Scheduling Concurrent Training 48 versus 72 h after Simulated Match Play: Effects on Neuromuscular Function and Fatigue.

INTRODUCTION: Scheduling concurrent training (CT) during the in-season microcycle in field-based team sport is driven by prematch and postmatch recovery. This study examined the neuromuscular function, fatigue, and soreness responses to CT administered 48 h (match day (MD) + 2) versus 72 h (MD + 3) after match. METHODS: Ten male recreational-level team sport athletes were monitored daily during two 5-d microcycles, which began with a simulated match (Soccer-specific Aerobic Field Test (SAFT90)) and CT performed either 48 or 72 h after match. Maximal voluntary force, quadriceps maximum EMG, voluntary activation, muscle contractile function (evoked twitch responses), muscle soreness, and fatigue were assessed immediately before and after the SAFT90, and every 24 up to 96 h after match. Outcome measures were also assessed immediately after CT. The CT consisted of an intermittent sprint protocol and a lower limb resistance training session separated by 1 h. RESULTS: Immediately after the SAFT90 in both conditions, maximal voluntary force was below baseline (mean change (Δ), -14.6% ± 10.0%; P = 0.03), recovering 48 h post. Quadriceps contractile function (Δ, -31.5% ± 11.4%; P = 0.003) and voluntary activation (Δ, -8.9 ± 6.2%; P = 0.003) were also hampered after the SAFT90, recovering 24 h post in both conditions. In addition, the SAFT90 elicited elevated levels of fatigue and muscle soreness that recovered 24 h after the SAFT90 before increasing at 72 and 96 h post in the MD + 2 and MD + 3 conditions, respectively. CONCLUSIONS: Recovery of fatigue was only observed at the end of the microcycle when CT was prescribed on MD + 2. Therefore, CT scheduled early (MD + 2) in the microcycle might avoid compromising forthcoming match preparation.

The Global Characterisation of a Drug-Dendrimer Conjugate - PEGylated poly-lysine Dendrimer.

The recent emergence of drug-dendrimer conjugates within pharmaceutical industry research and development introduces a range of challenges for analytical and measurement science. These molecules are very high molecular weight (100-200kDa) with a significant degree of structural complexity. The characteristics and quality attributes that require understanding and definition, and impact efficacy and safety, are diverse. They relate to the intact conjugate, the various building blocks of these complex systems and the level of the free and bound active pharmaceutical ingredient (API). From an analytical and measurement science perspective, this necessitates the measurement of the molecular weight, impurity characterisation, the quantitation of the number of conjugated versus free API molecules, the determination of the impurity profiles of the building blocks, primary structure and both particle size and morphology. Here we report the first example of a global characterisation of a drug-dendrimer conjugate - PEGylated poly-lysine dendrimer currently under development (AZD0466). The impact of the wide variety of analytical and measurement techniques on the overall understanding of this complex molecular entity is discussed, with the relative capabilities of the various approaches compared. The results of this study are an essential platform for the research and development of the future generations of related dendrimer-based medicines.

Improving retention-time prediction in supercritical-fluid chromatography by multivariate modelling.

The prediction of chromatographic retention under supercritical-fluid chromatography (SFC) conditions was studied, using established and novel theoretical models over ranges of modifier content, pressure and temperature. Whereas retention models used for liquid chromatography often only consider the modifier fraction, retention in SFC depends much more strongly on pressure and temperature. The viability of combining several retention models into surfaces that describe the effects of both modifier fraction and pressure was investigated. The ability of commonly used retention models to describe retention as a function of modifier fraction, expressed either as mass or volume fraction, pressure and density was assessed. Using the multivariate surfaces, retention-time prediction for isocratic separations at constant temperature improved significantly compared to univariate modelling when both pressure and modifier fractions were changed. The "mixed-mode" model with an additional exponential pressure or density parameter was able to predict retention times within 5%, with the majority of the predictions within 2%. The use of mass fraction and density further improves retention modelling compared to volume fraction and pressure. These variables however, do require extra computations.

Impact of Microcycle Structures on Physical and Technical Outcomes During Professional Rugby League Training and Matches.

