National Research Service Award Research Articles

Development of CRISPR/Cas9-Based Mouse Models for X-linked Alport Syndrome

Intro: Alport Syndrome (AS) is a rare hereditary disorder characterized by progressive renal impairment and sensorineural hearing loss, resulting from mutations in the genes that encode COL4 synthesis. X-linked Alport Syndrome (XLAS) is the most common form of AS, comprising an estimated 80% of all human AS cases (the others being various forms of autosomal recessive or autosomal dominant AS), and is caused by various mutations on the COL4A5 gene. However, to date, there are no animal models of XLAS. Methods: To facilitate comprehensive preclinical investigations and therapeutic developments, we established two novel mouse models of XLAS using CRISPR/Cas9 technology. Leveraging the robust C57Bl/6J mouse strain, we precisely introduced the L1655R and C1570S mutations, which represent the predominant human variants of XLAS observed in the Western United States. Discussion: Our innovative mouse models not only serve as invaluable tools for elucidating the underlying mechanisms of XLAS pathogenesis but also provide a platform for evaluating the effcacy of potential therapeutic interventions. The establishment of these faithful genetic replicas of human XLAS marks a significant advancement in the field, offering unprecedented opportunities for in-depth mechanistic studies and the development of targeted therapeutic strategies for this debilitating renal and auditory disorder. This research is supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Kidney Foundation of Utah and Idaho. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of aging on vasoconstrictor responses of diaphragm arterioles

Introduction: In health, the diaphragm is the most important inspiratory muscle and the only skeletal muscle that is continuously recruited throughout life. Because of the diaphragm’s importance to generating the exercise hyperpnea and wealth of evidence that diaphragm fatigue and exhaustion can impair exercise tolerance, we were interested in whether the exercise intolerance that attends older populations might be associated with altered diaphragm vasomotor control. While it is known that aging compromises peripheral skeletal muscle vasomotor control, it is not known if similar impairments are present in the respiratory muscle vasculature, namely the diaphragm. Therefore, we tested the hypothesis that advanced age decreases diaphragm arteriole contractile responses to alpha-adrenergic agonists. Methods: 4–5 month old Fischer rats and 22–24 month old Fischer rats were divided by age into two groups: young (Y) and old (O). In Y and O, diaphragm first order (1A) arterioles (~200μm diameter, ~2μm in length) were isolated, cannulated, and pressurized to develop spontaneous tone. Following this, contractile responses to cumulative doses of norepinephrine (NE) and phenylephrine (PE) (10−9 to 10−4M) were determined. Results and Conclusion: The physical characteristics of the two groups differed as O were heavier than Y. However, with respect to the diaphragm 1A arterioles, there were no differences in spontaneous tone development (%) or maximal diameter (μm) between Y (38 ± 4%; 132 ± 19μm) and O (37 ± 4%; 157 ± 14 μm; both P >0.05). In diaphragm 1A arterioles, the maximal vasoconstrictor response to NE was also not different between O and Y animals (O: 37 ± 8% vs. Y: 45 ± 1%; p > 0.05). However, the maximal vasoconstrictor response to PE was greater in Y compared to O (O: 29 ± 3% vs. Y: 39 ± 5%; p ≤ 0.05). These data support that aging may reduce a1 adrenergic receptor mediated vascular control, altering the vasoconstrictive responsiveness of diaphragm resistance vessels. We are increasing our group sizes to test the robustness of this conclusion. This work was supported by the Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants awarded to T.I.M and D.C.P. by Kansas State University College of Veterinary Medicine and the National Institute on Aging 1R15AG078060 awarded to B.J.B and D.C.P. A.G.H and K.M.S. were financially supported by Ruth L. Kirschstein National Research Service Awards 1F31HL167618-01 and 1F31HL170643-01, respectively. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NADPH oxidase-derived reactive oxygen species regulates adipose tissue lipolysis in humans

