit (ICU) who share the same environmental exposure are likely to be more similar with regard to risk factors predisposing to a NI than patients from different ICUs. We aimed to develop an analytical approach to account for both features and to use it to evaluate associations between patient- and ICU-level characteristics with both rates of NI and competing risks and with the cumulative probability of infection. Methods We considered a multicenter database of 159 intensive care units containing 109,216 admissions (813,739 admission-days) from the Spanish HELICS-ENVIN ICU network. We analyzed the data using two models: an etiologic model (rate based) and a predictive model (risk based). In both models, random effects (shared frailties) were introduced to assess heterogeneity. Death and discharge without NI are treated as competing events for NI. Results There was a large heterogeneity across ICUs in NI hazard rates, which remained after accounting for multilevel risk factors, meaning that there are remaining unobserved ICU-specific factors that influence NI occurrence. Heterogeneity across ICUs in terms of cumulative probability of NI was even more pronounced. Several risk factors had markedly different associations in the rate-based and risk-based models. For some, the associations differed in magnitude. For example, high Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were associated with modest increases in the rate of nosocomial bacteremia, but large increases in the risk. Others differed in sign, for example respiratory vs cardiovascular diagnostic categories were associated with a reduced rate of nosocomial bacteremia, but an increased risk. Conclusions A combination of competing risks and multilevel models is required to understand direct and indirect risk factors for NI and distinguish patient-level from ICU-level factors.

A comparison between conductive and infrared devices for measuring mean skin temperature at rest, during exercise in the heat, and recovery

Purpose: Skin temperature assessment has historically been undertaken with conductive devices affixed to the skin. With the development of technology, infrared devices are increasingly utilised in the measurement of skin temperature. Therefore, our purpose was to evaluate the agreement between four skin temperature devices at rest, during exercise in the heat, and recovery. Methods: Mean skin temperature (T̅sk) was assessed in thirty healthy males during 30 min rest (24.0± 1.2°C, 56 ± 8%), 30 min cycle in the heat (38.0 ± 0.5°C, 41 ± 2%), and 45 min recovery(24.0 ± 1.3°C, 56 ± 9%). T̅sk was assessed at four sites using two conductive devices(thermistors, iButtons) and two infrared devices (infrared thermometer, infrared camera). Results: Bland–Altman plots demonstrated mean bias ± limits of agreement between the thermistors and iButtons as follows (rest, exercise, recovery): -0.01 ± 0.04, 0.26 ± 0.85, -0.37 ± 0.98°C; thermistors and infrared thermometer: 0.34 ± 0.44, -0.44 ± 1.23, -1.04 ± 1.75°C; thermistors and infrared camera (rest, recovery): 0.83 ± 0.77, 1.88 ± 1.87°C. Pairwise comparisons of T̅sk found significant differences (p < 0.05) between thermistors and both infrared devices during resting conditions, and significant differences between the thermistors and all other devices tested during exercise in the heat and recovery. Conclusions: These results indicate poor agreement between conductive and infrared devices at rest, during exercise in the heat, and subsequent recovery. Infrared devices may not be suitable for monitoring T̅sk in the presence of, or following, metabolic and environmental induced heat stress.

Specificity and context in post-exercise recovery: It is not a one-size-fits-all approach

The concept of specificity of exercise prescription and training is a longstanding and widely accepted foundation of the exercise sciences. Simply, the principle holds that training adaptations are achieved relative to the stimulus applied. That is, the manipulation of training variables (e.g. intensity or loading, mode, volume and frequency) directly influences the acute training stimulus, and so the long-term adaptive response (Young et al., 2001; Bird et al., 2005). Translating this concept to practice then recommends that exercise be prescribed specific to the desired outcomes, and the more closely this is achieved, the greater the performance gain is likely to be. However, the cardiovascular and metabolic adaptations traditionally associated with long, slow distance training types, similarly achieved using high-intensity training methods (for a review see Gibala et al., 2012), highlights understanding of underlying physiology as paramount for effective training program design. Various other factors including illness, sleep and psychology also impact on the training stimulus (Halson, 2014) and must be managed collectively with appropriate post-exercise recovery to continue performance improvements and reduce overtraining and injury risks (Kenttä and Hassmén, 1998).