Postdoctoral Research Articles

Cell scientist to watch – Bhavani Shankar Sahu

ABSTRACT Bhavani Shankar Sahu is an Associate Professor at the National Brain Research Centre (NBRC) in Manesar, India. He completed his PhD in cell biology and molecular genetics at the Indian Institute of Technology Madras, studying links between neuroendocrine function and metabolic disease. Inspired to better understand fundamental mechanisms of neuropeptide secretion, he then completed postdoctoral research in Prof. Margaret Robinson's lab at the University of Cambridge, UK and with Prof. Alessandro Bartolomucci at the University of Minnesota and University of California San Diego, USA. In 2019, he returned to India to start his own lab at the NBRC, where he and his team investigate the biology of exocytosis in neurons and neuroendocrine cells from the molecular to the physiological level. We spoke with Bhavani over Zoom about his career journey, mentorship philosophy and enthusiasm for science outreach.

Power of introspection

This article presents the results of collaborative autoethnographical (CAE) research that investigated the group dynamics and processes of an undergraduate student, a post-doctoral fellow, and two senior administrators engaged in a students-as-partners (SaP) project. The CAE methodology allowed us to systematically, collaboratively, and iteratively analyze reflections on key takeaways from our experiences. We identified empowerment through learning and support and putting values into practice as essential components of working with SaP in mixed-role groups. This CAE research also substantiates the importance of negotiating existing power dynamics, as we identified a hierarchical structure within our group that we did not address during the actual project. Based on our group’s dynamics, we suggest that active reflection and communication regarding intersecting identities, shared and individual values, and an openness to learning and growth for all group members are critical components of working toward equitable partnerships with SaP.

Dynamic Home Care Routing and Scheduling with Uncertain Number of Visits per Referral

Despite the rapid growth of the home care industry, research on the scheduling and routing of home care visits in the presence of uncertainty is still limited. This paper investigates a dynamic version of this problem in which the number of referrals and their required number of visits are uncertain. We develop a Markov decision process (MDP) model for the single-nurse problem to minimize the expected weighted sum of the rejection, diversion, overtime, and travel time costs. Because optimally solving the MDP is intractable, we employ an approximate linear program (ALP) to obtain a feasible policy. The typical ALP approach can only solve very small-scale instances of the problem. We derive an intuitively explainable closed-form solution for the optimal ALP parameters in a special case of the problem. Inspired by this form, we provide two heuristic reduction techniques for the ALP model in the general problem to solve large-scale instances in an acceptable time. Numerical results show that the ALP policy outperforms a myopic policy that reflects current practice, and is better than a scenario-based policy in most instances considered. Funding: This work was supported by the Natural Sciences and Engineering Research Council of Canada [Grants RGPIN-2018-05225 and RGPIN-2020-210524] and by the Telfer School of Management SMRG Postdoctoral Research Fellowship Support [Grant 2020]. Supplemental Material: The electronic companion is available at https://doi.org/10.1287/trsc.2023.0120 .

The Africa non-communicable diseases (NCD) Open Lab: Impact of a portfolio of clinical studies to deepen the understanding of NCDs in sub-Saharan Africa.

Clinical research in sub-Saharan Africa (SSA) has often focussed on communicable diseases. However, with the increasing burden of non-communicable diseases (NCDs), there is a need for Africa-specific NCD research. GSK established the Africa NCD Open Lab in 2014. Three calls for proposals were advertised through various media channels. An external independent scientific advisory board, predominantly representing African scientists and NCD experts, reviewed and selected projects to receive funding. An additional programme in the Africa NCD Open Lab was designed to build statistical capability by supporting training initiatives. We assessed the impact of the Africa NCD Open Lab in three ways: scientific quality with impact; research training and professional development; and research environments. We captured metrics through regular reports/interactions with researchers; via a final report; and through exit interviews with principal investigators. Twenty projects in 11 African countries were funded; reports from 18 completed projects are available (data capture is ongoing). Overall, 139 articles have been published in peer-reviewed journals and other data have been presented at conferences and other forums. Most completed projects led to positive outcomes, such as further research, informing policy, or positively impacting clinical care, including three projects that saw changes to regional or national practice guidelines: the CREOLE study in Nigeria; the African Severe Asthma Program in Uganda; and the African Prospective Study on the Early Detection and Identification of Cardiovascular Disease and Hypertension in South Africa. Participation in the Africa NCD Open Lab led to the award of 34 grants related to or influenced by increased research capacity or experience. Significant professional development related to the projects also occurred with higher-level degrees being awarded, including 30 MScs, 30 PhDs, and nine postdoctoral fellowships. Through these projects, research capacity was strengthened across the region by equipping core research facilities, training research staff, strengthening research support services, and supporting the expansion of investigator networks. The completed Africa NCD Open Lab projects demonstrate high-quality research outcomes addressing important health challenges with potential benefits to African populations. Based on the success of the Africa NCD Open Lab, additional funding has been secured to extend the Open Lab initiative.

Identifying and Responding to Gaps in Services for International Graduate Students and Postdoctoral Fellows to Enhance the Graduate Community and Promote Quality Research Outcomes

The growing enrollment of international students in doctoral science, technology, engineering, and mathematics (STEM) programs in the U.S. has contributed to the need for an increase in specifically-designed programs, resources, and services that address the unique challenges of the international student population. When transitioning to the U.S. to pursue higher education, international students often experience obstacles, including language barriers, sociocultural challenges, and academic alienation, which significantly impacts the ability to successfully adapt to the new learning and living environment, oftentimes continuing throughout the duration of the program. Furthermore, international postdoctoral fellows engaged in STEM research fields may lack a sense of belonging due to similar barriers. The recruitment and retention of competitive international students and postdoctoral fellows directly impacts the quality and magnitude of research outcomes, while also enhancing and enriching the cultural diversity and experience of the graduate community. We use data collected from an international student and postdoctoral survey distributed to more than 65 students and 90 postdoctoral fellows as well as listening sessions to identify gap areas where additional resources are required and where modifications are needed to more productively serve the international population at Augusta University. Our findings indicate that international students and postdoctoral fellows experience more anxiety and isolation when they are not supported, personally, socially and academically, by their administrative leaders, faculty, peers, and surrounding community. Incoming students who were connected with current graduate students prior to their arrival to the U.S. experienced a greater level of support and were more confident and comfortable in their adjustment to society in the U.S. The implementation of additional events and experiences led by the responses of the international population, including research shadowing opportunities in on-campus laboratories, social gatherings connecting students and postdoctoral fellows of different cultures, writing circles and professional development workshops, and one-on-one sessions enhances community belonging and prioritizes personal and professional growth among this vulnerable student population. 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.

Increasing confidence in mentoring strategies for predoctoral and postdoctoral scholars through implementation of a mentoring academy

In support of the development of undergraduate researchers and the mentor development of predoctoral and postdoctoral scholars (Near Peers), a targeted near peer mentor training program was developed. Innovative and highly impactful early training experiences in research play a key role to inspire and motivate the next generation of scientists. Predoctoral and postdoctoral trainees are often asked to assist in the training and mentoring of short-term summer undergraduate researchers. Previous studies have shown that the efforts of these “near peers” to educate, train, and develop the summer undergraduate scholars have a positive impact on the undergraduate researchers. However, many near peer mentors have limited mentor training/experience and lack confidence in their ability to mentor undergraduate scholars. Thus, developing avenues and opportunities to train near peers on mentoring is vital to enhancing the experience of both the undergraduate researchers and near peers. The Kidney Undergraduate Research Experience (KURE) at the University of Alabama at Birmingham (UAB) engages undergraduate summer scholars with a wholistic research experience which includes an involved team of near peer mentors. A near peer mentor training program was developed with input from near peer focus groups, based on their interest in expanding their mentoring abilities and developing confidence in their role as a near peer mentor. The aim of this study was to determine whether this mentoring program was effective in increasing near peer mentoring abilities and confidence levels through quantitative and qualitative measures. Evaluations were given to near peers prior to, during, and at the end of the 6-month program (44 scholars started the program in Fall 2022 and 33 scholars completed all activities). We found that 83% of near peers expressed increased confidence in mentoring (n=24). Improvement in specific mentoring abilities were also assessed, such as “confidence in effectively communicating expectations” (pre- 4.02, ±0.82, n=44; post- 4.83 ±0.37, n=24; using a scale from 1 = not at all to 5 = very confident). We also identified the most impactful elements of the program including facilitation, topics, and platforms. Through assessing these aspects of the program, we determined essential and sustainable elements for future near peer mentor training programs. We conclude that a near peer mentor training program builds confidence and enhances the ability of predoctoral and postdoctoral scholars in their roles as near peer mentors. NIH R25DK115353 — 05S1. 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 progestinic drug, etonogestrel, rescues breathing in a rodent model of central chemoreflex impairment

The central CO2-chemoreflex is crucial in regulating breathing, providing stimulation at rest and during challenges to homeostasis. The retrotrapezoid nucleus (RTN), located in the ventral brainstem, is essential to this process. Impaired RTN function is linked to hypoventilation syndromes, including congenital central hypoventilation syndrome (CCHS) characterized by apnea and blunted responsiveness to inhaled CO2. Intriguingly, progestins stimulate breathing and CO2-chemoreflexes, and the high-affnity ligand etonogestrel (ETO) has been proposed as a treatment option in CCHS. Our objectives were to address two important gaps-in-knowledge: 1) assess ETO’s ability to “rescue” breathing in a rat model of central CO2-chemoreflex impairment, and 2) determine ETO’s site of action. We generated a model of CO2-chemoreflex impairment in adult female Sprague-Dawley rats using targeted injections of substance-P conjugated saporin toxin. Resulting RTN lesions were classified by the number of surviving chemosensitive neurons vs. non-surgical controls. We first administered systemic ETO (acute/vehicle:1h; or chronic/sham: 28 days) and measured resting ventilation and chemoreflexes (hypercapnia, 7.2% CO2; hypoxia, 10% O2) using whole-body plethysmography. In separate ongoing experiments, ETO was infused directly into target brain regions expressing progesterone receptors. We observed a positive relationship between lesion size and CO2-chemoreflex impairment (hypoxic response was intact). Twenty-one days of systemic ETO reversed CO2-impairment selectively in rats with moderate RTN lesions; large-lesion rats were refractory to ETO’s effects. These data suggest that ETO may enhance the CO2-chemoreflex by inducing neuroplasticity in brain structures with connectivity to surviving RTN chemosensitive neurons. Experiments are ongoing to parse out the site of ETO’s restorative action in the brain. T.A.J.:Canadian Lung Association BaO Fellowship, Canadian Institutes of Health Research (CIHR) Postdoctoral Fellowships. S.C.: Canadian Lung Association BaO Fellowship. S.P.: Women and Children’s Health Research Institute Innovation Grant, CIHR Project scheme grant. Nexplanon was kindly donated by Merck through the Investigator-Initiated Study Program (IISP # 55869; SP). 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.

Loss of nitric oxide bioavailability promotes arterial stiffening in a tissue transglutaminase-dependent manner

Although arterial stiffening undeniably contributes to cardiovascular disease, the cellular and molecular mechanisms leading to increased arterial stiffness are not well understood. Increased arterial stiffness in disease states such as obesity and type 2 diabetes typically coexists with indices of endothelial dysfunction including diminished nitric oxide (NO) bioavailability. In fact, it is commonly assumed that endothelial dysfunction precedes, and causally contributes to, arterial stiffening; yet the molecular underpinnings for this causal relationship remain elusive. We and others previously showed that activation of tissue transglutaminase (TG2), a crosslinking enzyme that also works as a G-protein and cellular scaffold, promotes cytoskeletal actin polymerization and stiffening of vascular smooth muscle cells (VSMC), thus contributing to whole-artery stiffening. It is also reported that NO is an endogenous inhibitor of TG2. Accordingly, herein, we tested the hypothesis that increased exposure of VSMC to NO suppresses TG2 activity and actin polymerization. Conversely, we hypothesized that loss of NO bioavailability promotes arterial stiffening, and that this effect is mediated by TG2 activity and downstream activation of LIM kinase (LIMK), an enzyme that modulates fibrillar (F)-actin severing via cofilin phosphorylation. All differences reported herein are significant at P<0.05. In support of our hypotheses, we show that treatment of cultured human aortic VSMC with the NO donor S-nitrosoglutathione (200μM for 2 hours) reduces TG2 activity (N-epsilon gamma glutamyl Lysine presence) and F-actin formation (phalloidin staining) induced by TG2 activation (dithiothreitol, 200μM). Furthermore, we show that exposure of mouse isolated mesenteric arteries to the NO synthase inhibitor L-NAME (300μM for 18 hours) causes an increase in arterial stiffness ( i.e., increased incremental modulus of elasticity) as determined using pressure myography and videomicroscopy. Of note, L-NAME-induced arterial stiffening is prevented when arteries are co-treated with a TG2 inhibitor (cystamine, 1mM) or LIMK inhibitor (LIMKi, 1μM). Taken together, these findings support the notion that NO is a key downregulator of TG2 activity and demonstrate that reduced NO bioavailability promotes arterial stiffening in a VSMC TG2-LIMK-F-actin dependent manner. As such, this work provides insight into plausible molecular mechanisms by which endothelial dysfunction leads to arterial stiffening. R01HL153264 (to LAML and JP), and MU Research Excellence Program (REP) postdoctoral fellowship (to FIRP). 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.

DNA methylation by DNMT3a drives endothelial inflammation and death during acute lung injury

Vascular endothelium, the inner most lining of all blood vessels, controls the transport of nutrients, protein, water, and leukocytes across the vessel wall and thereby maintains lung tissue and fluid homeostasis. Damage to endothelial integrity caused by mediagenic, inflammatory, or toxic agents results in pulmonary edema, inflammation, organ failure, and high lethality. A key pathogenic characteristic of vascular injury involves an excessive loss of endothelial cells (EC) accompanied by the generation of inflammatory cytokines. Genome-wide analyses of DNA methylation, the most enduring epigenetic modification of DNA, and its impact on EC transcriptomes and cellular functions, still lack comprehensive understanding. We demonstrate that lipopolysaccharide (LPS) elevated DNA methylation in EC within the lungs, primarily through a mechanism dependent on DNA methyltransferase 3a (DNMT 3a). Mice with endothelial cell-specific deletion of DNMT3a ( EC-DNMT3a−/−) promptly recovered from lung injury, attributed to a reduction of EC death. Furthermore, we observed that the deletion of DNMT3a in EC led to an increase in the expression of the apoptosis inhibitor B-cell lymphoma 2 (BCL2). Collectively, the data implies that inhibiting DNMT3a activity plays a crucial role in reshaping the EC methylome and promoting BCL-2 activity. This, in turn, facilitates the generation of reparative EC and contributes to the resolution of acute lung injury (ALI). These findings offer insights into potential targets for preventing ALI. American Heart Association (AHA) Postdoctoral Fellowship (19POST34450241). 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.

Carbon Dioxide as a Novel Blood Pressure Therapy for Neurogenic Orthostatic Hypotension

BACKGROUND: On standing, people with neurogenic orthostatic hypotension (nOH) experience plummeting blood pressure (>20/10mmHg), crippling symptoms, and syncope. Current blood pressure therapies lack effcacy and have serious side effects. In healthy people, increased inspired CO2 increases blood pressure. This raises the tantalizing possibility that increased inspired CO2 may effectively treat nOH patients. OBJECTIVE: We aimed to understand if increased inspired CO2 can increase standing blood pressure in nOH. METHODS: Seventeen patients with nOH (F=5; 71±8 years) completed three randomized sit-to-stand tests with inspired CO2 maintained 0mmHg (0CO2), +5mmHg (+5CO2) or +10mmHg (+10CO2) relative to baseline. Respiratory and hemodynamics (blood pressure, heart rate, stroke volume, cardiac output, systemic vascular resistance) were continuously measured. Data are reported as mean ± SD. The change (stand-sit) in respiratory and hemodynamics between 0mmHg, +5mmHg and +10mmHg were compared using a repeated measures ANOVA with a Bonferroni correction. RESULTS: Inspired CO2 caused a dose-dependent increase in standing blood pressure (0CO2:-43±25mmHg; +5CO2:-22±26mmHg; +10CO2:-4±30mmHg; p<0.001). Blood pressure changes were predominantly facilitated by increased stroke volume (0CO2:-10±10mL; +5CO2:-5±10mL; +10CO2:0.1±10mL;p<0.001) and cardiac output (0CO2:-0.2±0.6L/min; +5CO2:0.2±0.5L/min; +10CO2:0.7±0.5L/min; p<0.001) as neither the changes in heart rate (p=0.2) nor systemic vascular resistance (p=0.6) were different across CO2 conditions. Breathing CO2 decreased the percentage of time that standing systolic blood pressure dropped ≥20mmHg (0CO2:75±35%; +5CO2:48±38%; +10CO2:32±37%; p<0.001) and fewer patients met the blood pressure criteria for orthostatic hypotension (p=0.02). CONCLUSION: Our data indicates increased inspired CO2 can increase standing blood pressure in nOH patients by increasing stroke volume and cardiac output. CO2 may offer a novel pressor therapy for patients with nOH. This work was supported by a University of Calgary Seed Funding (S.R.R). J.R.B is supported by a Canadian Institutes of Health Research (CIHR) Fellowship, a Libin Cardiovascular Institute Post-Doctoral Fellowship in Women’s Cardiovascular Health, the Natural Sciences and Engineering Research Council of Canada (NSERC) Brain CREATE Program, and a Canadian Cardiovascular Society Fellowship. S.I.R. is supported by the NSERC Brain CREATE Program, an Alberta Graduate Excellence Scholarship, and an Alberta Strategy for Patient Oriented SUPPORT Unit scholarship. A.V.I is supported by a CIHR Fellowship and an Achievers in Medical Science Postdoctoral Fellowship. 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 Retrospective Analysis of Career Outcomes in Neuroscience.

What factors are associated with career outcomes among biomedical PhDs? Research to date has focused on drivers of interest in (and intention to pursue) various careers, especially during graduate school, but fewer studies have investigated participants' ultimate career outcomes. Even less is known about what factors matter for groups historically underrepresented in the US science, technology, engineering, and mathematics (STEM) workforce, such as women, some racial and ethnic groups, and persons with disabilities ( National Center for Science and Engineering Statistics (NCSES), 2021a). This study reports a new analysis of data from 781 PhD neuroscientists that were obtained from a retrospective survey ( Ullrich et al., 2021) to investigate the factors that influence the career sector in which neuroscience PhDs are employed and whether there were group differences according to social identity. We find evidence of academia as a "default path" for incoming PhD students, but interest in different careers increases over time. Those who remained in academia had greater acceptance of the structural aspects of academic careers, such as promotion and tenure processes, and greater faculty support during postdoctoral training. Conversely, prioritizing monetary compensation and/or varied work were associated with not being in academia, while a strong interest in research was positively associated with being in nonacademic research. Somewhat surprisingly, there were few interactions with gender, and no interactions with underrepresentation status, although perhaps this was due to lower statistical power for these analyses. Our findings also underscore the role of advisors, networking, and personal relationships in securing employment in STEM.

Acknowledgments

The CEC and ICMC Boards wish to thank our supporters and sponsors who have contributed to the 2023 CEC/ICMC Conference. CONFERENCE SPONSORS AND ADVERTISERS • Aerospace Fabrication and Materials • Cryofab, Inc • Cryogenic US LLC • Cryomech, Inc • Danaher Cryogenics LTD • FAMU-FSU College of Engineering • GenH2 • ICEoxford • National High Magnetic Field Laboratory • Scientific Instruments • Sumitomo (SHI) Cryogenics of America, Inc. • Texas Center for Superconductivity at the University of Houston EXHIBITORS • Ability Engineering Technology, Inc. • Aerospace Fabrication and Materials • Air Liquide Advanced Technologies • attocube systems Inc. • Barber-Nichols • Celadon Systems Inc. • Cryofab, Inc. • Cryomagnetics, Inc. • Cryomech, Inc. • CryoTechnics • CryoWorks • Cryoworld BV • Danaher Cryogenics LTD • FABRUM • FormFactor Inc. • Fujikura Ltd. • Honeywell • Hyper Tech • ICEoxford • Iris Technologies • Lake Shore Cryotronics • Lihan Cryogenics Co., Ltd. • Linde Kryotechnik AG • Omegaflex • Ratheon Technologies Research Center • Scientific Instruments • Stöhr Armaturen GmbH & Co.KG • Sumitomo (SHI) Cryogenics of America, Inc. • Sunpower, Inc. • Vacree Technologies Co., Ltd. • WSU HYPER Lab Producing the Open Access IOP Conference Series: Materials Science and Engineering (MSE) requires the dedicated effort of many individuals. The quality of the publication relies on the dedication of those who took time from their busy schedules to review and edit papers. The Boards wish to specifically acknowledge the efforts of Centennial Conferences for their editorial support, and the chief technical editors, John Weisend (CEC) and Balu Balachandran (ICMC). Their contributions to ensure a quality publication in a timely manner are greatly appreciated. The chief technical editors are indebted to the individual technical sub-editors for their time spent with reviewers and authors to get the final manuscripts ready for publication. The Boards also wish to thank all students and post-doctoral researchers from a number of universities for their service with the initial format checking of the papers submitted for peer review.CEC/ICMC 2023 was managed by Paula Pair and Centennial Conferences. Recognizing this is a tremendous team effort, the Boards would especially acknowledge, in addition to Paula, all her staff – Annett Cady, Andrea and Paul Crangle, Brion Jacobs, Andrew Kahl, Carrie Lian, Katherine Neilson, Alicia Orcutt, Sydney Pair, and Christina Tomhave – for the fantastic job they did wonderfully.We also would like to recognize the effort and dedication of the attendees in preparing their presentations and papers, and showing the professionalism and enthusiasm that makes the CEC/ICMC such a great Conference time after time.

Acknowledgments

The CEC and ICMC Boards wish to thank our supporters and sponsors who have contributed to the 2023 CEC/ICMC Conference. CONFERENCE SPONSORS AND ADVERTISERS • Aerospace Fabrication and Materials • Cryofab, Inc • Cryogenic US LLC • Cryomech, Inc • Danaher Cryogenics LTD • FAMU-FSU College of Engineering • GenH2 • ICEoxford • National High Magnetic Field Laboratory • Scientific Instruments • Sumitomo (SHI) Cryogenics of America, Inc. • Texas Center for Superconductivity at the University of Houston EXHIBITORS • Ability Engineering Technology, Inc. • Aerospace Fabrication and Materials • Air Liquide Advanced Technologies • attocube systems Inc. • Barber-Nichols • Celadon Systems Inc. • Cryofab, Inc. • Cryomagnetics, Inc. • Cryomech, Inc. • CryoTechnics • CryoWorks • Cryoworld BV • Danaher Cryogenics LTD • FABRUM • FormFactor Inc. • Fujikura Ltd. • Honeywell • Hyper Tech • ICEoxford • Iris Technologies • Lake Shore Cryotronics • Lihan Cryogenics Co., Ltd. • Linde Kryotechnik AG • Omegaflex • Ratheon Technologies Research Center • Scientific Instruments • Stöhr Armaturen GmbH & Co.KG • Sumitomo (SHI) Cryogenics of America, Inc. • Sunpower, Inc. • Vacree Technologies Co., Ltd. • WSU HYPER Lab Producing the Open Access IOP Conference Series: Materials Science and Engineering (MSE) requires the dedicated effort of many individuals. The quality of the publication relies on the dedication of those who took time from their busy schedules to review and edit papers. The Boards wish to specifically acknowledge the efforts of Centennial Conferences for their editorial support, and the chief technical editors, John Weisend (CEC) and Balu Balachandran (ICMC). Their contributions to ensure a quality publication in a timely manner are greatly appreciated. The chief technical editors are indebted to the individual technical sub-editors for their time spent with reviewers and authors to get the final manuscripts ready for publication. The Boards also wish to thank all students and post-doctoral researchers from a number of universities for their service with the initial format checking of the papers submitted for peer review.CEC/ICMC 2023 was managed by Paula Pair and Centennial Conferences. Recognizing this is a tremendous team effort, the Boards would especially acknowledge, in addition to Paula, all her staff – Annett Cady, Andrea and Paul Crangle, Brion Jacobs, Andrew Kahl, Carrie Lian, Katherine Neilson, Alicia Orcutt, Sydney Pair, and Christina Tomhave – for the fantastic job they did wonderfully.We also would like to recognize the effort and dedication of the attendees in preparing their presentations and papers, and showing the professionalism and enthusiasm that makes the CEC/ICMC such a great Conference time after time.

Cellular Mechanisms for pH Regulation and Energy Metabolism in Sharks and Rays

Similar to mammalian kidney distal tubule cells, specialized shark and ray gill epithelial cells maintain blood pH homeostasis. These shark and ray gill cells are either enriched with the Na+/K+-ATPase (NKA) and responsible for acid secretion or enriched for the vacuolar H+-ATPase (VHA) and responsible for base secretion. Moreover, both cell types contain abundant mitochondria and glycogen, and glycogen content decreases in active acid-base regulatory cells in vivo. Additional experiments with isolated gill cells in vitro show that glycogen coincides with the subcellular localization of NKA and VHA. Under control conditions, glycogen and NKA are at the cell membrane of acid-secreting cells while glycogen and VHA are in the cytoplasm of base-secreting cells. During exposure to alkalosis, NKA and glycogen localization remains unchanged in acid-secreting cells; however, both VHA and glycogen translocate to the cell membrane of base-secreting cells. Interestingly, this process is prevented by pharmacological inhibition of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC). This suggests a novel mechanism, a coupling of glycogen metabolism with acid-base regulation that sustains ATPase function during sudden periods of increased energy demand. Since VHA, sAC, and glycogen are evolutionarily widespread, this mechanism is likely to apply broadly to other organisms and systems, perhaps including chronic kidney disease. APS Porter Predoctoral Fellowship and National Science Foundation Postdoctoral Research Fellowship in Biology #1709911 to JR, National Science Foundation Grant IOS 1354181 to MT. 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.

EphB2 deficiency in hepatic stellate cells mitigates fibrogenesis in metabolic dysfunction-associated steatohepatitis by attenuating the TGFβ/SMAD signaling

Background & Aims: The receptor tyrosine kinase EphB2 is now emerging as a critical regulator of inflammation and fibrogenesis in metabolic dysfunction-associated steatohepatitis (MASH) though the underlying mechanism remain unclear. We aimed to investigate the role or mechanism by which EphB2 contributes in the progression of MASH fibrogenesis. Hypothesis: We hypothesized that EphB2 in hepatic stellate cells (HSCs) promotes MASH-liver fibrosis Methods: Single nuclear RNA-sequencing of liver from human and mouse MASH were analyzed for EphB2 expression. Human liver biopsies obtained from patients with MASH were used to assess EphB2 expression by immunohistochemistry. Primary human HSCs were stimulated with TGFβ with or without TGFβR/SMAD inhibitors (SB525334, CS208, and SIS3 at 1μM) and EphB2 expression assessed by qPCR, western blot and immunofluorescence staining. EphB2 was silenced in primary human HSCs using siRNA and the resulting HSCs assess for TGBβ-induced HSC-myofibroblasts transition. MASH models were developed in hepatocyte-specific EphB2 knockout and HSCs EphB2 knockout and wild-type mice fed a high fat diet for 26 weeks or a choline-deficient amino-acid defined (CDAA) high fat diet for 10 weeks. Statistical analysis was performed using GraphPad Prism and a p-value < 0.05 was considered significant. Results: The expression of EphB2 was 3-fold significantly upregulated in liver specimens from patients with advanced MASH and correlated with disease severity. EphB2 was modestly expressed in hepatocyte, but strongly induced in HSCs in human and mouse MASH. Hepatocyte-specific EphB2 deficiency does not inhibit the development of steatohepatitis and fibrosis in mice fed the CDAA-diet. TGFβ/SMAD signaling promotes an 8-fold increase of EphB2 expression in human HSCs while siRNA silencing of EphB2 in human HSCs was suffcient to abolished TGFβ-mediated HSCs activation. Interestingly, HSCs-specific EphB2 knockout mice developed 2.5 to 6-fold less fibrosis than wild-type controls in response to high fat diets feeding suggesting that EphB2 expression on HSCs is required for the development of fibrogenesis in MASH. Deletion of EphB2 in activated HSCs after fibrosis was established in mice was suffcient to mitigate MASH fibrosis. Conclusions: EphB2 is upregulated in human and mouse MASH. Upregulation of EphB2 in human HSCs is mediated via the TGFβ/SMAD signaling. HSCs expressing EphB2 regulate the development of MASH-fibrosis. Specific EphB2 inhibitors could potentially mitigates fibrogenesis in MASH, opening up an avenue for the treatment of MASH. Funding sources: NIDDK, NIAMS, APS postdoctoral fellowship. 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.

Co-transplantation of hiPSC-Derived Mesenchymal Stem Cells and Cardiomyocytes Enhances Cardiac Regeneration After Myocardial Infarction

Nearly a billion cardiomyocytes (CMs) are lost during an acute myocardial infarction (AMI) which eventually leads to heart failure. While most conventional treatments for AMI are only palliative, transplantation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) has been shown to improve cardiac function. However, the use of hiPSC-CMs has been limited by their poor engraftment and survival in the ischemic microenvironment post-MI. Since hiPSC-derived mesenchymal stem cells (hiPSC-MSCs) have been shown to promote cardioprotection via paracrine mechanisms, we hypothesized that co-transplantation of hiPSC-CMs and hiPSC-MSCs in ischemic hearts may improve cardioprotection and engraftment of transplanted hiPSC-CMs. We co-cultured hiPSC-CMs and hiPSC-MSCs and assessed the changes in morphology (TEM, ICC), gene expression (qRT-PCR), and cardiac function (MEA, and calcium transients) under normoxia and hypoxia (1% O2). We observed increased maturation (elevated β-MHC expression, higher amplitude of contraction, increased sarcomere alignment) in hiPSC-CMs cultured in hiPSC-MSC derived conditioned medium. On the other hand, we observed enhanced survival (TUNEL assay) of hiPSC-CMs co-cultured with hiPSC-CMs following hypoxic insult. Additionally, following intramyocardial transplantation of (a) hiPSC-CMs, (b) hiPSC-MSCs or (c) hiPSC-CMs + hiPSC-MSCs (3:1) in a mouse MI model, we observed improved cardiac function, LVEF (p < 0.01) and FS (p<0.01) in all three groups as compared to MI-only group. We also observed effcient hiPSC-CM engraftment (human-specific markers, cTnT and HNA) in both hiPSC-CM-only and hiPSC-CM + hiPSC-MSC groups at 4-weeks post-MI. Furthermore, we observed increased angiogenesis (α-SMA, CD31) in the hiPSC-MSC-only and hiPSC-CM + hiPSC-MSC groups, as compared to control and hiPSC-CM-only groups. Taken together, our results showed that co-transplantation of hiPSC-MSCs and hiPSC-CMs can be explored as a novel combinatorial therapy for myocardial regeneration post-MI. Funding: AHA postdoctoral fellowship to DS (916599), and NIH RO1 HLHL136232 and OSU Start-up funds to MK. 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.

Motor unit loss and neuromuscular junction degeneration precede clinically diagnosed sarcopenia

Background: Degeneration of motoneuron and neuromuscular junctions (NMJ) and loss of motor units (MUs) contribute to age-related muscle wasting and weakness, leading to sarcopenia. However, these features have not been adequately investigated in humans. This study aimed to compare neuromuscular system integrity and function across different stages of sarcopenia, with a particular focus on NMJ stability and MU properties. We hypothesized that progressive impairment of the neuromuscular system would be a hallmark of the progression of sarcopenia. Methods: We recruited 42 healthy young individuals (Y) (aged 25.98±4.6 yr; 57% females) and 88 older individuals (aged 75.9±4.7 yr; 55% females). The older group underwent a sarcopenia screening according to the revised guidelines of the European Working Group on Sarcopenia in Older People (EWGSOP2). In all groups, knee extensor muscle force was evaluated by isometric dynamometry, muscle size by ultrasound and MUP properties by intramuscular electromyography (iEMG). MU number estimate (iMUNE) was also obtained. Blood samples and muscle biopsies were collected to assess biomarkers of neuromuscular degeneration. Results: 39 older individuals were non-sarcopenic (NS), 31 pre-sarcopenic (PS) and 18 sarcopenic (S). A progressive decline in quadriceps force, cross-sectional area and appendicular lean mass was detected with the different stages of sarcopenia. Handgrip force and short physical performance battery (SPPB) scores were also progressively reduced. iEMG recordings showed increased near fiber segment jitter in NS, PS and S compared to the Y group, highlighting a decreased NMJ transmission stability. Elevated concentration of serum C-terminal agrin fragment and altered Cav3 protein expression further supported the finding of age-related NMJ instability in all the older groups. The iMUNE was lower in all older groups, confirming age-related loss of MUs. Motoneuron firing rate was decreased in S vs Y. Age-related increases in MU potential complexity were also observed. These observations were accompanied by increased muscle denervation and axonal damage, respectively, indicated by the increase in NCAM-positive fibres and the increase in serum concentration of neurofilament light chain. Notably, most of these MU and NMJ parameters did not differ in older individuals with or without sarcopenia. Conclusion: Alterations in MU properties, axonal damage, altered innervation profile and NMJ instability are signatures of neuromuscular system ageing. These neuromuscular impairments are accompanied by muscle atrophy and weakness, however, they appear to precede clinically diagnosed sarcopenia, as they are already detectable in older NS individuals. These are important parameters to monitor in order to counteract these impairments and slow down the onset and progression of sarcopenia. The present work was funded by the PRIN project ‘NeuAge’ (2017CBF8NJ_001) to MVN and MAP and the Milky Way Foundation, the Baxter Foundation and the Li Ka Shing Foundation to HMB. EM was supported by the Stanford Wu Tsai Human Performance Institute fellowship and Marie Skłodowska-Curie Actions postdoctoral fellowship. We also acknowledge co-funding from Next Generation EU to MVN, in the context of the National Recovery and Resilience Plan, Investment PE8 — Project Age-It: “Ageing Well in an Ageing Society”. This resource was co-financed by the Next Generation EU [DM 1557 11.10.2022]. The views and opinions expressed are only those of the authors and do not necessarily reflect those of the European Union or the European Commission. Neither the European Union nor the European Commission can be held responsible for them. 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.

Na/K-ATPase α1/Src Signaling Regulates Mitochondrial Metabolic Function and ROS Production in Human iPSC-derived Cardiomyocytes

Through an isoform specific mechanism, the Na/K-ATPase (NKA) α1 polypeptide-mediated regulation of Src kinase has emerged as a novel regulator of mitochondrial function in various cell types. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To investigate the importance of the proposed NKA/Src regulatory signaling on cardiac mitochondrial metabolic function independently from the classic Na/K-ATPase enzymatic function, we used a gene targeting approach in cardiomyocytes. Human induced pluripotent stem cells (hiPSC) expressing a Src-signaling null mutant (A420P) form of NKA α1 were generated using CRISPR/Cas9-mediated genome editing. NKA isoform protein expression and enzymatic activity remained unchanged. However, baseline levels of Src and ERK1/2 phosphorylation were decreased by 55% and 80%, respectively in the A420P hiPSC (n=3, p<0.05). Both the WT and A420P hiPSC differentiated into cardiomyocytes (iCM) as assessed by morphology, spontaneous cell contraction, marker gene expression, and subcellular striations. Cell metabolism assessed by Seahorse extracellular flux analysis revealed significant reductions in basal and maximal rates of mitochondrial respiration (50%), spare respiratory capacity (50%), ATP production (60%), and coupling effciency (20%) (n=4, p<0.05). ROS production was monitored in live cells by measuring the oxidation of the CM-H2DCFDA dye using a fluorescence microscope. The slope of the ROS production rate curve was dramatically reduced in A420P iCM (80%, n=3, p<0.05). Protein carbonylation levels, analyzed by ELISA as markers of oxidative damage, were also decreased in A420P iCM (n=4, p<0.05). Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This has potential significance in cardiometabolic diseases and suggests that targeting the NKA α1/Src signaling axis may offer a novel approach to address mitochondrial dysfunction. NIH Grant R15 HL145666, NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence, and American Heart Association Postdoctoral Fellowship — 22POST917776 — to Marco T. Pessoa. 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.

Deletion of Mitogen-inducible gene-6 (Mig6) in the Liver Alters Glucose Homeostasis and Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) Progression

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a spectrum of chronic liver disease that can progress from benign steatosis to metabolic dysfunction-associated steatohepatitis (MASH) - both of which, if left untreated, can progress to cirrhosis and hepatocellular carcinoma. The strongest risk factor for progression of MASLD to MASH is insulin resistance. Previous work in our lab identified that the adaptor protein mitogen-inducible gene-6 (Mig6) is increased in obese mice and that deletion of Mig6 in the liver (LKO) improves glucose tolerance and insulin action during hyperinsulinemic-euglycemic clamps in diet-induced obese mice. Mig6 is an endogenous feedback inhibitor of epidermal growth factor receptor activation (EGFR) and regulates cellular repair and survival. Thus, we sought to elucidate the extent to which loss of Mig6 modulates the progression of MASLD to MASH. We hypothesized that liver-specific loss of Mig6 during early MASLD improves whole-body glucose homeostasis and protects against MASLD-mediated liver damage. LKO mice and matched, control littermates (CON) were fed a MASH diet (40% fat, 40% carbohydrate, 2% cholesterol; CON n=13-19, LKO n=16-22) or a low-fat-matched diet (LFD; 10% fat, 70% carbohydrate; CON n=8-16, LKO n=16-21) for up to 40 weeks. Body weights were recorded weekly, and body composition assessment, glucose and insulin tolerance tests, and hepatic histological analysis were performed after 20-, 30- and 40-weeks on each diet. Compared to the MASH-fed CON mice, MASH-fed LKO mice had lower body weights starting at 10-weeks and persisting throughout the study. Body composition analysis established these differences in body weight were due to decreases in fat, but not lean, mass. Interestingly, whereas intraperitoneal glucose tolerance tests did not identify significant differences in glucose handling between the LFD and MASH fed mice, oral glucose tolerance was significantly improved in 10- and 20-week MASH-fed LKO mice, suggesting alterations in the incretin response in this MASH model. Lastly, histological analysis revealed MASH-fed LKO mice had a preservation of hepatic lipid zonation and blunted levels of circulating alanine aminotransferase (ALT) compared to MASH-fed CON, indicating less hepatic damage in LKO mice. These results suggest that liver-specific loss of Mig6 improves whole-body glucose handling, which in turn ameliorates hepatic damage during MASLD progression. In conclusion, Mig6 prevents, or at least delays, the progression of MASLD, which may reveal a new therapeutic area for the prevention and treatment of MASH. MS was supported by a National Cancer Institute Cancer Metabolism Training Program Postdoctoral Fellowship (T32CA221709). 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.

Sex Differences In The Phenotype Of Sleep-Disordered Breathing In C57BL/6J Mice

Sex differences in sleep-disordered breathing (SDB) are widely recognized. SDB is more prevalent in men, but women appear to exhibit more symptoms at lower levels of SDB severity, including insomnia symptoms. Mechanisms underlying sex differences in SDB are unknown. In this study, we compared the sleep architecture and breathing patterns during sleep between male and female mice and examined the effects of ovariectomy on SDB. We hypothesized that female and male mice differed in sleep and SDB, which are related to differences in carbon dioxide (CO2) chemosensitivity between sexes. Adult male (n=9) and female (n=10) C57BL/6J mice underwent full-polysomnography inside a whole-body plethysmography chamber. Arousals from sleep and apneas were manually scored. Breathing variability was assessed with Poincaré plots of minute ventilation. Hypercapnic ventilatory response (HCVR) was assessed during wakefulness and NREM sleep by acute administration of 8% CO2. In female mice, we performed bilateral ovariectomy and repeated sleep studies after 14 days. We found that arousal index was 55% increased in females compared to males, but sleep fragmentation was mainly attributed to spontaneous arousals in both groups. Surprisingly, apnea severity was significantly increased in female mice compared to males (32.4±6.7 vs 11.8±4.5, P=0.001). Breathing instability was also increased in females. HCVR was positively correlated with apnea index (r=0.55, P=0.05) and was significantly augmented in females compared to males. Ovariectomy did not affect sleep fragmentation, but attenuated CO2 sensitivity and improved breathing stability during sleep. Our results have suggested different SDB phenotypes between male and female C57BL/6J mice. SDB in female mice is associated with a hyperarousal state and increased HCVR compared to males. An exacerbated CO2 responsiveness related to ovarian hormones in females is an important determinant of differences in SDB severity and respiratory patterns between male and female sex. American Heart Association Postdoctoral Fellowship Award 828142 (to L.J.K.), NHLBI grant 2R01 HL133100-05, NIH R01 HL128970, and R01 HL13892 (to V.Y.P.), 1K23HL155730 and American Academy of Sleep Medicine Foundation 238-BS-20 (to L.V.P.). 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.