There is mounting evidence suggesting that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) regulate physiological processes, as NOX has been shown to promote increases in lipolysis in cultured adipocytes. PURPOSE: To determine the mechanisms by which NOX stimulates adipose tissue lipolysis. METHODS: Young healthy females (n = 17; mean ± SD; age: 21.7 ± 5 years; body mass index: 22.2 ± 3.8 kg/m2) participated in this study. Participants underwent microdialysis and hyperinsulinemic-euglycemic clamp (clamp) procedures. Three microdialysis probes were inserted into subcutaneous abdominal adipose tissue to monitor local adipose tissue ROS (H2O2) production and lipolysis (as indicated by dialysate glycerol). To determine whether NOX stimulates lipolysis via distinct signaling pathways, microdialysis probes were perfused with either isoproterenol (Iso; β-adrenergic agonist to stimulate canonical lipolysis signaling) or atrial natriuretic peptide (ANP to stimulate non-canonical lipolysis signaling), then perfused with a local NOX inhibitor (apocynin). A control probe was perfused with apocynin but without any agonists of lipolysis. The microdialysis procedures were then repeated under hyperinsulinemic conditions during the same study visit. RESULTS: In the fasted state, dialysate H2O2 concentrations were lower in the control and Iso probes upon apocynin perfusion (control probe without apocynin: 1.06 ± 0.5 μM, with apocynin: 0.62 ± 0.2 μM; p = 0.005; Iso probe without apocynin: 1.01 ± 0.4 μM, with apocynin: 0.66 ± 0.2 μM; p = 0.003). Apocynin perfusion did not alter dialysate H2O2 concentrations in the ANP probe under the fasted state (p = 0.11). Dialysate glycerol concentrations did not change upon apocynin perfusion under any of the microdialysis probe conditions in the fasted state (p > 0.05). During the clamp, apocynin perfusion resulted in lower dialysate H2O2 concentrations in the three conditions (control probe without apocynin: 1.22 ± 0.6 μM, with apocynin: 0.75 ± 0.3 μM; p = 0.03; Iso probe without apocynin: 1.55 ± 0.8 μM, with apocynin: 0.79 ± 0.3 μM; p = 0.01; ANP probe without apocynin: 1.55 ± 0.6 μM, with apocynin: 0.92 ± 0.3 μM; p = 0.03). Dialysate glycerol concentrations were lower in the Iso probe upon apocynin perfusion during the clamp (Iso probe without apocynin: 124.7 ± 38 μM, with apocynin: 90.9 ± 25 μM; p = 0.003). CONCLUSIONS: This study presents adipose tissue NOX as a source of in vivo ROS production in human participants. In addition, we reveal a novel mechanism of NOX in adipose tissue function whereby NOX exerts increases in β-adrenergic-mediated lipolysis during hyperinsulinemia. Our findings highlight the roles of ROS as signaling mediators and suggest that an imbalance between NOX activity and insulin action on adipose tissue could negatively affect systemic fatty acid metabolism. Porter Pre-doctoral Fellowship from the American Physiological Society and an NIH National Research Service Award (F31HL154642). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Impact of Aging and Biological Sex on Diaphragm Hyperemia and Blood Flow Distribution

INTRODUCTION: Age-related respiratory muscle dysfunction (i.e., the diaphragm) diminishes physical capacity and increases the susceptibility to respiratory failure. The diaphragm dysfunction associated with old age is characterized by decreased diaphragm muscle mass and strength, and increased fatiguability. While inadequate perfusion and diminished microvascular function are well recognized to play a central role in age-related peripheral skeletal muscle dysfunction, the impact of aging on diaphragm hyperemia and blood flow distribution, and whether sex differences exist, has not been investigated. METHODS: Young (Y; 4-6 months) and Old (O; 22-24 months) Fischer 344 rats (n=34) were divided into four groups: 1) Young female (YF; n=8), 2) Young male (YM; n=8), 3) Old female (OF; n=9), and 4) Old male (OM; n=9). Fluorescent microspheres were used to assess bulk and regional diaphragm blood flow at rest and during electrically-induced 1Hz contractions. RESULTS: There were no age-related or sex differences in quiescent diaphragm blood flow (BF) or vascular conductance (VC) ( P>0.05). Age-related differences during 1Hz contractions: The increase in total diaphragm BF was significantly lower in OM (98 ± 11 ml/min/100g) compared to YM (141 ± 7 ml/min/100g; p<0.05). Total and regional diaphragm BF was not different in F rats ( P>0.05); however, medial costal diaphragm VC was decreased in OF (1.2 ± 0.1 ml/mmHg/min/100g) compared to YF (1.8 ± 0.2 ml/mmHg/min/100g; p<0.05). Sex differences during 1Hz contractions: Total diaphragm BF and VC were similar in YF and YM ( P > 0.05). While both ventral and medial costal diaphragm BF responses were not different between YF and YM, dorsal costal diaphragm BF was higher in YF (144 ± 27 ml/min/100g) versus YM (84 ± 11 ml/min/100g; p<0.05). Total and regional (ventral, medial, and dorsal costal) diaphragm BF was significantly higher in OF compared to OM ( p<0.05). CONCLUSION: These data demonstrate that aging impaired the ability of the diaphragm to augment BF in response to contractile activity in M rats and decreased medial costal diaphragm VC during contractions in F rats with no significant effect on diaphragmatic hyperemia. In regards to sex differences, these data suggest that both YF and OF may have a greater regional recruitment of the diaphragm compared to YM and OM. The data herein provide a putative mechanism for age-related diaphragm contractile dysfunction and future studies are needed to investigate the impact of aging and biological sex on diaphragm vascular function. National Institute of Aging 1R15AG078060, Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute 1F31HL167618-01 and 1F31HL170643-01, and Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants at Kansas State University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Novel Approach for Assessing Mitochondrial Respiration in Freshly Isolated Glomerular and Tubular Fractions from Whole Mouse Kidney

