Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

US Food and Drug Administration regulatory reviewer disagreements and postmarket safety actions among new therapeutics.

OBJECTIVES: To examine the association between regulatory reviewer disagreements and postmarket safety actions among novel therapeutics approved by the US Food and Drug Administration (FDA) between 2011 and 2015. Disagreements among FDA reviewers regarding the recommendation for a novel therapeutic's approval, its safety, the indicated patient population and/or other parameters of the drug's approval are common. However, the implications of such disagreements-particularly with respect to postmarket safety actions-are poorly understood. DESIGN: Cross-sectional study. SETTING: All novel therapeutics approved by the FDA between January 2011 and December 2015. PARTICIPANTS: None. MAIN OUTCOME MEASURES: Postmarket safety actions defined as new label warnings/increased warning severity, FDA safety communications and safety-related therapeutic withdrawals after the original regulatory approval. RESULTS: Among 174 novel therapeutics approved by the FDA between 2011 and 2015, 42 (24%) had at least one regulatory reviewer disagreement. Altogether, 156 instances of disagreement were observed. Following market approval, a total of 253 postmarket safety actions were taken by the FDA among all new therapeutics, with at least one postmarket safety action identified for 98 (56.3%) of the 174 novel therapeutic approvals. Overall, therapeutics that were the subject of disagreement during the FDA's review had fewer safety actions following approval compared with therapeutics in which no disagreement was observed (38.1% vs 62.1%; RR 0.61, 95% CI 0.41 to 0.92; p=0.006). Therapeutic approvals containing at least one reviewer disagreement also more often carried a black box warning at the point of approval (47.7% vs 31.1%; RR 1.53, 95% CI 1.02 to 2.30; p=0.05). CONCLUSIONS: This investigation of regulatory reviewer disagreements and postmarket safety actions among new therapeutics suggests that disagreements among regulatory reviewers may lead to important pre-emptive actions, potentially mitigating the need for postmarket safety actions to be taken.

Extracellular Vesicles as Predictors of Individual Response to Exercise Training in Youth Living with Obesity.

BACKGROUND: Exercise is associated with health benefits, including the prevention and management of obesity. However, heterogeneity in the adaptive response to exercise training exists. Our objective was to evaluate if changes in extracellular vesicles (EVs) after acute aerobic exercise were associated with the responder phenotype following 6-weeks of resistance training (RT). METHODS: This is a secondary analysis of plasma samples from the EXIT trial (clinical trial#02204670). Eleven sedentary youth with obesity (15.7 ± 0.5 yrs, BMI ≥95th percentile) underwent acute exercise (60% HRR, 45 min). Blood was collected at baseline [AT0 min], during [AT15-45 min], and 75 min post-recovery [AT120], and EVs purified using size exclusion chromatography from extracted plasma. Afterward, youth participated in 6-weeks RT and were categorized into responders or non-responders based on changes in insulin sensitivity. RESULTS: We assessed EV biophysical profile (size, zeta potential, protein yield, and EV subtype protein expression) in a single-blind fashion. Overall, there was a general increase in EV production in both groups. Average EV size was larger in responders (~147 nm) vs. non-responders (~124 nm; p < 0.05). EV size was positively associated with absolute change in Matsuda index (insulin sensitivity) following RT (r = 0.44, p = 0.08). EV size distribution revealed responders predominantly expressed EVs sized 150-300 nm, whereas non-responders expressed EVs sized 50-150 nm (p < 0.05). At baseline, responders had ~25% lower TSG101, ~85% higher MMP2 levels. EV protein yield was higher in responders than non-responders at AT15 (p < 0.05). CONCLUSIONS: Our data suggest that youth with obesity that respond to RT produce larger EVs that are TSG101+ and CD63+, with increased EV protein yield during acute exercise.

Irisin response to acute moderate intensity exercise and high intensity interval training in youth of different obesity statuses: A randomized crossover trial.

Limited data exist regarding the impact of an acute bout of exercise with varying intensities on irisin levels in the youth of different obesity statuses. The objectives were to (1) compare an acute bout of moderate continuous intensity (MCI) exercise and an acute bout of high-intensity interval training (HIIT) on irisin response in youth with different obesity statuses and, (2) investigate whether changes in irisin levels are correlated with exploratory outcomes. A randomized crossover design study was conducted on 25 youth aged 12-18 years old. Participants were classified as either healthy weight (BMI percentile <85; n = 14) or overweight/obese (BMI percentile ≥85; n = 11). Participants performed an MCI exercise session at 50% of heart rate reserve for 35 min and a HIIT exercise session for 35 min, with intervals every 5 min increasing from 50% heart rate reserve to 85-90% for 2 min. Irisin was measured using an enzyme-linked immunoabsorbent assay from plasma sampling obtained throughout the exercise (at times 0, 7, 14, 21, 28, and 35 min). A time effect was observed throughout the HIIT session [F(1,5) = 6.478, p < 0.001]. Bonferonni post-hoc analysis revealed significant differences in irisin levels post-exercise (35 min) compared to times 7, 14, 21, and 28 min. Irisin increased during HIIT (81.0% ± 71.3; p = 0.012) in youth with a healthy weight. No differences were observed for youth living as overweight or with obesity. Overall, HIIT elicits a higher peak irisin response compared to MCI exercise training in youth.

Exploring the Food and Drug Administration's review and approval of Entresto (sacubitril/valsartan).

Federal regulatory agencies such as the United States Food and Drug Administration review pharmacological evidence to ensure the safety and efficacy of new and repurposed pharmaceuticals prior to market approval. The discussions, disagreements and procedural decisions contained within such reviews offer unique insight into a pharmaceutical's strengths, weaknesses and opportunities, yet are often overlooked as a significant source of pharmacological information for research and development. To highlight the value of such resources, we present a case study on Entresto, a first-in-class angiotensin receptor-neprilysin inhibitor for the treatment of heart failure with reduced ejection fraction, and explore the regulatory rationale underlying its market approval. Using information extracted from Entresto's online approval package at Drugs@FDA, we explore some of the procedural complexities underlying market approval of new pharmaceuticals, discuss the broad pharmacological implications contained within regulatory agency grey literature, and highlight opportunities for future therapeutic development.

Effects of sprint interval training on substrate oxidation in adults living with and without obesity: The i-FLEX study.

Metabolic flexibility is the ability to adapt substrate oxidation according to metabolic demand. Exercise increases fat oxidation responses in individuals living with obesity; however, limited research exists on the relationship between substrate oxidation and insulin sensitivity after sprint interval training (SIT). The primary objective was to investigate changes in substrate oxidation at rest and during submaximal exercise, and in insulin sensitivity after 4 weeks of SIT in individuals living with or without obesity. The secondary objective was to investigate correlations between changes in substrate oxidation and insulin sensitivity following SIT. Adults living with obesity (n = 16, body mass index (BMI) = 34.1 kg/m2  ± 3.8) and without obesity (n = 18, BMI = 22.9 kg/m2  ± 1.6) took part in a 4-week SIT intervention. Participants completed three sessions of SIT per week, consisting of repeated sets of a 30-s Wingate separated by 4 m of active recovery. Substrate oxidation at rest and during submaximal exercise was measured using VCO2 /VO2 . Insulin sensitivity was calculated using the Matsuda index. No difference in substrate oxidation at rest was observed for either group (p > 0.05), while a significant increase in fat oxidation was observed in individuals living with obesity [F(1,31) = 14.55, p = 0.001] during the submaximal exercise test. A significant decrease in insulin sensitivity was observed among individuals without obesity [F(1,31) = 5.010, p = 0.033]. No correlations were observed between changes in substrate oxidation and insulin sensitivity (p > 0.05). Following SIT, individuals living with obesity increased submaximal fat oxidation compared to individuals without obesity. No correlations were observed between substrate oxidation and insulin sensitivity. Thus, SIT impacts fat oxidation during exercise in individuals living with obesity while having no such influence on insulin sensitivity.

Impact of acute circuit training on irisin in younger and older overweight adults.

Studies show aerobic exercise increases irisin and leads to health benefits. The impact of circuit training (CT) on irisin in overweight younger and older adults is unknown. The objectives were to determine whether, during an acute bout of CT, changes in irisin differed between overweight younger and older adults, and if irisin is associated with body composition, fitness level, or muscle strength. Inactive, overweight adults aged between 19-35 (25.9 ± 5.0; n = 15) and 60-75 years (67.7 ± 4.1; n = 14) participated in this study. The primary exposure variable was an acute bout of CT (12-15 repetitions; 65-70% of 1-repetition maximum; 3 loops). The primary outcome measure was the concentration of irisin determined by ELISA before, during, and after exercise. Repeated-measures analyses showed no effect of time on irisin levels during acute CT, and no interaction effect between age and time (p > 0.05). No associations were observed between changes in irisin and body composition, fitness, or strength (p > 0.05). In conclusion, acute CT does not increase irisin in overweight individuals, and irisin is not associated with the measured outcomes. Further studies are needed to elucidate the release of irisin by different types of exercise across the lifespan. This trial was registered at clinicaltrials.gov (NCT03715088). Novelty: Younger and older adults show a similar irisin response to an acute bout of circuit training. Irisin response is not associated with measures of body composition, cardiorespiratory fitness, or muscle strength.

Transcriptome network data in larval zebrafish (Danio rerio) following exposure to the phenylpyrazole fipronil.

Fipronil is a phenylpyrazole pesticide that is used in both residential and agricultural applications. Fipronil is detected in run-off and water systems that are near areas in which the pesticide has been applied. The pesticide acts to antagonize gamma aminobutyric acid receptors, leading to over-excitation in the central nervous system. Fipronil has relatively high toxicity to fish, but the mechanisms underlying the toxicity are not well understood in embryonic stages. Zebrafish embryos were exposed to a single concentration of fipronil for 48 h at ∼3-4 h-post-fertilization. Following a 7-day depuration phase, transcriptome and behavioral analyses were conducted. Transcriptomics identified neural processes as those differentially expressed with different doses of fipronil (0.2 µg, 200 µg and 2 mg fipronil/L). Gene networks associated with astrocyte differentiation, myelination, neural tube development, brain stem response, innervation, nerve regeneration, astrocyte differentiation, among other pathways were altered with exposure. In addition, miRNA-related events are disrupted by fipronil exposure and genes associated with primary or pri-miRNA processing were increased in larval fish exposed to the pesticide. These data present putative mechanisms associated with neurological impacts at later ages of zebrafish. This is important because it is not clear how early exposure to pesticides like fipronil affect central nervous system function and organisms later in life.

Residual molecular and behavioral effects of the phenylpyrazole pesticide fipronil in larval zebrafish (Danio rerio) following a pulse embryonic exposure.

Pesticides are typically applied to crops as acute applications, and residual effects of such intermittent exposures are not often characterized in developing fish. Fipronil is an agricultural pesticide that inhibits γ-amino-butyric acid (GABA) gated chloride channels. In this study, zebrafish (Danio rerio) embryos were exposed for 48 h (starting at ~3 h post fertilization, hpf) to various concentrations of fipronil (0.02 µg/L up to 4000 µg/L). Following this acute exposure, a subset of fish was transferred to clean water for a 7-day depuration phase. We hypothesized that a pulse exposure to fipronil during critical periods of central nervous system development would adversely affect fish later in life. After a 48 hour pulse exposure, survival was reduced in embryos exposed to 2 µg fipronil/L or greater. However, there was no further mortality during the depuration phase, nor were there changes in body length nor notochord length in larvae 9 dpf (days post-fertilization) compared to controls. Additional experiments were carried out at higher concentrations over 96 h (up to 4 dpf) to also elucidate developmental effects and teratogenicity of fipronil (43.7 µg/L up to 4370 µg/L). Fipronil at these higher concentrations significantly impacted the development of zebrafish, and the following morphometric and teratogenic effects were observed in 4 dpf fish; reduced body length, yolk sac and pericardial edema, reduced midbrain length, reduced optic and otic diameter, and truncation of the lower jaw. In depurated fish, we hypothesized that there would exist residual effects of exposure at the molecular level. Transcriptome profiling was therefore conducted on 9 dpf depurated larvae exposed initially for 48 h to one dose of either 0.2 µg/L, 200 µg/L or 2000 µg/L fipronil. The expression of gene networks associated with glycogen and omega-3-fatty acid metabolism were decreased in larvae exposed to each of the three concentrations of fipronil, suggesting metabolic disruption. Moreover, transcriptomics revealed that fipronil suppressed gene networks related to light-dark adaptation, photoperiod sensing, and circadian rhythm. Based on these data, we tested fish for altered behavioral responses in a Light-Dark preference test. Larvae exposed to >200 µg fipronil/L as embryos showed fewer number of visits (20-30% less) to the dark zone compared to controls. Larvae also spent a lower amount of time in the dark zone compared to controls, suggesting that fipronil strengthened dark avoidance behavior which is indicative of anxiety. This study demonstrates that a short pulse exposure to fipronil can affect transcriptome networks for metabolism, circadian rhythm, and response to light in fish after depuration, and these molecular responses are hypothesized to be related to aberrant behavioral effects observed in the light-dark preference test.

Moderate and severe hypoxia elicit divergent effects on cardiovascular function and physiological rhythms.

KEY POINTS: In the present study, we provide evidence for divergent physiological responses to moderate compared to severe hypoxia, addressing an important knowledge gap related to severity, duration and after-effects of hypoxia encountered in cardiopulmonary situations. The physiological responses to moderate and severe hypoxia were not proportional, linear or concurrent with the time-of-day. Hypoxia elicited severity-dependent physiological responses that either persisted or fluctuated throughout normoxic recovery. The physiological basis for these distinct cardiovascular responses implicates a shift in the sympathovagal set point and probably not molecular changes at the artery resulting from hypoxic stress. ABSTRACT: Hypoxia is both a consequence and cause of many acute and chronic diseases. Severe hypoxia causes hypertension with cardiovascular sequelae; however, the rare studies using moderate severities of hypoxia indicate that it can be beneficial, suggesting that hypoxia may not always be detrimental. Comparisons between studies are difficult because of the varied classifications of hypoxic severities, methods of delivery and use of anaesthetics. Thus, to investigate the long-term effects of moderate hypoxia on cardiovascular health, radiotelemetry was used to obtain in vivo physiological measurements in unanaesthetized mice during 24 h of either moderate (FIO2=0.15) or severe (FIO2=0.09) hypoxia, followed by 72 h of normoxic recovery. Systolic blood pressure was decreased during recovery following moderate hypoxia but increased following severe hypoxia. Moderate and severe hypoxia increased haeme oxygenase-1 expression during recovery, suggesting parity in hypoxic stress at the level of the artery. Severe but not moderate hypoxia increased the low/high frequency ratio of heart rate variability 72 h post-hypoxia, indicating a shift in sympathovagal balance. Moderate hypoxia dampened the amplitude of circadian rhythm, whereas severe disrupted rhythm during the entire insult, with perturbations persisting throughout normoxic recovery. Thus, hypoxic severity differentially regulates circadian blood pressure.

Central-acting therapeutics alleviate respiratory weakness caused by heart failure-induced ventilatory overdrive.

Diaphragmatic weakness is a feature of heart failure (HF) associated with dyspnea and exertional fatigue. Most studies have focused on advanced stages of HF, leaving the cause unresolved. The long-standing theory is that pulmonary edema imposes a mechanical stress, resulting in diaphragmatic remodeling, but stable HF patients rarely exhibit pulmonary edema. We investigated how diaphragmatic weakness develops in two mouse models of pressure overload-induced HF. As in HF patients, both models had increased eupneic respiratory pressures and ventilatory drive. Despite the absence of pulmonary edema, diaphragmatic strength progressively declined during pressure overload; this decline correlated with a reduction in diaphragm cross-sectional area and preceded evidence of muscle weakness. We uncovered a functional codependence between angiotensin II and ß-adrenergic (ß-ADR) signaling, which increased ventilatory drive. Chronic overdrive was associated with increased PERK (double-stranded RNA-activated protein kinase R-like ER kinase) expression and phosphorylation of EIF2α (eukaryotic translation initiation factor 2α), which inhibits protein synthesis. Inhibition of ß-ADR signaling after application of pressure overload normalized diaphragm strength, Perk expression, EIF2α phosphorylation, and diaphragmatic cross-sectional area. Only drugs that were able to penetrate the blood-brain barrier were effective in treating ventilatory overdrive and preventing diaphragmatic atrophy. These data provide insight into why similar drugs have different benefits on mortality and symptomatology, despite comparable cardiovascular effects.

Assessment of hypoxia and TNF-alpha response by a vector with HRE and NF-kappaB response elements.

Hypoxia and inflammatory cytokine activation (H&I) are common processes in many acute and chronic diseases. Thus, a single vector that responds to both hypoxia and inflammatory cytokines, such as TNF-alpha, is useful for assesing the severity of such diseases. Adaptation to hypoxia is regulated primarily by hypoxia inducible transcription factor (HIF alpha) nuclear proteins that engage genes containing a hypoxia response element (HRE). Inflammation activates a multitude of cytokines, including TNF-alpha, that invariably modulate activation of the nuclear factor kappa B (NF-kB) transcription factor. We constructed a vector that encompassed both a hypoxia response element (HRE), and a NF-kappaB responsive element. We show that this vector was functionally responsive to both hypoxia and TNF-alpha, in vitro and in vivo. Thus, this vector might be suitable for the detection and assessment of hypoxia or TNF-alpha.