Body Fluid Estimation Via Segmental Multi-Frequency Bioelectrical Impedance Analysis Following Acute Resistance Exercise

Segmental multi-frequency bioelectrical impedance analysis (S-MFBIA) estimates body composition and fluids by passing electrical currents through the body and can separate the body into distinct segments. The minimum required abstention from exercise before S-MFBIA is unclear. PURPOSE: The purpose of this study was to monitor changes in total body water (TBW), intracellular water (ICW), and extracellular water (ECW) estimated via S-MFBIA following acute, localized bouts of resistance exercise (RE). METHODS: Thirty-two female (n = 18; age: 22.7 ± 1.4 y; height: 167.5 ± 7.5 cm; body mass: 66.6 ± 14.5 kg; body fat: 30.3 ± 6.2%) and male (n= 14; age: 24.2 ± 2.9; height: 178.7 ± 5.3; body mass: 85.7 ± 7.8 kg; body fat: 19.6 ± 6.9%) resistance-trained volunteers completed three randomly assigned conditions in a crossover design. Each RE protocol (REUPPER or RELOWER) consisted of three exercises and began with two warm-up sets of 12-15 repetitions per exercise. This was followed by a RE circuit of 5 sets of 10 repetitions per exercise with a one-minute rest interval between circuits. In the resting (REST) condition, participants did not complete any physical activity. S-MFBIA was performed at five timepoints: pre-exercise, immediate post-exercise, 15-, 30-, and 60-minutes post-exercise. Data were analyzed using linear mixed-effects models with a random intercept for participant. In all models, REST was the reference condition, and pre-exercise was the reference time point. RESULTS: Although body mass did not differ between conditions, condition by time interactions were observed for TBW, ICW, and ECW (p\u3c0.001 each), with the higher values observed at post-exercise time points in REUPPER as compared to the REST condition. Mean differences between REUPPER and REST for TBW, ICW, and ECW ranged from 0.6-1.0 kg, 0.4-0.6 kg, and 0.2-0.4 kg, respectively. Conversely, RELOWER did not alter fluid estimates. CONCLUSION: An acute increase in TBW, ICW, and ECW is detected by S-MFBIA after a single bout of upper body, but not lower body, RE. This could be due to the smaller initial diameter and greater relative change in diameter of the arms as compared to legs. Due to the potential of artificial body fluid changes, users should avoid exercise – particularly upper body exercise – prior to S-MFBIA assessments

Acute Resistance Exercise Influences Bioelectrical Impedance Analysis Segmental Fat Mass Estimates

Bioelectrical impedance analysis (BIA) is an attractive tool for routine assessment of human body composition. However, there is also concern regarding how some variables, particularly exercise, may affect its measurements and therefore limit the conditions under which this technology can provide useful body composition data. PURPOSE: The purpose of this study was to determine if acute, localized resistance exercise (RE) compromises the validity of BIA segmental fat mass (FM) estimates. METHODS: In a crossover design, 32 healthy, resistance trained adults (18 F, 14 M; age: 23.4 ± 2.3 y; height: 172.4 ± 8.7 cm; body mass: 74.9 ± 15.3 kg; body fat: 25.6 ± 8.4%) completed three conditions in a randomized order: lower-body resistance exercise (L), upper-body resistance exercise (U), and rest (R). The RE protocol included a warm-up consisting of 2 sets of 12-15 repetitions of 3 upper-body exercises (U), or 3 lower-body exercises (L), followed by 5 sets of 10 repetitions per exercise, with 1-minute rest intervals. The R condition involved no exercise. BIA (InBody 770) was completed immediately pre- and post-exercise and at 15-, 30-, and 60-minutes post-exercise. The effects of the acute RE session on BIA estimates of total and segmental FM were analyzed using linear mixed-effects models with condition and time specified as within-subject factors and a random intercept for participant. In all models, the reference groups were R for condition and the pre-exercise time point for time. RESULTS: Condition by time interactions were observed for total and segmental FM. Examination of model coefficients indicated that most condition by time interactions were attributable to differences in the U condition across time relative to the reference group (i.e., R condition at baseline). In relation to the reference group, mean decreases of 0.75 to 1.25 kg for total FM, 0.38 to 0.58 kg for trunk FM, 0.27 to 0.47 kg for leg FM, and 0.15 to 0.22 kg for arm FM were observed in the U condition (p≤0.001 for all). In contrast, no changes across time were observed in the L condition. CONCLUSION: These findings suggest that an acute bout of localized RE influences BIA total and segmental FM estimates to an extent that can compromise accurate interpretation of the results. These data corroborate the need for a period of rest from physical activity, particularly upper body RE, prior to BIA body composition assessment

Multiple Conformers in Active Site of Human Dihydrofolate Reductase F31R/Q35E Double Mutant Suggest Structural Basis for Methotrexate Resistance*