Intro: Understanding mitochondrial function within specific renal compartments is crucial for unraveling the intricate metabolic processes underlying kidney physiology and pathophysiology. In this study, we present a novel method for accurately measuring mitochondrial respiration in freshly isolated glomerular and tubular fractions derived from dissected kidneys. Methods: Employing a differential adhesive sieving technique, we isolated glomerular and tubular fractions from freshly harvested whole kidneys (n=2) and subsequently utilized a standard substrate-inhibitor titration protocol to assess mitochondrial respiration in the Oroboros Oxygraph O2K (Oroboros Instruments, Innsbruck AT). Results: Notably, our findings revealed distinct state 3 respiration rates in various fractions, with the 70 μm tubule fraction exhibiting a rate of 2133.029 pmol/sec/mgprotein, the 40 μm tubule fraction displaying a rate of 522.4116 pmol/sec/mgprotein, the first glomerular fraction demonstrating a rate of 407.7911 pmol/sec/mgprotein, and the second tubular fraction manifesting a rate of 362.3286 pmol/sec/mgprotein. Moreover, these fragments exhibited differential substrate preferences, as the addition of succinate increased state 3 respiration by ~90% in the 70 μm tubule fraction. In contrast, succinate increased respiration by ~30% in the 40 μm tubule and glomerular fractions. Discussion: These ongoing experiments provide compelling evidence supporting the feasibility and reliability of our approach in quantifying mitochondrial respiration within isolated renal fractions. Our methodology not only enables precise measurements of mitochondrial function in distinct nephron segments but also lays the groundwork for further investigations into the physiological and pathological implications of mitochondrial dynamics in kidney health and disease. This research is supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Kidney Foundation of Utah and Idaho. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Myocardial Infarction-induced Heart Failure on Diaphragm Arteriolar Vasorelaxation

INTRODUCTION: Diaphragm atrophy and contractile dysfunction are prevalent in heart failure (HF), diminishing physical capacity and quality of life. The delivery of blood flow and oxygen necessary to support the metabolic demand of contractions is largely governed by the resistance vasculature (i.e., arterioles) with the endothelium playing a central role in arteriolar vasorelaxation. Adequate perfusion and blood flow distribution, and thus resistance vessel vasodilation, is a major determinate of diaphragm contractile function. While the mechanistic bases of HF-induced diaphragm dysfunction continue to be elucidated, the impact of HF on diaphragm vasomotor control has not been investigated. METHODS: Adult (6 mo) Sprague-Dawley rats (n=7) were divided into two groups: 1) Healthy Control (HC; n=4) and 2) MI-induced heart failure (HF; n=3). First-order arterioles (1A) from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O and allowed to equilibrate for 60 min to develop spontaneous tone. Thereafter, endothelium-dependent and -independent vasorelaxation responses to cumulative doses of acetylcholine (ACh; 10−9 to 10−4M) and sodium nitroprusside (SNP; 10−9 to 10−4M) were determined. RESULTS: There were no differences in spontaneous development (%) or maximal diameter (μm) between HC (20 ± 4%; 178 ± 11 μm) and HF (18 ± 2%; 184 ± 29 μm) diaphragm 1As ( P > 0.05). Maximal ACh-mediated relaxation (%) was significantly reduced in HF rats compared to HC rats (52 ± 2 versus 68 ± 5%; P < 0.05). Maximal endothelial-independent vasorelaxation with SNP, was not different between HC and HF rats (71 ± 3 versus 73 ± 1%; P > 0.05). CONCLUSION: The preliminary data herein demonstrate that HF impaired endothelial function and vasomotor function in the medial costal diaphragm resistance vasculature. This suggests that, in HF, there may be a redistribution of blood flow to less effcient regions of the diaphragm (e.g., ventral costal) to sustain contractile function and compensate for the diminished medial costal vasomotor function demonstrated herein. Studies including a larger sample size are warranted to investigate the impact of HF on regional blood flow distribution and resistance vessel function in the diaphragm. Such investigations would yield novel mechanistic insights into HF-induced diaphragm dysfunction. National Institute of Aging 1R15AG078060, Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute 1F31HL167618-01, and Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants at Kansas State University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tumor Oxygenation in an Orthotopic Model of Breast Cancer: Impact of Dietary Nitrate Supplementation

Introduction: Breast cancer (BC), the predominant non-melanoma cancer among women, is projected to escalate to more than 3 million new cases and ~1 million fatalities globally by 2040. Chronic and fluctuating low oxygen (O2) conditions within tumors favor malignant growth, decreased responsiveness to therapeutic interventions, and reduced patient survivability. Treatment strategies to combat tumor hypoxia are desperately needed. Dietary nitrate supplementation via beetroot juice (BRJ), which increases nitric oxide (NO) production through the nitrate-nitrite-NO pathway, has been demonstrated to increase skeletal muscle blood flow, improve mitochondrial effciency, and reduce metabolic demands. Specifically, BRJ improves interstitial oxygen pressures (PO2 is) in acidic, low oxygen environments such as that found in the tumor microenvironment. Therefore, we hypothesized that BRJ supplementation would increase PO2 is in BC tumors. Methods: Female Fischer 344 retired breeder rats (6-8 months old) were used in this investigation. Adenocarcinoma cells (MAT B III at 6 x 103) were injected into the mammary duct and tumor growth was monitored over 2 weeks. Once tumors reached ~5 mm in diameter, rats were randomly assigned to consume BRJ (1 mmol/kg/day; BRJ, n=3) or water (CON; n=3) for 5 days. Under pentobarbital anesthesia, tumor PO2 is measurements were obtained using phosphorescence quenching. Results: Tumor growth rate was decreased in BRJ compared to CON rats (0.5 ± 0.1 vs. 1.0 ± 0.1 mm/day; p<0.05). Heart rate and mean arterial pressure were not different between groups (P>0.05). Blood lactate was significantly decreased in BRJ compared to CON rats (0.5 ± 0.1 vs. 1.2 ± 0.1 mmol/L, p<0.05). There was an increase in mean tumor PO2 is in BRJ vs. control rats (6.9 ± 1.3 vs. 3.7 ± 0.47; p<0.05). Conclusions: This study offers promising preliminary insights into the potential of dietary nitrate supplementation as an adjunctive approach to BC treatment to increase tumor PO2 is and reduce tumor growth rate. Future and ongoing studies will increase the sample size, use fluorescent microspheres to measure tumor blood flow, and explore the potential for BRJ and other NO agonists to combat tumor hypoxia and the skeletal muscle dysfunction associated with cancer progression. This research was funded by the following sources: the Johnson Cancer Research Center Innovative Research Award and Sustained Momentum for Investigators with Laboratories Established (SMILE) Grants at Kansas State University, the National Institute on Aging (Grant 1R15AG078060), and Ruth L. Kirschstein National Research Service Awards from the National Heart, Lung, and Blood Institute (Grants 1F31HL167618-01 and 1F31HL170643-01). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Early life consumption of a "processed" diet high in advanced glycation end products impairs memory function and alters the gut microbiome in rats

Recent evidence indicates that “ultra-processed” foods constitute ~65% of total energy intake among children in the U.S. The impacts of excessive processed food consumption during development are poorly understood, in part because there are neither clear definitions of, nor a robust classification system accounting for, the complexity of food processing variables and how they interrelate in final food products. Heat treatment, a common form of food processing, can induce the chemical Maillard reaction and lead to foods rich in advanced glycation end products (AGEs). The neurocognitive implications of high levels of AGE consumption, particularly during early life, are unknown. Here, we used a rat model to evaluate the effects of adolescent consumption of a heat-treated diet high in AGEs on cognition and the gut microbiome in adulthood. In a series of studies, rats received either an otherwise healthy AGE-rich diet (AGE rats; AIN-93G diet heated at 160ºC for 60min) or a non-AGE-rich diet (CTL rats; AIN-93G diet without heat treatment) during adolescence (postnatal days 26-60). Metabolic, behavioral, and gut microbiome assessments were performed in adulthood. Results revealed that AGE rats exhibited hippocampal (HPC)-dependent memory deficits relative to CTL rats in the absence of altered body weight, body composition, glucose tolerance, and behavioral measures of anxiety. Administering a drug to break down AGEs (alagebrium; 1mg/kg/day) concomitantly with the heat-treated diet prevented memory impairments, supporting a causal role of AGEs in early life diet-induced HPC dysfunction. Microbiome sequencing analyses revealed that AGE rats had substantially reduced abundance of the bacterial genus Lactococcus relative to controls, and Lactococcus abundance was strongly correlated with memory performance. Administration of Lactococcus lactis by oral gavage (109 colony forming units/day) from PN 26-40 during exposure to the heat-treated diet rescued the AGE diet-induced memory impairments. Collective findings from this work identify a connection between a specific food processing variable (i.e., increased dietary AGEs through heat treatment), the gut microbiome, and impaired neurocognition. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases under grant DK123423 (awarded to SEK and AAF); the National Institute on Aging through a Postdoctoral Ruth L. Kirschstein National Research Service Award under grant F32AG077932 (awarded to AMRH); and a National Science Foundation Graduate Research Fellowship (awarded to LT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sulforaphane improves vasodilatory reactivity in diaphragm arterioles from rats with pulmonary hypertension

Pulmonary hypertension (PH) is a chronic, progressive disease characterized by pulmonary vascular remodeling, respiratory muscle and cardiac impairments, and exercise intolerance. Specifically, the diaphragm displays compromised contractile and vascular function, precluding it from meeting the increased metabolic demands of PH-induced chronic hyperpnea. Oxidative stress has been implicated in vascular dysfunction and PH pathology, therefore our objective was to target the antioxidant mediator Nrf2 (Nuclear factor erythroid 2-related factor 2) using sulforaphane (SFN) to determine whether this could improve vascular function in the diaphragm. We hypothesized that incubation with SFN would increase vasodilatory responsiveness in diaphragm arterioles from PH rats. Methods: Female Sprague-Dawley rats were administered monocrotaline (MCT; 50 mg/kg, i.p.) to induce PH. Disease progression was monitored via echocardiography. Once animals developed PH (~3 wks), the diaphragm was removed and first-order arterioles from the medial costal diaphragm were isolated, cannulated, and pressurized at 90 cmH2O. After a 60 min equilibration period and spontaneous tone development, endothelium-dependent and -independent vasorelaxation responses to cumulative doses of acetylcholine (ACh) and sodium nitroprusside (SNP) (10−9 to 10−4 M) were determined. These vessels were subsequently incubated with SFN (10 μM, 20 min), then ACh and SNP dose-response measurements were repeated. Results: All PH rats displayed morphometric and echocardiographic criteria for disease (i.e., right ventricular hypertrophy and reduced pulmonary artery acceleration time). Incubation with SFN improved endothelium-dependent and -independent vasodilation (p<0.05). Maximal vasodilatory responses with SFN were 14% and 16% higher for ACh (30±2 vs 34±3%; P=0.02) and SNP (52±2 vs 60±2%; P=0.03), respectively. However, vasodilatory reactivity remains substantially lower than that of healthy animals. Conclusions: These findings suggest that Nrf2 activation using SFN may improve vascular function in the diaphragm with PH. Future investigations will focus on chronic administration of SFN to PH rats to determine the therapeutic potential of this compound in vivo. The results of these studies provide insight into novel pharmacological strategies to improve diaphragm vascular and contractile function, with the potential to help mitigate dyspnea and exercise intolerance and enhance life quality for PH patients. Sustained Momentum for Investigators with Laboratories Established Grants (TIM & DCP); National Institute on Aging 1R15AG078060 (BJB & DCP); Ruth L. Kirschstein National Research Service Awards 1F31HL167618-01 (AGH) and 1F31HL170643-01 (KMS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acute Hypoxia Following Disuse Atrophy in Aged Mice Enhances Cellular Remodeling and Whole-body Strength During Early Recovery

Age-related impairments in muscle recovery following disuse atrophy are associated with altered macrophage metabolism and blunted inflammatory function. HIF-1α is a key regulator of macrophage metabolism and inflammatory function. Therefore, we investigated the effects of acute hypoxia on muscle recovery following disuse in aging. We hypothesized that transient hypoxic exposure following disuse in aged mice would enhance macrophage metabolism and inflammatory status, resulting in improved muscle cellular remodeling and recovery. Accordingly, old (~20 months) and young adult male mice (~4 months) were exposed to acute (24 hours) normobaric hypoxia (8% O2) immediately following 14-days hindlimb unloading. We characterized muscle phenotype and function during early recovery (4- and 7-days) with and without hypoxia exposure. We found that acute hypoxia stimulated the expression of inflammatory (TNF-α, VEGF p<0.01) and glycolytic transcripts (PFK-1 p<0.05), LDH, PDH p=0.08) downstream of HIF-1α in aged skeletal muscle under basal conditions. Following disuse atrophy, transient hypoxic exposure enhanced macrophage inflammatory function, indicated by increased phagocytosis capacity (p<0.01), and promoted early skeletal muscle cellular remodeling events, including enhanced muscle satellite cell content (p<0.05). Furthermore, these effects facilitated the recovery of whole-body strength in aged mice indicated by a return to baseline values (p>0.05). The results of this investigation suggest that hypoxic exposure following disuse atrophy promoted inflammatory macrophage function, muscle cellular remodeling, and enhanced early recovery in aged mice. These findings elucidate the potential benefits of hypoxia in manipulating macrophage function to ameliorate age-related deficits in muscle recovery following disuse atrophy. This research was supported by R01AG076075 (MJD) and the National Institutes of Health Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Interventions to support fellowship application success among predoctoral physician-scientists.

A critical element of physician-scientist training is the development and practice of core competencies that promote success in research careers. The ability to develop compelling training and research proposals is one such foundational skill. The NIH Ruth L. Kirschstein National Research Service Award (NRSA) individual fellowship for dual-degree students (F30, F31, or F31-Diversity) creates an ideal opportunity to provide formal instruction in grant-writing skills to physician-scientists early in training. In the guided process of preparing a predoctoral fellowship application, students learn to formulate clear short- and long-term research and training goals; construct a comprehensive, well-reasoned, and rigorous proposal; become familiar with funding agency priorities; and gain strategic insights into the peer review system. Beyond building scientific writing skills, the application process for an NRSA F30 or F31 is an opportunity for trainees to strengthen mentor-mentee relationships, identify learning opportunities key to their scientific development, and build effective research and mentoring teams. These skills also apply to developing future postdoctoral mentored K applications or faculty research program grants. Here, we outline key features of the structured proposal development training developed for students in the Yale MD-PhD Program and review outcomes associated with its implementation.