PURPOSE: To assess the impact of microcycle (MC) structures on physical and technical performances in rugby league training and matches. METHODS: Thirty-four professional rugby league players were monitored during all training sessions and matches across a single season wherein 2 different competition-phase MC structures were implemented. The first MC structure involved the first session on match day (MD) + 2 and the main stimulus delivered MD - 3, and the second structure delayed all sessions by 1 day (first session on MD + 3 and main session MD - 2; MC structure in the second half of the season). Physical output was quantified via relative total speed (in meters per minute), high-speed running (per minute; ≥4.0 m·s-1), and very-high-speed running (per minute; ≥5.5 m·s-1), measured using a global positioning system (10 Hz) in addition to accelerometer (100 Hz) metrics (PlayerLoad per minute and PlayerLoadslow per minute]) during training and matches. Technical performance (number of runs, meters gained, tackles made and missed) was recorded during matches. Generalized linear mixed models and equivalence tests were used to identify the impact of MC structure on physical and technical output. RESULTS: Nonequivalent increases in meters per minute, high-speed running per minute, and PlayerLoad per minute were observed for the first training stimulus in MC structure in the second half of the season with no practical difference in midcycle sessions observed. The MC structure in the second half of the season structure resulted in increased high-speed running per minute and decreased PlayerLoadslow per minute during MD with no differences observed in technical performance. CONCLUSIONS: Delaying the first training stimulus of the MC allowed for greater training load accumulation without negative consequences in selected match running and technical performance measures. This increased MC load may support the maintenance of physical capacities across the in-season.

Acute Neuromuscular Response to Team Sports-Specific Running, Resistance, and Concurrent Training: A Crossover Study.

PURPOSE: This study aimed to examine the changes in muscle contractile function, voluntary activation, and muscle damage after lower limb resistance training (RT), intermittent sprint exercise, and concurrent training (CT). METHODS: Ten male, recreational team sport athletes with a history of RT participated in a randomized crossover study involving an intermittent sprint protocol (ISP), lower limb RT, and CT (ISP and RT separated by 1 h). Before (PRE), immediately after (POST), 24 h and 48 h after each exercise condition, quadriceps muscle activation, voluntary activation, muscle contractile function (evoked twitch responses), creatine kinase, muscle soreness, and Profile of Mood States (POMS)-fatigue were recorded. RESULTS: Quadriceps contractile function was hampered in all conditions, with a significantly greater decline observed POST RT (58.4% ± 18.0%) and CT (54.8% ± 8.6%) compared with ISP (35.9% ± 10.7%, P < 0.05), recovering at 48 h after all exercise conditions. POMS-fatigue ratings increased at POST in all conditions with CT and ISP eliciting the greatest increase, returning to baseline 48 h after all exercise conditions. Quadriceps muscle soreness remained elevated from PRE at 48 h after all exercise conditions. No changes across time were observed for voluntary activation and quadriceps surface EMG amplitude after any exercise condition. The volume and load lifted in the RT session was unaffected by previous intermittent exercise (ISP) in CT. CONCLUSIONS: RT impairs contractile function, which is not exacerbated when performed 1 h after the ISP. Contractile function after all exercise conditions displayed the same recovery profile (48 h) despite the postexercise decrement being smaller after the ISP compared with RT and CT. Previous intermittent sprint exercise does not negatively affect the volume of exercise performed in a lower limb RT session.

The Hyperhydration Potential of Sodium Bicarbonate and Sodium Citrate.

Buffering agents have not been comprehensively profiled in terms of their capacity to influence water retention prior to exercise. The purpose of this investigation was to profile the fluid retention characteristics of sodium bicarbonate (BIC) and sodium citrate (CIT) to determine the efficacy of these buffering mediums as hyperhydrating agents. Nineteen volunteers (13 males and six females; age = 28.3 ± 4.9 years) completed three trials (randomized and cross-over design). For each trial, a baseline measurement of body mass, capillary blood, and urine was collected prior to ingestion of their respective condition (control condition [CON] = 25 ml/kg artificially sweetened water; BIC condition = CON + 7.5 g/L of sodium in the form of BIC; CIT condition = CON + 7.5 g/L of sodium in the form of CIT). The fluid loads were consumed in four equal aliquots (0, 20, 40 and 60 min; fluid intake was 1.972 ± 361 ml [CON]; 1.977 ± 360 ml [BIC]; 1.953 ± 352 ml [CIT]). Samples were recorded at 20 (body mass and urine) and 60 min (blood) intervals for 180 min. Blood buffering capacity (HCO3-) was elevated (p < .001) in both BIC (32.1 ± 2.2 mmol/L) and CIT (28.9 ± 3.8 mmol/L) at 180 min compared with CON (25.1 ± 1.8 mmol/L). Plasma volume expansion was greater (p < .001) in both BIC (8.1 ± 1.3%) and CIT (5.9 ± 1.8%) compared with CON (-1.1 ± 1.4%); whereas, total urine production was lower in BIC and CIT at 180 min (BIC vs. CON, mean difference of 370 ± 85 ml; p < .001; CIT vs. CON, mean difference of 239 ± 102 ml; p = .05). There were no increases observed in body mass (p = .9). Under resting conditions, these data suggest BIC and CIT induce a greater plasma hypervolemic response as compared with water alone.

Fatigue, pain, and the recovery of neuromuscular function after consecutive days of full-body resistance exercise in trained men.

PURPOSE: This study measured the self-reported level of fatigue, pain, and neuromuscular function of the knee extensor muscles over a three-day period that included two consecutive days of full-body resistance exercises. METHODS: 10 resistance-trained men performed two consecutive days of full-body resistance exercise. Muscle activation (electromyography and voluntary activation), contractility, and presynaptic inhibition of Ia afferents (homosynaptic and GABA mediated presynaptic inhibition) for the quadriceps were examined from femoral and posterior tibial nerve stimulation. RESULTS: Fatigue and pain were elevated after Day 1, and were not reduced to pre-exercise levels at the start of Day 2 (p < 0.05). Maximal voluntary torque (- 51.4 Nm, 95% CI = 12.4-90.4 Nm, p = 0.005) and rate of torque development (- 469 Nm.s-1, 95% CI = 109-829 Nm.s-1, p = 0.006) were reduced after Day 1, had recovered by Day 2, and did not change after the second training session. The maximal amplitude and rate of rise for the quadriceps twitch were reduced after both training sessions (p < 0.01), with recovery 24 h each session. The maximal amplitude and rate of early muscle activation were reduced after Day 1 (p < 0.01), but no changes were observed for voluntary activation, H-reflex size and shape, or measures of Ia presynaptic inhibition. CONCLUSION: Resistance exercise in the presence of elevated fatigue and pain from a previous training session does not worsen recovery, or lead to significant alterations in quadriceps neuromuscular function. Reduction in muscle contractility, in the absence of declines in muscle activation, does not lead to decreased voluntary torque.

The Impact of Individualizing Sodium Bicarbonate Supplementation Strategies on World-Class Rowing Performance.

Contemporary meta-analyses have generally demonstrated a positive effect of sodium bicarbonate (NaHCO3) supplementation on exercise performance. However, despite these claims, there is limited data on contrasting individualized and standardized timing of NaHCO3 ingestion prior to exercise to further enhance performance outcomes. Purpose: To determine whether NaHCO3 ingestion timing impacts 2,000-m rowing time-trial (TT) performance in elite-level rowers (Senior National team including Olympic/World Championships level) adhering to their own individualized pre-race strategies (e.g. nutrition, warm-up, etc.). Methods: Twenty three (n = 23) rowers across two research centers (using the exact same methods/protocols) completed three trials: NaHCO3 loading profile at rest to determine the individual's time-to-peak bicarbonate concentration [ HCO 3 - ], followed by two randomized 0.3 g·kgBM-1 NaHCO3 supplementation experimental trials conducted at different time points [consensus timing (CON): TT performed 60 min post-NaHCO3 ingestion; and individualized peak (IP): TT performed at the rower's individual peak [ HCO 3 - ] determined from the profiling trial post-NaHCO3 ingestion]. Results: There was a significant mean difference of +2.9 [± 0.4 mmol·L-1 HCO 3 - for IP vs. CON (95% CI 2.0 to 3.8 mmol·L-1); p = 0.02; d = 1.08] at pre warm-up, but not immediately prior to the TT (post warm-up). Performance times were significantly different between IP (367.0 ± 10.5 s) vs. CON (369.0 ± 10.3 s); p = 0.007; d = 0.15). Conclusions: The present study demonstrated a small but significant performance effect of an individualized NaHCO3 ingestion strategy. Similarities after warm-up between pre-TT sHCO 3 - values (CON ~ + 5.5 mmol·L-1; IP ~ + 6 mmol·L-1), however, would suggest this effect was not a result of any meaningful differences in blood alkalinity.