Methotrexate is a slow, tight-binding, competitive inhibitor of human dihydrofolate reductase (hDHFR), an enzyme that provides key metabolites for nucleotide biosynthesis. In an effort to better characterize ligand binding in drug resistance, we have previously engineered hDHFR variant F31R/Q35E. This variant displays a >650-fold decrease in methotrexate affinity, while maintaining catalytic activity comparable to the native enzyme. To elucidate the molecular basis of decreased methotrexate affinity in the doubly substituted variant, we determined kinetic and inhibitory parameters for the simple variants F31R and Q35E. This demonstrated that the important decrease of methotrexate affinity in variant F31R/Q35E is a result of synergistic effects of the combined substitutions. To better understand the structural cause of this synergy, we obtained the crystal structure of hDHFR variant F31R/Q35E complexed with methotrexate at 1.7-Å resolution. The mutated residue Arg-31 was observed in multiple conformers. In addition, seven native active-site residues were observed in more than one conformation, which is not characteristic of the wild-type enzyme. This suggests that increased residue disorder underlies the observed methotrexate resistance. We observe a considerable loss of van der Waals and polar contacts with the p-aminobenzoic acid and glutamate moieties. The multiple conformers of Arg-31 further suggest that the amino acid substitutions may decrease the isomerization step required for tight binding of methotrexate. Molecular docking with folate corroborates this hypothesis

Barriers and Co-Designed Strategies for the Implementation of Negative Pressure Wound Therapy in Acute Paediatric Burn Care in Australia: A Mixed Method Study

BackgroundPaediatric burn injuries pose a major clinical problem worldwide and result in significant morbidity. Early adjunctive application of negative pressure wound therapy (NPWT) significantly improves time to healing by re-epithelialisation in children with burns. This treatment strategy has not been consistently adopted as part of acute paediatric burn care.MethodsThis investigation used a sequential mixed methods design to identify and explore barriers to the implementation of adjunctive NPWT in acute paediatric burn care. An online questionnaire was developed and disseminated to healthcare professionals within four major paediatric hospitals in Australia, each with a dedicated burns service. Specific barrier data were coded according to the Consolidated Framework for Implementation Research (CFIR). Semi-structured interviews were then conducted with senior clinicians across the four participating hospitals to tailor implementation strategies to local contexts. A stakeholder consensus meeting was then conducted to consolidate implementation strategies and local processes.ResultsA total of 63 healthcare professionals participated in the online questionnaire, and semi-structured interviews were conducted with nine senior burn clinicians. Two interviews were also conducted with parents and caregivers of paediatric burn patients who had received adjunctive NPWT as part of their acute burn treatment within the last 12-months. This investigation identified eight implementation barriers across all five CFIR domains then co-designed targeted strategies to address these identified barriers. Barriers included lack of available resources, limited access to knowledge and information, individual stage of change (which describes clinicians’ readiness or enthusiasm to change practice), patient needs and resources, limited knowledge and beliefs about the intervention, lack of external policies and incentives, intervention complexity, and poor planning of the intervention implementation.ConclusionThere are multiple and inter-related contextual characteristics that influence the uptake of adjunctive NPWT into acute paediatric burn settings in Australia. Results from this investigation will be used within a multi-state stepped-wedge cluster randomised controlled trial. In order to implement adjunctive NPWT into clinical practice for the acute treatment of paediatric burn injuries, additional resources, education, training, and updates to policies and guidelines are required. It is anticipated that adjunctive NPWT, in conjunction with tailored implementation strategies, will enhance adoption and sustainability.Trial RegistrationThis trial was prospectively registered with the Australian and New Zealand Clinical Trials Registry (ANZCTR) on the 1st of February 2022 – registration number ACTRN12622000166774

Barriers and co-designed strategies for the implementation of negative pressure wound therapy in acute pediatric burn care in Australia: A mixed method study

PurposePediatric burn injuries are a global clinical issue causing significant morbidity. Early adjunctive negative pressure wound therapy improves re-epithelialization rates in children with burns, yet adoption in acute burn care is inconsistent. This investigation aimed to determine barriers to the implementation of adjunctive negative pressure wound therapy for the acute management of pediatric burns and co-design targeted implementation strategies.MethodsA sequential mixed methods design was used explore barriers to adjunctive negative pressure wound therapy implementation in acute pediatric burn care. An online questionnaire was disseminated to healthcare professionals within four major Australian pediatric hospitals, each with a dedicated burns service. Barriers were coded according to the Consolidated Framework for Implementation Research (CFIR). Semi-structured interviews with senior clinicians tailored implementation strategies to local contexts. A stakeholder consensus meeting consolidated implementation strategies and local processes.ResultsSixty-three healthcare professionals participated in the questionnaire, and semi-structured interviews involved nine senior burn clinicians. We identified eight implementation barriers across all five CFIR domains then co-designed targeted strategies to address identified barriers. Barriers included lack of available resources, limited access to knowledge and information, individual stage of change, patient needs and resources, limited knowledge and beliefs about the intervention, lack of external policies, intervention complexity, and poor implementation planning.ConclusionMultiple contextual factors affect negative pressure wound therapy uptake in acute pediatric burn settings. Results will inform a multi-state stepped-wedge cluster randomized controlled trial. Additional resources, education, training, updated policies, and guidelines are required for successful implementation. It is anticipated that adjunctive negative pressure wound therapy, in conjunction with tailored implementation strategies, will enhance adoption and sustainability.Trial registrationAustralian and New Zealand Clinical Trials Registry: ACTRN12622000166774. Registered 1 February 2022

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe