bluuebunny/crossref_metadata_2025 huggingface dataset:
Carotid Baroreflex Responsiveness in Patients with Heart Failure with a Preserved Ejection Fraction
Authors: Francisco Michael , Alpenglow Jeremy , Bunsawat Kanokwan , Iacovelli Jarred , Ma Christy , Ryan John , Quencer Keith , Kaufman Claire , Wray Walter
Published: May 2022

The carotid baroreflex (CBR) plays an important role in blood pressure regulation by modulating heart rate (HR) and vasomotor tone. In humans, CBR function can be investigated non‐invasively using the variable pressure neck collar, which alters carotid sinus transmural pressure through application of positive and negative pressure within the neck chamber. Using this technique, derangements in CBR function have been identified in a variety of patient populations, but whether patients with heart failure with a preserved ejection fraction (HFpEF) suffer from disease‐related changes in CBR responsiveness has yet to be determined. We evaluated beat‐to‐beat changes in HR and mean arterial pressure (MAP) in response to 5‐sec pulses of neck pressure (NP, +40 Torr) and neck suction (NS, ‐80 Torr) to simulate carotid hypotension and hypertension, respectively in 5 patients with HFpEF (69±3 yrs and BMI: 38±4) and 4 healthy, older controls (68±2 yrs and BMI: 26±3). Resting HR was lower in patients with HFpEF compared to control subjects (77±7 vs. 68±6 bpm, respectively; p<0.005), while MAP was similar between groups (88±2 vs. 88±5mmHg, HFpEF vs controls). Following NP, a disease‐related decrement in CBR responsiveness was evident, with diminished changes in HR (2±1 vs 9±1 bpm, HFpEF vs controls, p=0.041) and MAP (4±2 vs 8±1 mmHg, p=0.095) in the HFpEF group. Similarly, changes in both HR (‐6±1 vs ‐8±1 bpm, HFpEF vs controls, p=0.21) and MAP (‐5±1 vs ‐8±1 mmHg, HFpEF vs controls, p=0.16) in response to carotid hypertension provoked by NS were reduced in patient with HFpEF. Though preliminary in nature, these initial findings appear to indicate a decline in carotid baroreflex‐mediated cardiac and vasomotor control in patients with HFpEF that is likely to contribute significantly to autonomic nervous system dysregulation in this patient group.
The Impact of Short‐Term Tetrahydrobiopterin (BH4) Supplementation on Peripheral Vascular Function in Heart Failure with Preserved Ejection Fraction (HFpEF)
Authors: Bunsawat Kanokwan , Broxterman Ryan M. , Jarrett Catherine L. , Craig Jesse C. , Francisco Michael A. , Alpenglow Jeremy K. , Iacovelli Jarred J. , Ma Christy L. , Ryan John J. , Wray D. W.
Published: May 2022

Background

Patients with heart failure with preserved ejection fraction (HFpEF) exhibit macro‐ and microvascular dysfunction in both the upper and lower limbs that may be mediated by a decline in nitric oxide (NO) bioavailability. Tetrahydrobiopterin (BH4) is a vital cofactor for NO synthase (NOS) activity, the principal enzyme for NO production. BH4 insufficiency contributes to NOS uncoupling, which promotes cellular oxidative stress and limits NO production. While evidence supports the efficacy of BH4 supplementation to reverse NOS uncoupling in other patient populations, the utility of this intervention to restore vascular health has not been examined in HFpEF. We tested the hypothesis that short‐term BH4 supplementation would improve peripheral vascular function in patients with HFpEF.

Methods

Five patients with HFpEF (3F/2M; 72±8 years; 31.7±6.1 kg/m2) participated in a randomized, double‐blind crossover study, having consumed either BH4 (Sapropterin, 10 mg/kg) or placebo for 10 days, separated by a 14‐day washout period. Peripheral vascular function was assessed via flow‐mediated dilation (FMD; an index of macrovascular function) and reactive hyperemia (RH, an index of microvascular function) in both the arm (brachial artery, BA) and leg (popliteal artery, PA) at the beginning (pre) and end (post) of each treatment period.

Results

There were no baseline hemodynamic differences between trials (p>0.05). BA FMD was unchanged following either trial (pre vs. post; BH4: 3.2±2.5% vs. 4.0±1.9%, Δ 0.8±1.4%; Placebo: 2.9±1.9% vs. 2.2±2.2%, Δ ‐0.7±1.1%; p>0.05). Likewise, BA RH was unchanged following either trial (pre vs. post; BH4: 399±206 ml vs. 387±161 ml; Placebo: 242±126 ml vs. 174±149 ml; p>0.05). In contrast, PA FMD improved following the BH4 trial, but was unchanged in the placebo trial (pre vs. post; BH4: 1.5±1.2% vs. 2.5±0.8%; Placebo: 1.6±1.1% vs. 1.3±1.3%; p=0.03). Changes in %FMD for the PA were greater in BH4 compared to placebo (∆ values: 1.0±1.0% vs. ‐0.4±0.4%; p=0.03). No improvements were observed for PA RH (pre vs. post; BH4: 533±257 ml vs. 561±191 ml; Placebo: 682±284 ml vs. 656±107 ml; p>0.05).

Conclusion

These findings identify a potential for short‐term BH4 supplementation to improve lower limb macrovascular function in patients with HFpEF. This apparent vascular plasticity suggests that drugs targeting discreet points in the NO pathway may be efficacious in mitigating the disease‐related decline in vascular health in this patient group.
HoxB8‐conditional Neutrophil Progenitor Transplant Provides a Therapeutic Benefit for S. aureusPulmonary Infection in a Murine Model of Chronic Granulomatous Disease (CGD)
Authors: Hinman Kristina , Cohen Joshua , Mundy Miles , Lefort Craig
Published: May 2022

CGD is a congenital disease marked by neutrophil dysfunction leading to increased infectious susceptibility. Despite prophylactic treatment with antimicrobial agents, outcomes for CGD patients are troubling with a typical lifespan of 30‐40 years due to breakthrough infections. As we strive to stay ahead of the impacts of drug‐resistant microbes, it is reasonable to consider cellular therapeutics which replace immune cells to treat CGD and other immunodeficiencies. We hypothesize that a conditionally‐immortalized neutrophil progenitor cell line has a translational application as a therapy for immune compromised hosts. This cell line was first generated by Wang, et al. through inducing ectopic expression of the transcription factor HoxB8 in murine bone marrow stem cells. Induction of HoxB8 leads to exponential growth of cells, while withdrawal leads to differentiation into neutrophil‐like cells. We have found that unconditioned transplant of HoxB8 progenitors lead to robust engraftment in the bone marrow. Engrafted progenitor cells differentiate in vivointo donor‐derived neutrophils found circulating in the peripheral blood. Donor neutrophils are also recruited to the lungs of mice infected with Staphylococcus aureus. Using a novel fluorescent killing assay, we demonstrate that donor‐derived neutrophils can kill pulmonary S. aureus both in vitro and in vivo with comparable efficiency to wild type host neutrophils. Strikingly, our results find that treatment with HoxB8‐conditional progenitors improves survival of CGD animals infected with S. aureus from 0% to >50%. This work demonstrates the potential of conditionally‐immortalized neutrophil progenitors as a novel cellular therapy in the context of neutrophil dysfunction.
Pharmacological Inhibition of Cullin Neddylation Improves Hepatic Insulin Sensitivity
Authors: Gu Lijie , Chen Cheng , Matye David J. , Hasan Mohammad N. , Clayton Yung‐Dai , Li Tiangang
Published: May 2022

Background and significance

Hepatic insulin resistance is a hallmark feature of non‐alcoholic fatty liver disease (NAFLD) and type‐2 diabetes (T2D) and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress‐sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin‐RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin‐like protein called Nedd8 to a cullin scaffold.

Aim

To investigate the role of CRLs in regulating hepatic insulin signaling in NAFLD.

Methods

Neddylation inhibitor MLN4924 was used to study its effect on insulin sensitivity in liver cells. Hyperinsulinemic‐euglycemic clamp was used to assess MNL4924 effect on insulin sensitivity in vivo. siRNA‐mediated screening was used to identify the Cullin members that mediates IRS protein turnover in liver cells. Co‐immunoprecipitation was performed to study Cullin and IRS interaction. Glucose homeostasis and insulin sensitivity were studied in mice with AAV‐mediated liver specific knockdown of Cul1 or Cul3.

Results

Neddylation inhibition delays CRL‐mediated IRS protein turnover to prolong insulin action in cultured liver cells. Consistently, acute MLN4924 treatment rapidly decreases hepatic glucose production without causing hypoglycemia in mice, while chronic MLN4924 treatment completely normalizes hyperglycemia in obese mice independent of obesity. Hyperinsulinemic‐euglycemic clamp study revealed that MLN4924 significantly increased insulin sensitivity and improved hepatic glucose output. In vitro siRNA based screening showed that knockdown of Cul1 or Cul3, but not other cullin family members, stabilized IRS protein and increased insulin sensitivity in AML12 cells. Consistently, Co‐immunoprecipitation demonstrated IRS interaction with Cul1 and Cul3. AAV‐mediated liver specific knockdown of Cul1 in mice improved insulin sensitivity, lowered fasting glucose, and prevented Western diet‐induced obesity and NAFLD. In contrast, AAV‐mediated liver Cul3 knockdown only decreased fasting glucose in chow‐fed mice but not Western diet‐fed mice.

Conclusion

These findings suggest that pharmacological inhibition of cullin neddylation is a novel therapeutic strategy for hepatic insulin sensitization in NAFLD and T2D. This effect may be at least in part mediated by hepatic Cul1 and Cul3 neddylation.
Long‐term myocardial DNA methylation and metabolomic responses to exertional heat stroke in mice
Authors: Murray Kevin O. , Brant Jason O. , Kladde Michael P. , Clanton Thomas L.
Published: May 2022

Epidemiological studies indicate that exposure to exertional heat stroke (EHS) can increase the risk of all‐cause cardiovascular and systemic disease and promote early mortality. Using a preclinical mouse model of EHS, we previously characterized profound DNA methylation changes in immune cells at 30 d of recovery and severe cardiometabolic disturbances in the ventricular myocardium (VM) at 9‐14 d. These observations indicate that a single exposure to EHS can result in long‐term epigenetic and metabolic alterations. However, whether a long‐term epigenetic responses occur in the VM and or whether cardiometabolic perturbations persist is unknown.

PURPOSE

To determine if long‐term alterations to (1) DNA methylation and (2) the metabolome occur within the VM after 30 d of recovery from EHS and (3) whether epigenetic alterations predict imbalances within the metabolome.

METHODS

Sixteen C57BL6 female mice underwent a standardized EHS protocol using a forced running wheel (environmental temp: 37.5°C, 40% humidity) or a matched exercise control trial (EXC) (22.5°C). The EHS mice achieved peak core temps of ~42.4°C, accompanied by transient loss of consciousness. Mice were sacrificed after 30 d of recovery; left ventricles were isolated and frozen in liquid nitrogen for DNA methylation sequencing and untargeted metabolomics.

RESULTS

Following 30 d of recovery from EHS, more than 6,000 differentially methylated cytosines (DMCs) were observed within promoter regions; 900 differentially methylated regions (DMRs) were also observed (i.e ≥ 5 differentially DMCs within 300 base pairs) at (Q < 0.05) compared to EXC. Pathway analysis of promoter DMRs indicated changes in genes that modulate basic cell functions, DNA binding, transcription, and metabolism. Cardiometabolic perturbations were found to largely resolve compared to observations at 9‐14 d. Some metabolic disturbances remained, e.g. reductions in α‐ketoglutarate and increases in several lipid intermediates. These remnant alterations are known to be common in early onset heart failure and chronic kidney disease. Repressive DMCs (P < 0.05) were observed within transcriptional regulatory regions of enzymes that regulate α‐ketoglutarate, e.g. isocitrate dehydrogenase 1 & 2 (IDH1 & IDH2), suggesting a potential link with metabolomic findings.

CONCLUSIONS

A single EHS episode results in a robust epigenetic response in VM when observed at 30 d of recovery, whereas all but a subset of metabolites within the VM had fully recovered. These long‐term molecular changes may induce altered or maladaptive physiological outcomes in response to future environmental or medical challenges.
Intrarenal Renin‐Angiotensin System and its Role in Angiotensin II‐Induced Hypertension: Insights from Computational Modelling
Authors: Smith Delaney , Layton Anita
Published: May 2022

The kidney expresses all and independently regulates most elements of the renin‐angiotensin system (RAS). Importantly, the inappropriate activation of intrarenal RAS, as indicated by the local accumulation of angiotensin II (Ang II), has been linked to Ang II‐induced hypertension. Various Ang II and angiotensin type 1 receptor (AT1R)‐dependent positive feedback systems have been documented in the literature. However, their functional importance to the overactivation of the intrarenal RAS have yet to be lineated. The goal of this study is to identify the key mechanisms actuating the intrarenal accumulation of Ang II during hypertension induced by Ang II infusion. We hypothesize that the upregulation of tubular epithelial AT1Rs is the primary mediator of intrarenal RAS overactivation following Ang II infusion. To test this hypothesis, computational models of the intrarenal RAS and the AT1R‐blocker Losartan were created, coupled, and used to simulate Ang II‐infusion experiments in the absence and presence of this anti‐hypertensive therapy. Model simulations indicate that total renal Ang II concentrations rise once Ang II starts accumulating within tubular epithelial cells and that the upregulation of local AT1R expression, not Ang II production, is required to produce this effect. Indeed, the removal of this positive feedback and the administration of Losartan both prevented the intrarenal accumulation of Ang II. Since the simulated onset of Ang II accumulation in tubular epithelial cells coincides exactly with the experimental inception of Ang II‐dependent hypertension, we conclude that the stimulation of sodium reabsorption mediated by local Ang II is likely key to the development of Ang II‐induced hypertension.
Elucidating the Role of the Hsc70 Chaperone System in SNARE Complex Formation
Authors: Bogoian‐Mullen Antonia , Bhasne Karishma , Clerico Eugenia M. , Gierasch Lila M.
Published: May 2022

Neurons communicate through neurotransmitters that are released into the synaptic cleft. For this release to occur a synaptic vesicle must fuse with the plasma membrane in the presynaptic terminal of neurons. Fusion requires the formation of the SNARE complex, which is composed of four helices from three proteins: one helix from syntaxin, one helix from synaptobrevin, and two from SNAP‐25 (one from the C‐terminal and one from the N‐terminal). This fusion process is tightly regulated and requires an Hsp70 chaperone system including the constitutive cytoplasmic Hsp70, Hsc70, and its co‐chaperones CSPɑ (a J protein) and Hsp110 (a nucleotide‐exchange factor). However, the molecular details of this system are still poorly understood. SNAP‐25 is a disordered protein that is prone to aggregation and premature folding. Therefore, we hypothesize that SNAP‐25 is chaperoned by Hsc70 to ensure that it remains in a state that is competent for SNARE complex formation. Using a peptide array of SNAP‐25, we identified three most probable binding sites of Hsc70 on SNAP‐25. We took the advantage of the fact that the fully formed SNARE complex is SDS‐resistant and used SDS‐PAGE to monitor successful reconstitution of the SNARE complex in vitro. Our preliminary data support our hypothesis in that the addition to Hsc70 facilitates the formation of the SNARE complex. Our goal is to elucidate in detail the molecular interactions of Hsc70 and CSPɑ with the SNARE complex components, notably SNAP‐25, in the formation of the SNARE complex and the initial stages of synaptic vesicle fusion.
Obese Animal Serum Derived Vesicles Alter Gene Expression in C2C12 Myotubes
Authors: Pitzer Christopher , Paez Hector G. , Ferrandi Peter J. , Mohamed Junaith S. , Alway Stephen E.
Published: May 2022

Purpose

Regulation of metabolism by extracellular vesicles (EV’s) is an emerging interest due to the ability of vesicle‐bound cargo to alter gene expression. C2C12 myotubes are an in vitro model of murine skeletal muscle fibers. The skeletal muscle is the primary site of glucose disposal in the body. Obesity is known to decrease insulin‐stimulated glucose uptake in the muscle. The purpose of this project was to determine the changes in gene expression caused by serum‐derived extracellular vesicles of an obese animal relative to normal healthy controls. Due to the known association of obesity and dysregulated metabolism, we hypothesized that EV’s isolated from serum of an obese animal would negatively alter metabolically important gene expression.

Methods

Serum was collected from obese, but not diabetic (body weight 42 g blood glucose 131 mg/dL) and age‐matched non obese/diabetic control (body weight 26g blood glucose 115 mg/dL), mice and extracellular vesicles were precipitated using a commercially available precipitation reagent (Exoquick, System Biosciences Paolo Alto CA). Fully differentiated myotubes (5 days in differentiation media) were incubated with EV’s (n=4 per group) protein matched to a concentration of 37.5 µg/ml in exosome‐free differentiation media (DMEM supplemented with 1% exosome free FBS and 1% anti‐anti) for 24 hours. Total RNA was isolated using Trizol and cDNA synthesis was performed prior to real‐time PCR experiments.

Results

NR4A1 mRNA expression was significantly increased in obese animal exosome treated myotubes relative to controls (15% increase P=0.016). NR4A2 also tended to increase in obese animal exosome treated myotubes (31% increase P= 0.059) compared to controls. The expression of ND4, a mitochondrially encoded subunit of complex I of the electron transport system, mRNA tended to decrease (11% decrease P=0.091) in the obese animal EV treated myotubes compared to controls.

Conclusions

These data indicate a potential role of obesity‐derived extracellular vesicles in altering metabolic processes of skeletal muscle. The dose of exosomes used in this work was relatively low, therefore more research is warranted to determine if additional effects would be seen at physiological exosome concentrations.
Diurnal Variation in Motivation, Cocaine Value, and In Vivo Dopamine Release
Authors: Iacino Melody C‐L , Stowe Taylor A. , Pitts Elizabeth G. , Ferris Mark J.
Published: May 2022

Circadian rhythms have been shown to influence a number of psychiatric disorders, like substance use disorder (SUD). For example, individuals with evening chronotypes (i.e. night owls) express higher vulnerability to drug‐craving and drug‐taking behaviors.

Similarly, the mesolimbic dopamine (DA) system, which is an essential circuit in these behaviors, exhibits diurnal rhythms in activity and function. Although diurnal rhythms have been documented in both drug‐taking and DA dynamics, it remains unclear whether these variations in DA confer with variations in‐drug taking. Here, we utilized in vivo fast scan cyclic voltammetry and a behavioral economic analysis of cocaine self‐administration to examine time‐of‐day variations in in vivo DA release, motivation, and cocaine value. We hypothesize that the degree to which the magnitude of mesolimbic DA release in the nucleus accumbens (NAc) varies across time will correspond diurnal variations found in motivation to take cocaine. We found that in vivo DA release was increased across the dark cycle in comparison to time‐points in the light cycle. We found no difference in cocaine intake in rats across time‐of‐day when cocaine “cost” was low and easy to obtain (Q0). However, we found that rats in the mid‐point of their dark cycles were more persistent in maintaining high cocaine intake as the “cost” or effort to maintain blood‐levels increased within their session in comparison to rats midway through their light cycle. These novel data suggest that specific time‐points across the dark cycle are associated with corresponding increases in DA and motivation to self‐administer cocaine under high, but no low, cost conditions. Understanding this dynamic modulation of diurnal variations may provide another therapeutic avenue for the circadian intervention of SUDs as well as target time‐points at which current pharmacotherapies might be most effective.
A Test of Altitude‐Related Variation in Aerobic Metabolism of Andean Birds
Authors: Gutierrez‐Pinto Natalia , Londoño Gustavo A. , Chappell Mark A. , Storz Jay F.
Published: May 2022

Endotherms at high altitude face the combined challenges of cold and hypoxia. Cold increases thermoregulatory costs, and hypoxia may limit both thermogenesis and aerobic exercise capacity. Consequently, in comparisons between closely related highland and lowland taxa, we might expect to observe consistent differences in basal metabolism (BMR), maximal metabolism (MMR), and aerobic scope. Previous broad‐scale comparative studies of birds have revealed no association between BMR and native elevation; however, altitude effects may be detectable in fine‐grained comparisons between pairs of ecologically similar, closely related species that are native to different elevations. Moreover, it remains unknown if exercise‐induced MMR and aerobic scope exhibit consistent patterns of altitude variation. To evaluate whether there is an association between altitude and metabolic rates, we tested for altitude‐related variation in aerobic metabolism in 10 Andean passerines representing five pairs of closely related species with contrasting elevational ranges. We used a paired‐lineage design such that the five pairwise comparisons were phylogenetically independent. In our small sample of Andean passerines, we found a significant effect of elevation on mass‐corrected BMR, MMR, and aerobic scope in most but not all cases. BMR and MMR were significantly higher for the high‐elevation species in three pairs, two of which also had a significantly higher aerobic scope at high‐elevation. One pair did not exhibit significant elevational differences in BMR, MMR, or aerobic scope. Overall, we could not document a uniformly consistent elevational trend in the species analyzed.
Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in the Absence and Presence of Chronic Kidney Disease
Authors: Heitman Kylie , Czaya Brian , Campos Isaac , Yanucil Christopher , Kentrup Dominik , Westbrook David , Faul Christian
Published: May 2022

Background

Chronic Kidney Disease (CKD) is a public health epidemic that is associated with elevated serum levels of phosphate (hyperphosphatemia) as well as skeletal muscle atrophy, but this interconnection is poorly understood. Elevated phosphate (Pi) has direct effects on smooth muscle cells and induces vascular calcification. Pi also induces the production of the hormone fibroblast growth factor (FGF) 23 in bone. We wanted to test if Pi induces atrophy and/or FGF23 expression in skeletal muscle cells. Furthermore, we analyzed skeletal muscle on a functional, histological and molecular level in three models of hyperphosphatemia – two CKD models, i.e. mice with global deletion of collagen 4a3 (Col4a3‐/‐) and wildtype mice receiving an adenine‐rich diet, as well as wildtype mice on a high Pi diet with normal kidney function. Finally, we determined the effect of a low Pi diet on skeletal muscle in Col4a3‐/‐ mice.

Methods

C2C12 myotubes were treated with 1‐5 mM Pi for 24 hours, followed by qPCR expression analysis of atrophy genes (atrogenes), including MT1, Trim63, and Fbox32, as well as FGF23. Furthermore, we studied Col4a3‐/‐ mice receiving normal chow or a 0.2% Pi diet at 10 weeks of age. We also analyzed C57Bl/6 mice receiving an adenine‐rich (0.2%) diet for 14 weeks or a 3% Pi diet for 3 months. We analyzed grip strength, hindlimb area by MRI, muscle wet weight, cross‐sectional area of individual muscle fibers immuno‐labeled with anti‐laminin by fluorescence microscopy, and expression levels of atrogenes by qPCR and of FGF23 by qPCR and ELISA.

Results

Pi treatments increased the expression levels of atrogenes as well as FGF23 in C2C12 myotubes. In the three mouse models, grip strength and cross‐sectional area of myofibers were significantly reduced, and the expression levels of atrogenes were significantly elevated when compared to respective controls. Furthermore, we detected elevations in the mRNA and protein levels of FGF23 in the hindlimb muscles of all models. Additionally, the two CKD models showed significant reductions in muscle weight and hindlimb area. Administration of a 0.2% Pi diet protected Col4a3‐/‐ mice from developing skeletal muscle atrophy.

Conclusion

Elevated Pi induces myotube atrophy and FGF23 expression in vitro. Mouse models with hyperphosphatemia not only develop skeletal muscle atrophy, but also produce FGF23 in skeletal muscle tissue in the presence and absence of CKD, and a low Pi diet protects the skeletal muscle in CKD mice. Pharmacological approaches targeting Pi uptake or excretion, or inhibition of Pi’s direct actions on tissues might alleviate various CKD‐associated pathologies. Future studies need to determine whether skeletal muscle‐derived FGF23 contributes to tissue injury or is protective against Pi‐induced damage.
Glucocorticoids differentially regulate the skeletal muscle transcriptome in response to acute aerobic exercise at distinct times of day
Authors: Dunlap Kirsten , Laskin Grant , Waddell David , Black Adam , Vied Cynthia , Steiner Jennifer , Gordon Bradley S.
Published: May 2022

Adaptations to the skeletal muscle following long term aerobic exercise training are mediated at least in part by transcriptional changes that occur in response to each bout of exercise. There is evidence that glucocorticoids induce transcriptional changes in the skeletal muscle in response to a bout of aerobic exercise, but the extent of these changes and whether time of day exercise affects the glucocorticoid transcriptional response is unknown. The purpose of this study was to define changes to the skeletal muscle glucocorticoid transcriptome in response to acute aerobic exercise at different times of day. A secondary purpose was to define the role of Regulated in Development and DNA Damage 1 (REDD1) in mediating these changes as REDD1 is not only a transcriptional target of the glucocorticoid receptor, but it can also modulate glucocorticoid receptor transcriptional activity. A list of glucocorticoid target genes whose expression is altered in the skeletal muscle at various times of day in response to acute exercise was generated by comparing published genomic data sets from rodents subjected to acute aerobic exercise in the light or dark cycles to published genomic data sets from C2C12 myotubes treated with Dexamethasone. The role of glucocorticoid receptor signaling to the genomic changes was assessed in exercised mice treated with RU‐486. The role of REDD1 in mediating the changes to the glucocorticoid transcriptome was assessed in mice lacking REDD1. Glucocorticoids modulated expression of numerous genes in the skeletal muscle in response to acute aerobic exercise in the light and dark cycles, including those that are a target of, or contribute to, ubiquitin conjugation. The extent of changes to the glucocorticoid transcriptome was greater when exercise was conducted in the dark cycle compared to the light cycle. Only some genes encoding proteins involved in ubiquitin conjugation were induced by exercise at both times of day. REDD1 is required for the induction of these genes, whereas those only induced in the light cycle did not require REDD1. The time of day at which aerobic exercise is conducted dictates the extent of changes to the glucocorticoid transcriptome in skeletal muscle. REDD1 is a transducer mediating the time‐of‐day changes to the glucocorticoid transcriptome in skeletal muscle.
Deletion Of G‐Protein‐Coupled Receptor 75 Prevents Hypoxia‐Induced Pulmonary Vasoconstriction and Hypertension
Authors: D'Addario Catherine , Kitagawa Atsushi , Matsumura Shun , Zhang Frank , Zeldin Darryl , Gruzdev Artiom , Schwartzman Michal , Gupte Sachin A.
Published: May 2022

Pulmonary Hypertension (PH) is a cardiopulmonary disease estimated to affect between 20 million to 70 million individuals globally, with poor prognosis and inadequate treatment. Recent studies showed the orphan G Protein Coupled Receptor 75 (GPR75) is upregulated in the pulmonary vessels of PH patients, especially in females. Therefore, we hypothesized that Gpr75knock out (Gpr75KO) mice will be protected from developing PH. To test our hypothesis, first we determined the vasoreactivity of isolated intra‐lobar pulmonary arteries from wild‐type (WT) and GPR75KO mice induced by KCl (WT=6; Gpr75KO=8), U46619 (WT=8; Gpr75KO=8), ZD7288 (WT=7; Gpr75KO=7), and Endothelin (WT=7; Gpr75KO=8) to test their vasoreactivity. The Gpr75KO as compared with wild‐type pulmonary arteries contracted less to all the contractile agents. Next, we determine if Gpr75KO mice were protected from developing hypoxia‐induced PH. We placed 3‐month‐old female Gpr75KO (n=8) and control wild‐type (C57BL/C) mice (n=18) in a hypoxic chamber with 10% Oxygen or at atmospheric oxygen for 5 weeks. At the end of 5 weeks, we performed right heart catheterizations. Gpr75KO mice developed less (P<0.05) right ventricle systolic pressure (RVSP: 24.3±1.2 mmHg) and right ventricle diastolic pressure (RVDP: 2.0±0.0 mmHg) than the wild‐type mice under hypoxia (RSVP and RDVP of 56.6±3.5 mmHg and 9.8±0.9 mmHg, respectively). Since GPR75 is a G‐protein coupled receptor to determine secondary messengers that potentially contribute to regulating contractile function of pulmonary arteries, we measured cAMP and IP3 in lungs of wild‐type and GPR75KO mice. While we found no difference in the IP3 levels between the WT and Gpr75KO groups, cAMP levels were significantly higher in Gpr75KO compared to WT mice. These results suggest GPR75 plays a major role in the development of PH by potentially attenuating cAMP‐dependent signaling and concomitantly augmenting pulmonary constriction in response to hypoxia.
SUMO Regulation of Bir1 in Mitotic Progression
Authors: Stabile Haley , Cepec Julie , Valachovic Tori , Thu Yee Mon
Published: May 2022

Organisms utilize many mechanisms in order to preserve their genomic integrity when faced with DNA damage or chromosomal instability. One such mechanism is protein sumoylation, in which E1, E2, and E3 ligases covalently link small ubiquitin‐like modifier (SUMO) peptides or chains to target proteins. The goal of this research is to examine the contribution of Bir1 sumoylation to genome stability in Saccharomyces cerevisiae. Bir1 is the target of one of the three S. cerevisiae E3 SUMO ligases, Mms21, and is a subunit of the chromosomal passenger complex (CPC). The CPC mediates the spindle assembly checkpoint (SAC) to establish chromosome bi‐orientation during mitosis. Therefore, it has previously been hypothesized that the sumoylation of Bir1 influences mitotic progression. In order to explore this hypothesis, BIR1 was genetically modified to mimic conditions in which Bir1 is constantly sumoylated (BIR1‐SuON) or cannot be sumoylated (BIR1‐SuOFF). The BIR1‐SuOFF mutants appeared to be more resistant to DNA damage caused by methyl methanesulfonate (MMS) and ultraviolet radiation than the BIR1‐SuON mutant, as well as the control strain (BIR1‐SuC). BIR1‐SuOff mutants phenocopied the temperature sensitive mutant of Ipl1, ipl1‐321, suggesting that mitotic checkpoint regulation may be affected in BIR1‐SuOff mutants. However, cell cycle analyses of BIR1‐SuOff mutants revealed that mitotic checkpoint activation was comparable to the control strain, at least in response to treatment with nocodazole, an agent that depolymerizes microtubules. We will further characterize the status of mitotic checkpoint activation in BIR1 mutants by examining Pds1 degradation and cell cycle analyses in the presence of other genotoxic agents.
Changes in the Non‐Coding Transcriptome of Short‐Term Glucose‐Challenged Human Glomerular Epithelial Cells May Give Insights into Early Molecular Events of Diabetic Kidney Disease
Authors: Tsotakos Nik , Castaneira Ryan , Rice Josie , Kreiser Savannah , Morris Daniel
Published: May 2022

Diabetic kidney disease is a complication of diabetes that takes years, sometimes decades, to develop. Our previous work on a podocyte‐like cell line showed that phenotypic changes following exposure to high glucose, such as downregulation of podocalyxin and nephrin, are gradual and reversible. We also identified changes in the expression of long non‐coding RNAs (lncRNA) MEG3, MEG8, and H19 in human glomerular epithelial cells (HGEC) chronically cultured in high glucose levels. In the present study, we aim to identify lncRNA that are dysregulated shortly after exposure of cells to high glucose and the cellular events that are associated with such dysregulation. Through a qPCR array, we identified approximately 20 upregulated and 20 downregulated lncRNA in HGEC that were exposed to high glucose for 2 weeks. These changes precede the terminal loss of podocyte markers and may provide insights to novel biomarkers for early‐stage diabetic nephropathy. Additionally, ectopic expression of MEG3, a lncRNA upregulated in HGEC chronically treated with high glucose, modulates autophagy by inducing changes in p62/SQSTM1 protein levels.
Functional Diversity and Structural Analysis of SAM‐dependent Aminobutanoyl Transferases
Authors: Shoemaker Madeline R. , Jim Trevor T. , McFarlane Jeffrey S.
Published: May 2022

Aminobutanoyl transferases (ABT) catalyze the transfer of 2‐aminobutanoate from S‐adenosyl‐L‐methionine (SAM) in the production of a variety of natural products. Nicotianamine (NA), a metal‐chelator found in plants, is a classic example and is biosynthesized by nicotianamine synthase through the condensation of three aminobutanoyl moieties from three successive SAM molecules with the first moiety formed into an azetidine ring. Although NA is common in plants, fungal, archaeal and bacterial species have also been shown to produce NA‐like molecules using ABTs. Examples have been characterized from the bacterium S. aureus and the archaeal species M. thermautotrophicus. Structural and functional data remains limited, but existing data suggests a diversity of products using one or more SAM moieties and/or additional amino acid substrates. We used Enzyme Function Initiative (EFI) tools to create sequence similarity and genome neighborhood networks to explore the diversity of ABT‐containing operons and to guide our predictions for the functional roles of distinct ABTs. We also used multi‐sequence alignments of our EFI dataset and structural alignments of the two existing ABT crystal structures to identify structural motifs that may be important to function. We have identified five functional ABT classes producing distinct product types. Example hypotheses from our analysis include the identification of consecutive asparagines deep in the active site pocket that appear to predict processivity and the presence of a serine/alanine substitution that correlates with stereoselectivity. To begin testing hypotheses developed from our bioinformatic analysis, we have selected four ABTs for heterologous expression and purification. We have successfully purified a carboxyazetidine‐forming ABT from P. aeruginosa and have begun crystallization trials. Purification of additional ABTs and the development of an HPLC‐based activity assay is ongoing. Through the characterization of ABTs selected from our bioinformatic analysis we expect to establish rules for predicting processivity, carboxyazetidine ring formation and the use of amino acid substrates by uncharacterized ABTs and to expand the limited available structural data using X‐ray crystallography.
The Effects of Tail Truncations of Pre‐Messenger RNA Splicing Protein Dib1
Authors: McGrath Virginia , Hernandez Jonathan , Turner Caitlyn , Lee Grace , Maeder Corina
Published: May 2022

The spliceosome is a multi‐megadalton ribonucleoprotein complex that catalyzes the removal of non‐coding introns and the ligation of coding exons from pre‐messenger RNA. This process is crucial in the transformation of pre‐messenger RNA into messenger RNA, a template that is readable by the ribosome and allows for the production of a functional protein. Significantly, the spliceosome is constructed anew on every pre‐mRNA. This construction entails over 140 protein factors that associate and dissociate throughout the splicing cycle, causing the conformational rearrangements and transesterification reactions necessary to yield the mature RNA template. Therefore, rather than a singular composition of proteins and RNAs being known as “the spliceosome,” this term actually refers to several different complexes which are distinct in their makeup and their role within the splicing cycle. Construction of a catalytic splicing complex occurs after a series of early assembly steps. Amongst the proteins involved in these early assembly steps is a 16.8 kDa protein, Dib1, that is present in the pre‐catalytic spliceosome but absent from the catalytically active conformation. The loss of Dib1 at this critical juncture hints at a potential role helping to facilitate the transition from pre‐catalytic to catalytic. Previous work on Dib1 has revealed that not only is it required for splicing, but the human ortholog, hDim1, has been shown to possess autocleavage activity in vitro where the last 14 amino acids are cleaved from its C‐terminal tail. Thus, we are interested in whether the C‐terminus impacts the function of the protein as it pertains to its role in splicing. In order to elucidate the importance of this region, we have constructed a series of C‐terminal truncations of varying lengths within Dib1 in order to assess any resulting splicing defects. These defects have been monitored through the use of growth assays and splicing assays to ascertain not only the overall importance of this region, but also the specific length required to ensure splicing abilities similar to wildtype Dib1 within the model organism S. cerevisiae. Overall, we observe that Dib1 can tolerate a large deletion before affecting cell growth and splicing.
Chronic High‐Rate Pacing Induces Heart Failure with Preserved Ejection Fraction‐Like Phenotype in Obese Ossabaw Swine
Authors: Tune Johnathan D. , Goodwill Adam G. , Baker Hana E. , Dick Gregory M. , Warne Cooper M. , Bailey Chastidy A. , Klasing Jessica A. , Russell Jacob J. , McCallinhart Patricia E. , Trask Aaron J. , Bender Shawn B.
Published: May 2022

The lack of pre‐clinical large animal models of heart failure with preserved ejection fraction (HFpEF) remains a growing, yet unmet obstacle to improving understanding of this complex condition. This study tested the hypothesis that chronic, high‐rate pacing induces a HFpEF‐like phenotype in obese Ossabaw swine. Swine were fed standard chow or an excess calorie, high‐fat, high‐fructose diet for ~18 weeks. Obese swine were implanted with a pacemaker to increase heart rate to 180 beats/min for 4 weeks (obese HF). Compared to lean swine, chronic pacing did not affect baseline (un‐paced) blood pressure or heart rate, but increased heart weight (P= 0.03), fibrosis (P< 0.001), and tended to reduce capillary density (P= 0.06). Cardiac output was increased in obese HF (P= 0.02) and associated with ~45% increase in ventricular stroke volume (P= 0.01), elevated end‐diastolic pressure (25 ± 2 mmHg; P< 0.001), and a normal ejection fraction of 56 ± 7% (P= 0.57). Baseline coronary blood flow was increased in obese HF (P= 0.03); however, MVO2 (P= 0.48) and coronary venous PO2 (P= 0.14) remained unchanged. Hemorrhage studies revealed impairment of the chronotropic response (59 ± 10 to 158 ± 19 bpm in lean control vs. 70 ± 5 vs. 84 ± 5 bpm in obese HF; P< 0.001) and augmented reductions in coronary blood flow (0.39 ± 0.03 to 0.19 ± 0.04 ml/min/g in lean control vs. 0.60 ± 0.05 to 0.21 ± 0.04 ml/min/g in obese HF) as blood pressure decreased from 109 ± 3 to 42 ± 1 mmHg in lean control vs. 102 ± 4 to 42 ± 1 mmHg in obese HF swine. These findings support that chronic high‐rate pacing of obese Ossabaw swine induces key phenotypic features of the human HFpEF condition and provides a distinct preclinical tool for future mechanistic and therapeutic study.
Loss of Estrogen‐Related Receptor Alpha (ESRRA) Impairs Neuronal Mitochondrial Function and Results in Disturbed Sleep and Neural Oscillatory Patterns
Authors: Dickey Jacob E. , Saito Kenji , Toth Brandon , Deng Guorui , Cui Huxing
Published: May 2022

ESRRA is an orphan nuclear receptor enriched in metabolically active tissues and has been known to affect the transcription of a set of genes that regulate mitochondrial bioenergetics in peripheral organs. Of note, ESRRA is also abundantly expressed in the brain, but the functional role of brain ESRRA remains poorly understood. To test the role of ESRRA on energy metabolism in neurons, we performed the Seahorse assay in mouse primary cortical neurons with or without ESRRA expression and found that neurons lacking ESRRA have significantly reduced cellular respiration compared to control neurons, indicating that loss of ESRRA decreases mitochondrial oxidative capacity and thus neuronal energy production. We have previously reported that global ESRRA null (KO) displayed markedly reduced locomotor activity (home cage vibration plate p<0.05, wheel running p<0.001), suggesting that these mice do display an energy deficit as a result of this manipulation. Based on the commonly appreciated role of sleep in the replenishment of energy stores for normal brain function, we hypothesized that reduced brain energy levels caused by genetic loss of ESRRA would lead to hypersomnia and diminished neural oscillations, which may underlie hypolocomotion seen in ESRRA KO mice. To test this hypothesis, wireless electroencephalography/electromyography (EEG/EMG) devices were implanted in a cohort of ESRRA KO and wildtype (WT) mice and 24hr sleep state was recorded and scored. Sleep architecture analysis from 24‐hour wireless EEG/EMG recording revealed that ESRRA KO mice exhibit significantly increased non‐rapid eye movement (NREM) (p<0.05) sleep as well as disrupted circadian pattern of rapid eye movement (REM) (p<0.01) sleep compared to WT littermates. Power spectral analysis further revealed that high range gamma (40‐100Hz) (p<0.001) and alpha (9‐13Hz) (p<0.05) oscillations are significantly decreased while low range gamma (25‐40Hz) (p<0.05) and beta (13‐25Hz) (p<0.05) oscillations are increased in ESRRA KO mice. Our findings not only reveal a previously unappreciated role of ESRRA in regulating neuronal mitochondrial function, sleep‐wake cycle, and neural oscillations, but also support a novel concept that a single molecular cause of diminished brain energy levels can prolong sleep duration and results in aberrant REM sleep pattern.
Vagus Nerve Sensory Neurons Respond Distinctly to Specific Inflammatory Mediators
Authors: Huerta Tomas S. , Haider Bilal , Adamovich‐Zeitlin Richard , Chavan Sangeeta S. , Tracey Kevin J. , Chang Eric H.
Published: May 2022

The peripheral nervous system communicates with the immune system in response to injury or infection. In the case of inflammation, sensory signals travel up to the brain via the vagus nerve, which is a major pathway for neuro‐immune communication. While previous work from our group showed that cytokines administered systemically were associated with specific patterns of vagus nerve activity, we did not know how individual vagal sensory neurons encoded this immune information.

Here, we use in vivo calcium imaging to monitor the neural activity of individual vagal sensory neurons in response to specific inflammatory mediators. Using mice that express the calcium sensor GCaMP6f in glutamatergic neurons, we imaged neurons of the nodose ganglion in situ using a 1‐photon miniature microscope (Miniscope). During imaging, the inflammatory mediators tumor necrosis factor (TNF) and interleukin 1β (IL‐1β) were applied directly to the vagus nerve. The raw fluorescence data was analyzed with a Python‐based calcium imaging analysis (CaImAn) software and a customized pipelines to output the neural activity (change in fluorescence: ∆F) of individually detected neurons. An average of 69±16.3 neurons were recorded per 1 hour imaging session.

Our results reveal that specific vagal sensory neurons respond to differentially to specific immune mediators. The average amplitude, integral, and delay of TNF‐responsive neurons was significantly higher than IL‐1β‐responsive cells (TNF vs IL‐1β, p < 0.01). This result suggests that TNF application triggers responses later and with higher firing rates in responsive neurons compared to IL‐1β. Neuronal responses to IL‐1β had a significantly higher number of peaks when compared to TNF responses (IL‐1β vs TNF, p < 0.05). When compared to responses for the TRPV1‐specific agonist capsaicin, the cytokine responses were found to have significantly lower number of peaks (capsaicin vs IL‐1β, p < 0.05), which suggests that immune mediators produce different neural activity than canonical activators. Taken together, our findings demonstrate that individual vagal sensory neurons encode information about distinct inflammatory stimuli and provide insight into the neural sensing of inflammation. Further investigation into this type of neuro‐immune signaling may identify novel neural targets for the treatment of inflammatory disorders.
Characterizing F.nucleatumcomplement resistance
Authors: Williams Kevin J. , Nguyen Tam , Slade Daniel
Published: May 2022

Fusobacterium nucleatum (Fn) is a Gram‐negative, anaerobic, oral bacterium that disseminates from the mouth through the bloodstream where it can cause tissue infections, preterm birth, and adverse outcomes in multiple cancers. However, how Fn survives the barrage of immune system attacks while in the bloodstream is wholly unknown. We hypothesized that Fn must be resistant to clearance by the human complement system; a complex network of serum proteins that act to label bacteria and viruses for clearance by immune cells, or directly kill Gram‐negative bacteria by depositing large protein complexes that result in membrane pores and cell death. To test this hypothesis, we incubated Fn with pooled human sera (PHS) with active complement components to determine resistance levels for multiple bacterial strains that are clinically relevant in disease. Here we present varying complement resistance levels in highly similar Fn strains and outline our prediction for the bacterial genes that are driving this immune resistance. These studies have the potential to uncover Fn outer membrane proteins that inactivate the complement system to facilitate their survival and passage throughout the human body. Our goal is to therapeutically target these Fn proteins by disrupting their immune interactions using host‐generated antibodies (vaccine), thereby allowing both the innate and adaptive immune branches to clear these bacteria before they can establish infection and disease.