Topical Sodium Bicarbonate: No Improvement in Blood Buffering Capacity or Exercise Performance.

PURPOSE: To assess the efficacy of a topical sodium bicarbonate (0.3 g/kg body weight NaHCO3) application (PR lotion; Amp Human) on blood buffering capacity and performance in recreationally active participants (study A) and moderately trained athletes (study B). METHODS: In Study A, 10 participants completed 2 experimental trials: oral NaHCO3 (0.3 g/kg body weight + placebo lotion) or PR lotion (0.9036 g/kg body weight + oral placebo) applied 90 minutes prior to a cycling task to exhaustion (30-s sprints at 120% peak power output with 30-s rest). Capillary blood was collected and analyzed for pH, bicarbonate, and lactate every 10 minutes throughout the 90-minute loading period and postexercise at 5, 10, and 15 minutes. In Study B, 10 cyclists/triathletes completed 2 experimental trials, applying either PR or placebo lotion 30 minutes prior to a cycling performance task (3 × 30-s maximal sprints with 90-s recovery). Capillary blood samples were collected at baseline, preexercise, and postexercise and analyzed as per study A. RESULTS: In Study A, pH and bicarbonate were significantly elevated from baseline after 10 minutes in the oral NaHCO3 condition and throughout recovery compared with no elevation in the PR lotion condition (P < .001). No differences in cycling time occurred between PR lotion (349 [119] s) and oral NaHCO3 (363 [80] s; P = .697). In Study B, no differences in blood parameters, mean power (P = .108), or peak power (P = .448) were observed between conditions. CONCLUSIONS: PR lotion was ineffective in altering blood buffering capacity or enhancing performance in either trained or untrained individuals.

Changes in Fatigue Are the Same for Trained Men and Women after Resistance Exercise.

PURPOSE: To measure changes in fatigue and knee-extensor torque in the 48 h after trained men and women completed a full-body resistance exercise session. METHODS: Eight trained women (mean ± SD: age, 25.6 ± 5.9 yr; height, 1.68 ± 0.06 m; mass, 71.0 ± 8.6 kg) and eight trained men (age, 25.5 ± 6.2 yr; height, 1.79 ± 0.05 m; mass, 86.4 ± 9.8 kg) performed a full-body resistance exercise session based on real-world athletic practice. Measurements were performed before and after the exercise session, as well as 1, 24, and 48 h after the session. Fatigue and pain were measured with standardized self-report measures. Maximal isometric contractions with the knee extensors and superimposed femoral nerve stimulation were performed to examine maximal torque, rate of torque development, voluntary activation, and muscle contractility. Two sets of 10 isokinetic contractions (60°·s) with the knee extensors were performed during the protocol with use of near-infrared spectroscopy to assess muscle oxygenation. EMG were recorded from two quadriceps muscles during all isometric and isokinetic contractions. RESULTS: Fatigue was increased from baseline for both sexes until 48 h after training (P < 0.001). Maximal torque and evoked twitch amplitudes were similarly reduced after exercise for men and women (P < 0.001). Voluntary activation and EMG amplitudes were unchanged after the training session. Muscle oxygenation was 13.3% ± 17.4% (P = 0.005) greater for women during the isokinetic repetitions, and the values were unchanged after the training session. CONCLUSIONS: This is the first study to show similar changes in the fatigue reported by trained men and women in the 48 h after a training session involving full-body resistance exercises. Sex differences in muscle oxygenation during exercise do not influence the reductions in muscle force, activation, or contractility after the training session.

Biological maturation and match running performance: A national football (soccer) federation perspective.

OBJECTIVES: To examine the influence of maturation and its interaction with playing position upon physical match performances in U15 footballers from a national federation. DESIGN: Observational study. METHODS: 278 male outfield players competing in a national tournament were assessed for somatic maturity and match physical performances according to playing position. Stature, sitting height, and body mass were measured and entered into an algorithm to estimate the age at peak height velocity (APHV). Players match movements were recorded by Global Positioning System devices (10 Hz), to determine peak speed, and total- (TD), low-speed running (LSR; ≤13.0 km h-1), high-speed running (HSR; 13.1-16.0 km h-1), very high-speed running (VHSR; 16.1-20.0 km h-1) and sprint distances (SPR; >20.0 km h-1) expressed relative to match exposure (m min-1). RESULTS: Linear-mixed models using log transformed response variables revealed a significant contribution of estimated APHV upon TD (1.01; 95% CI: 0.99-1.02 m·min-1; p < 0.001), HSR (1.05; 95% CI: 0.98-1.13 m min-1; p < 0.001) and VHSR (1.07; 95% CI: 1.00-1.14 m min-1; p = 0.047). An increase by one year in APHV was associated with an increase of 0.6, 5.4 and 6.9% in TD, HSR and VHSR respectively. No effects of APHV were observed for LSR, SPR, and peak speed. Further, no APHV effects were observed relative to players' field position. CONCLUSIONS: Later maturing players covered substantially more higher-intensity (HSR and VHSR) running in matches, irrespective of playing position. The greater match intensity of later maturing players may inform talent identification and athletic development processes within a national federation.

Scheduling of training and recovery during the in-season weekly micro-cycle: Insights from team sport practitioners.

The aim of this study was to determine the in-season micro-cycle scheduling strategies used in professional team sport with particular reference to the reasoning and perceptions that underpin current practice. An online survey was completed by 35 practitioners from professional collision (C; Australian rules football: n = 9; rugby league: n = 6; rugby union: n = 2) and non-collision (NC; soccer; n = 18) sports. Respondents identified a common 48 h post-match recovery period, with few scheduling training stimuli during this period (NC: range: 0-17%; C: range: 0-47%). The majority (C: 78%; NC: 89%) of resistance training sessions were concurrent in nature, often delivering resistance training in the afternoon following field-based training (C: 72%; NC: 92%). NC practitioners were less satisfied with the maintenance of strength, power and hypertrophy (33-44%) versus C sports (59-76%), reflected by the lower frequency of prescription reported (C: 2.4 ± 0.6; NC: 1.5 ± 0.9; p = .003). Cold water immersion following both matches and daily training was highly prevalent, as was the adoption of self-report tools to monitor recovery. The congested and concurrent nature of training prescription during the micro-cycle, together with the universal adoption of routine cold water immersion warrants further investigation to inform evidence-based prescription and scheduling of training and recovery modalities in professional team sports.

Weed hygiene practices in rural industries and public land management: Variable knowledge, patchy implementation, inconsistent coordination.

Weed management science and practice largely focuses on eradicating, containing and reducing existing weed populations; the focus is on plants in situ. More recently, the redefinition of biosecurity to include weeds has seen greater attention paid to preventing the introduction of weeds to previously uninfested areas within countries. Thus weed hygiene has come to the fore, with a growing number of publications recommending a diverse range of practices to minimise the spread of weeds across farm, regional and state boundaries. Yet little is known about the uptake of weed hygiene practices. The aim of this paper is to evaluate the extent to which best practice weed hygiene is being implemented on, across and along private and public lands. Telephone interviews were conducted with 54 private and public land managers, weed contractors, and agricultural transport operators in New South Wales, Australia. Vehicle hygiene was commonly undertaken across all stakeholder groups when it was consistent with other goals, requirements or norms. Other practices, such as sequencing harvesting from least to most weedy paddocks or including weed hygiene clauses in contracts were often known, but rarely practiced because of the onerous labour and financial costs or concerns about social etiquette. Individual commitment to weed hygiene efforts were also undermined by intra and inter-organisational coordination challenges. Public debate and assessment are needed on the benefits and costs to society of weed hygiene compared to in situ weed control to determine where best to invest limited time and resources.

Opportunities for adaptive online collaboration to enhance rural land management.

Cross-property cooperation has the potential to enhance the effectiveness of environmental management actions that cut across property boundaries. Online tools can facilitate this and overcome barriers to landholder engagement in collaborative management. However, collaborative online tools need to be designed and tailored to users' needs and values, and landholder participation in the development process is critical to ensuring uptake and long-term use. This article presents a case study from the Central Tablelands region of New South Wales, Australia, where landholders have been involved in participatory development of a new online collaboration tool. The case study results highlight the significance of issues such as internet access, privacy, technical proficiency and differing stakeholder objectives. A landholder survey identified mapping and the uploading of monitoring data as important functions for the online tool, but these were not rated as highly as functions relating to data security, sharing settings and key term searches. Consequently, we recommend that a future online collaboration tool for the region is not framed specifically as a mapping or citizen science tool, but rather as an adaptive collaboration and communication tool that can incorporate a variety of data types and formats and be modified over time in line with changing landholder needs.

Acute attenuation of fatigue after sodium bicarbonate supplementation does not manifest into greater training adaptations after 10-weeks of resistance training exercise.

PURPOSE: In two concurrent studies, we aimed to a) confirm the acute effect of 0.3 g·kg-1 body weight (BW) sodium bicarbonate (NaHCO3) supplementation on central and peripheral mechanisms associated with explosive power (Study 1) and b) determine whether chronic NaHCO3 supplementation would improve the adaptive response of the neuromuscular system during a 10-week resistance training program (Study 2). METHODS: Eight resistance trained participants volunteered after providing written consent. The experimental design consisted of a week of baseline testing, followed by ten weeks of training with progress measures performed in Week 5. Study 1 involved neuromuscular measurements before and after the leg extension portion of a power based training session performed in Week 1. Changes in maximal torque (MVT) and rates of torque development (RTD), along with other variables derived from femoral nerve stimulation (e.g. voluntary activation, neural recruitment) were analysed to determine the extent of fatigue under NaHCO3 or placebo conditions. Changes in these same variables, coupled with functional 1-repetition maximum leg extension strength, were measured in Study 2 from baseline (Week 0) to Week 5, and again at Week 10. RESULTS AND CONCLUSION: In Study 1, we observed a decline after the leg extension task in both MVT (~ 30%) and rates of torque production (RTD) irrespective of acid-base status, however the decline in maximal RTD (RTDMAX) was nearly 20% less in the NaHCO3 condition when compared to placebo (mean difference of 294.8 ± 133.4 Nm·s-1 (95% CI -583.1 to -6.5 Nm, p < 0.05)). The primary finding in Study 2, however, suggests that introducing NaHCO3 repeatedly during a 10-week RT program does not confer any additional benefit to the mechanisms (and subsequent adaptive processes) related to explosive power production.

The fatigue of a full body resistance exercise session in trained men.

OBJECTIVES: We examined the fatigue and recovery for 48h following a full-body resistance exercise session in trained men. DESIGN: Experimental cross-sectional study. METHODS: Eight resistance trained men volunteered to participate (mean±SD; age 27.0±6.0 years, height 1.79±0.05m, weight 81.8±6.8kg, training experience 7.8±5.0 years). Fatigue and pain was measured before, after, 1h post, 24h and 48h post the full-body resistance exercise session, which was based on in-season models used in contact team sports (e.g. AFL, NRL). Other measures included maximal torque and rate of torque development, central motor output (quadriceps muscle activation, voluntary activation, H-reflexes), and muscle contractility (evoked twitch responses). Linear mixed-model ANOVA procedures were used for data analysis. RESULTS: Fatigue, soreness, and muscle pain did not return to pre-exercise levels until after 48h rest. Quadriceps maximal torque and muscle contractility were reduced from pre-exercise (p<0.01), and did not return to pre-exercise levels until 24h. Early rates of torque development and muscle activation were unchanged. The amplitude and slope of the normalized quadriceps H-reflex was higher immediately after exercise (p<0.05). CONCLUSIONS: Full-body resistance exercise including multiple lower limb movements immediately reduced maximal torque, muscle contractility, and increased pain. While recovery of voluntary and evoked torque was complete within a day, 48h rest was required for fatigue and pain to return to baseline. Maximal voluntary effort may be compromised for lower-limb training (i.e. sprinting, jumping) prescribed in the 48h after the session.

Dysbindin Deficiency Modifies the Expression of GABA Neuron and Ion Permeation Transcripts in the Developing Hippocampus.

The neurodevelopmental factor dysbindin is required for synapse function and GABA interneuron development. Dysbindin protein levels are reduced in the hippocampus of schizophrenia patients. Mouse dysbindin genetic defects and other mouse models of neurodevelopmental disorders share defective GABAergic neurotransmission and, in several instances, a loss of parvalbumin-positive interneuron phenotypes. This suggests that mechanisms downstream of dysbindin deficiency, such as those affecting GABA interneurons, could inform pathways contributing to or ameliorating diverse neurodevelopmental disorders. Here we define the transcriptome of developing wild type and dysbindin null Bloc1s8sdy/sdy mouse hippocampus in order to identify mechanisms downstream dysbindin defects. The dysbindin mutant transcriptome revealed previously reported GABA parvalbumin interneuron defects. However, the Bloc1s8sdy/sdy transcriptome additionally uncovered changes in the expression of molecules controlling cellular excitability such as the cation-chloride cotransporters NKCC1, KCC2, and NCKX2 as well as the potassium channel subunits Kcne2 and Kcnj13. Our results suggest that dysbindin deficiency phenotypes, such as GABAergic defects, are modulated by the expression of molecules controlling the magnitude and cadence of neuronal excitability.

The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin.

AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms.

Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65-85; 100-135; 160-250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC-MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose (r² = 0.45; P = 0.002), acetyl-carnitine (r² = 0.50; P = 0.001), glutamine (r² = 0.59; P = 0.0002); and negative correlations with taurine (r² = 0.74; P < 0.0001), fumarate (r² = 0.45; P = 0.003), aspartate (r² = 0.59; P = 0.0002), alanine (r² = 0.66; P < 0.0001) and phosphocholine (r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.

Protective Effect of Creatine Elevation against Ischaemia Reperfusion Injury Is Retained in the Presence of Co-Morbidities and during Cardioplegia.

AIMS: Ischaemic heart disease is most prevalent in the ageing population and often exists with other comorbidities; however the majority of laboratory research uses young, healthy animal models. Several recent workshops and focus meetings have highlighted the importance of using clinically relevant models to help aid translation to realistic patient populations. We have previously shown that mice over-expressing the creatine transporter (CrT-OE) have elevated intracellular creatine levels and are protected against ischaemia-reperfusion injury. Here we test whether elevating intracellular creatine levels retains a cardioprotective effect in the presence of common comorbidities and whether it is additive to protection afforded by hypothermic cardioplegia. METHODS AND RESULTS: CrT-OE mice and wild-type controls were subjected to transverse aortic constriction for two weeks to induce compensated left ventricular hypertrophy (LVH). Hearts were retrogradely perfused in Langendorff mode for 15 minutes, followed by 20 minutes ischaemia and 30 minutes reperfusion. CrT-OE hearts exhibited significantly improved functional recovery (Rate pressure product) during reperfusion compared to WT littermates (76% of baseline vs. 59%, respectively, P = 0.02). Aged CrT-OE mouse hearts (78±5 weeks) also had enhanced recovery following 15 minutes ischaemia (104% of baseline vs. 67%, P = 0.0007). The cardioprotective effect of hypothermic high K+ cardioplegic arrest, as used during cardiac surgery and donor heart transplant, was further enhanced in prolonged ischaemia (90 minutes) in CrT-OE Langendorff perfused mouse hearts (76% of baseline vs. 55% of baseline as seen in WT hearts, P = 0.02). CONCLUSIONS: These observations in clinically relevant models further support the development of modulators of intracellular creatine content as a translatable strategy for cardiac protection against ischaemia-reperfusion injury.