How to write a peer-polished proposal in 15 weeks

Creating a meticulously crafted proposal requires a strategic approach and systematic planning. An overview of a semester-long graduate course on how to write successful NIH grant applications will be provided. Particular emphasis is given to developing proposals for the Ruth L. Kirschstein Predoctoral Individual National Research Service Award (https://researchtraining.nih.gov/programs/fellowships/F31) or to disease-based foundations. The writing process involves drafting components in several key phases. The initial four weeks focus on understanding the proposal requirements, identifying the target audience, organizing a brilliant biosketch and remarkable resources and environment pages, and establishing a clear hypothesis and specific aims. An extensive literature review is conducted in the subsequent two weeks to contextualize the proposal, identify a gap in knowledge, and stress the significance and innovation of the proposed work. Weeks 7 and 8 are devoted to the development of a robust research approach and methodology, including data collection, analysis techniques, expected outcomes, potential challenges, and alternative approaches. The review process, refinement and enhancement take center stage for the remaining weeks. Peer review and feedback mechanisms are incorporated to iteratively improve each proposal's coherence, logic, and persuasiveness. This systematic 15-week timeline emphasizes iterative refinement through peer input, ensuring a polished proposal ready for submission.

Recommendations for Writing a National Institutes of Health Individual Predoctoral Fellowship (F31) Training Grant in Dissemination and Implementation Science.

Early career training is an essential component of building the future of dissemination and implementation (D&I) science. The United States National Institutes of Health Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship (F31) award offers a mechanism for doctoral students to acquire specialized D&I training and mentorship, to pursue independent research in D&I science, and to receive financial support for their graduate studies. Due to scarce resources on preparing early career grant proposals focused on D&I science, this commentary offers guidance to doctoral students on developing a successful application for a specific type of early career proposal - the NIH F31 mechanism. We offer strategies for the research and training components based on themes identified across six funded F31 grant applications and on our experiences in the application process (grants funded from 2019-2020). We are from diverse fields (clinical psychology, school psychology, public health, and epidemiology) with varied research foci (global health, mental health, and infectious diseases). Applications were funded on both the F31 and F31-Diversity mechanisms. Funded F31 research projects included dissemination strategies, treatment adaptions, piloting new methods, and pre-implementation inquiry. Common training goals included developing content expertise in D&I science, understanding specific D&I science methodologies, learning strategies for working with community partners, and enhancing knowledge of analytic methods. D&I training activities included journal clubs, meeting with consultants, attending D&I science trainings, and attending conferences. Mentored research training is essential for learning D&I science methods and developing research-practice partnerships as students.

Editorial introductions.

Editorial introductions.

0930 What explains high self-reports of poor sleep quality in Native Hawaiian/Pacific Islanders?

Abstract Introduction High rates of poor sleep quality have been reported among patients of Native Hawaiian/Pacific Islander (NHPI) race. Unrestful sleep might be attributable to short sleep times or to elevated rates of obstructive sleep apnea among NHPIs. We aimed to evaluate factors associated with self-reported poor sleep quality among NHPIs in a nationwide, representative community sample. Methods Using the 2014 National Health Interview Survey (NHIS), we used logistic regression to model the odds of individuals reporting that they did not awake feeling rested at least two out of the prior seven days. NHPIs from the NHPI-NHIS and non-NHPIs from the general sample were compared. Age, sex, BMI, smoking status, report of heavy drinking, employment status, marital status, and reported average hours of sleep time were evaluated in terms of their effects on self-reported sleep quality. Results Data from 36,697 non-NHPI and 2,590 NHPI individuals was available for analysis. There were significant differences in age, BMI, marital and current employment status between NHPIs vs. non-NHPIs. NHPI patients reported less sleep (mean 6.86±1.41 hours) in contrast to non-NHPIs (mean 7.12±1.42 hours) (P< 0.001). NHPIs were more likely to report two or more days awakening not feeling rested (Odds Ratio (OR) 1.22, 95% confidence interval (95CI) 1.12–1.32) as compared to non-NHPIs. After adjusting for demographics, BMI, alcohol, smoking, and marital and employment status, the relationship persisted (OR 1.17, 95CI 1.07-1.28). When additionally controlling for sleep time, which was strongly associated with the likelihood of awakening unrested (OR 3.1 95CI 2.9-3.2 for six or fewer average hours of sleep), there was no significant relationship between race and likelihood of awakening unrested (OR 1.02, 95CI 0.93-1.13). Findings were similar when evaluating different thresholds of unrestful nights per week. Conclusion NHPIs are more likely to awake feeling unrested than non-NHPIs. The higher rate of unrestful sleep is associated with higher reports of short (6 hours or less) sleep time. Therefore, future research to improve sleep quality in NHPIs should also investigate the cultural and social factors leading to short sleep time. Support (if any) BWL: NIH Ruth L. Kirschstein National Research Service Award 5T32HL105321 and the ATS ASPIRE Fellowship

Induced Blood Flow Oscillations at 0.1 Hz Protects Oxygenation of Severely Ischemic Tissue

Background: Early interventions that improve vital organ perfusion will reduce the number of lives lost from blood loss injuries. We have shown that generating 10 second (~0.1 Hz) fluctuations or “oscillations” in arterial pressure and blood flow during simulated hemorrhage protects cerebral tissue oxygenation. Lower body negative pressure (LBNP) was used to both simulate hemorrhage, and induce the hemodynamic oscillations in these previous studies. However, the magnitude of cerebral tissue ischemia is limited to 20-30% with LBNP due to the onset of pre-syncopal symptoms. To examine the effect of 0.1 Hz hemodynamic oscillations on blood flow delivery and tissue oxygenation of severely ischemic tissues, we developed a limb ischemia model. Hypothesis: Oscillatory arterial pressure and blood flow will attenuate reductions in brachial artery blood flow and forearm tissue oxygenation in a severely ischemic limb. Methods: Nine healthy human subjects (5M, 4F; 27.2 ± 4.1 y) completed two experimental protocols separated by ≥48 h. In both conditions, ischemia of the forearm was induced with a pneumatic cuff on the upper arm to decrease brachial artery (BA) blood velocity by ~70-80% from baseline. In the oscillation condition (OSC), 0.1 Hz oscillations in mean arterial pressure (MAP) and BA blood flow were then induced by inflating and deflating bilateral thigh cuffs every 10 seconds (0.1 Hz) throughout the forearm ischemia period. In the control condition (CON), the thigh cuffs were in place, but were inactive throughout the forearm ischemia period. BA blood flow was measured via duplex ultrasound, forearm muscle tissue oxygenation (SmO2) was measured via near infrared spectroscopy, and arterial pressure was measured via finger photoplethysmography. Results: The magnitude of forearm ischemia, indexed by the reduction in BA blood velocity, was matched between protocols (CON: -75.2 ± 8.4 % vs. OSC: -78.3 ± 7.8 %, p=0.20). Power spectral density of 0.1 Hz oscillations in MAP (CON: 19.4 ± 22.8 mmHg2 vs. OSC: 716.8 ± 514.6 mmHg2; p<0.001) and BA blood velocity (CON: 0.7 ± 1.0 cm/s2 vs. OSC: 10.6 ± 7.1 cm/s2, p=0.02) were greater with oscillatory thigh cuff compression compared with the control condition. While oscillatory thigh cuff compression during forearm ischemia had no effect on absolute MAP (CON: 94.3 ± 6.6 mmHg vs. OSC: 94.4 ± 10.8 mmHg, p=0.99), BA blood flow (CON: 9.7 ± 5.8 ml/min vs. OSC: 9.5 ± 7.3 ml/min, p=0.82), or BA conductance (CON: 0.10 ± 0.06 ml/min/mmHg vs. OSC: 0.09 ± 0.06 ml/min/mmHg, p=0.39), the reduction in SmO2 was attenuated (CON: -38.7 ± 8.3 % vs. OSC: -28.4 ± 9.7 %; p=0.04). These data provide further evidence for the use of 0.1 Hz hemodynamic oscillations as a therapeutic intervention for conditions associated with severe vital organ ischemia such as hemorrhage, stroke, myocardial infarction, and sepsis. National Institutes of Health/National Institute on Aging T32 Ruth Kirschstein Institutional National Research Service Award (T32AG020494); American Heart Association Transformational Project Award (19TPA34910073) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Microbiota engage a HDAC3 rheostat to shape ILC3 metabolism and function in the gut

Abstract Group 3 innate lymphoid cells (ILC3s) respond to microbial colonization of the intestine to coordinate tissue health. This process involves interpretation of host- or microbial-derived signals that must be tightly regulated to preserve immunologic homeostasis. Here we examine a key epigenomic regulator that is responsive to microbial-derived factors, histone deacetylase 3 (HDAC3), and find that lineage-specific deletion results in reduced ILC3s and increased susceptibility to intestinal damage. In contrast, the lineage-specific deletion of an essential corepressor, NCOR1, results in elevated ILC3s and enhanced protection from intestinal damage. This indicates that HDAC3 mediates both a supportive deacetylase-independent role and a repressive deacetylase-dependent role in ILC3s. To explore genes promoted by HDAC3, we performed RNA sequencing and found that HDAC3 deficient ILC3s exhibit impaired transcription of genes involved in polyamine synthesis including the polyamine rate limiting enzyme ornithine decarboxylase 1 (ODC1) and spermine oxidase (SMOX). At steady state, ILC3s uniquely express high levels of ODC1 and SMOX compared to other lymphocytes, and transcription of these genes is influenced by IL-23 and the presence of the microbiota. Overall, this indicates that HDAC3 is a rheostat of ILC3 functionality in the intestine, with the hypothesis that HDAC3/NCOR1 limit tissue protective ILC3 responses, and NCOR1 independent HDAC3-mediated transcription of polyamine synthesis genes promotes ILC3 tissue protective responses. Supported by NIDDK National Research Service Award (NRSA) T32 Diversity Administrative Supplement (NOT-DK-21-016), R01AI143842, R01AI123368, R01AI145989, U01AI095608, R21CA249274, R01AI162936, R01CA274534 and the NIAID Mucosal Immunology Studies Team (MIST), an Investigators in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund, Linda and Glenn Greenberg, the Dalton Family Foundation, and the Roberts Institute for Research in IBD. G.F.S. is a CRI Lloyd J. Old STAR.

PD18-03 THE EFFECTS OF PARENTAL-AGE DIFFERENCES ON ADVERSE PERINATAL OUTCOMES

You have accessJournal of UrologyCME1 Apr 2023PD18-03 THE EFFECTS OF PARENTAL-AGE DIFFERENCES ON ADVERSE PERINATAL OUTCOMES Victor Yu, Joemy Ramsay, Joshua John Horns, and James Hotaling Victor YuVictor Yu More articles by this author , Joemy RamsayJoemy Ramsay More articles by this author , Joshua John HornsJoshua John Horns More articles by this author , and James HotalingJames Hotaling More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003273.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The effects of advanced maternal age on perinatal outcomes have been well-documented, with several studies demonstrating a significant risk for women over 35 years. Other studies have identified potential associations between advanced paternal age and adverse perinatal outcomes. This study aimed to expand upon such findings by assessing the risks associated with the tertiary variable of parental-age differences. METHODS: A retrospective cohort analysis was performed using data compiled from the National Vital Statistics System for 21,021,091 US births between 2012-2018. Logistic regression controlling for demographic and health-related covariates was used to assess odds of low birthweight, very low birthweight, preterm birth, very preterm birth, small size for gestational age, low 5-minute APGAR score, congenital defects, and chromosomal anomalies. RESULTS: Increased parental-age differences were associated with significant risks for all adverse outcomes, aside from congenital defects, even when controlling for maternal age. Restricting maternal age to the reference range of 25-29 years, infants born to fathers aged 5-8 years younger (n=87,032) had 19% (OR 1.19, 95% CI 1.17 – 1.21) higher odds of having any adverse perinatal outcome. Conversely, infants born to fathers aged >16 years older (n=100,971) had 11% (OR 1.11, 95% CI 1.09 – 1.13) higher odds of having any adverse perinatal outcome. CONCLUSIONS: Large differences in parental age are associated with adverse birth outcomes and exhibit a bimodal distribution. Older mothers paired with younger fathers had the highest risks, even when maternal age was below 35 years. Potential mechanisms as to why younger fathers may elevate outcome risks include sperm genetic or epigenetic instability, lower socioeconomic status, unstable social and familial environments, or precarious lifestyle factors associated with this age group. Clinically, parental-age differences should be considered alongside maternal and paternal age when assessing risks of adverse perinatal outcomes for potential parents. Source of Funding: NIH Ruth L. Kirschstein National Research Service Award (NRS) institutional training grant (T35DK103596) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e503 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Victor Yu More articles by this author Joemy Ramsay More articles by this author Joshua John Horns More articles by this author James Hotaling More articles by this author Expand All Advertisement PDF downloadLoading ...

“I Quit”: Schedule Volatility as a Driver of Voluntary Employee Turnover

Problem definition: Employers across many sectors of the economy have been fast to adopt variable work scheduling policies. The cost of this flexibility for employers is usually borne by employees, for whom unstable work schedules create several disruptions. In the context of home healthcare, we examine how employer-driven volatility in nurses’ schedules impacts their decision to voluntarily leave their job. Methodology/results: Using an instrumental variables approach, we causally identify the effect of schedule volatility on nurses’ voluntary turnover. We begin by constructing an operational measure of schedule volatility using time-stamped work log data from one of the largest home health agencies in the United States. Because this measure may be endogenous to the worker’s decision to quit, we instrument for schedule volatility using paid days off taken by other nurses in the same branch. We find that higher levels of schedule volatility substantially increase a worker’s likelihood of quitting. Specifically, a one-standard-deviation increase in schedule volatility increases the average worker’s propensity to quit on a given day by more than threefold. Translated into yearly terms, 30 days of high schedule volatility over the course of the year increases the average worker’s probability of quitting that year by 20%. Our policy simulations of counterfactual scheduling policies suggest that excess schedule volatility can explain a significant portion of voluntary turnover, and some interventions have the potential to substantially reduce workers’ daily propensity to quit. Managerial implications: This work contributes to the understanding of the extent to which employees value control over their own work schedules and are averse to volatile work schedules that are dictated by employers. Especially in the current environment where there is a growing emphasis on work-life balance and employee-driven flexibility, finding a way to support stable schedules could be important for employers to attract and retain workers. Funding: This work was supported by the National Research Service Award Postdoctoral Fellowship, the Wharton Dean's Research Fund, the Agency for Healthcare Research and Quality [T32 Grant 5T32HS26116], and the Claude Marion Endowed Faculty Scholar Award. Supplemental Material: The e-companion is available at https://doi.org/10.1287/msom.2023.1205 .

Ruth L. Kirschstein National Research Service Award—Short‐term research training (NIH)

Ruth L. Kirschstein National Research Service Award—Short‐term research training (NIH)