bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Insulin Resistance in Male Hepatic Androgen Receptor Knockout Mice Fed High Fructose Diet
Authors: Carr Kiana R. , Wilson Andre , Levy Josephine , Falzarano Claire , Lofton Taylor , Andrisse Stanley
Published: May 2022

Introduction

Insulin resistance is a common hepatic dysfunction amongst individuals suffering from diabetes or obesity and affects 10‐25% of the general population1. The intracellular signaling pathway is reliant on every step being executed correctly. It is evident that excess dietary fats and sugars will result in obesity and likely diabetes. This cellular machinery can be altered when there is a build‐up of lipids which impact molecular insulin signaling. Our objective is to determine if chronic intake of fructose by male liver androgen receptor knockout (Liv‐ARKO) mice will show cascade alteration resulting in increased resistance to insulin when compared to male Liv‐ARKO mice on a control diet. We hypothesized that LivARKO mice on HFrD would show impaired insulin action when compared to control and chow diet mice.

Methods

We placed male Liv‐ARKO mice on high fructose (HFrD), Control (Research Diets Inc), and Chow diets. We performed insulin tolerance tests (ITT), glucose tolerance tests (GTT), and pyruvate tolerance tests (PTT) after 1‐2 months of being on diets. The mice were later sacrificed, and their tissues were extracted for further tests. To assess differences in insulin action that may arise from increased fructose some mice were given a dose of 0.5 U/kg insulin before being sacrificed to further investigate insulin signaling proteins. BCA protein assays were done to ensure that protein concentration in each sample was standardized. Western blots were performed using tissue from liver to measure molecular insulin action, via p‐AKT presence.

Results

Male mice on control diet for 2‐months showed improved glucose tolerance and enhanced pyruvate tolerance when compared to male mice on control diet for 1 month. Mice on 2‐month control diet showed impaired insulin sensitivity compared to mice on the same diet for 1 month.

Male LivARKO mice displayed no insulin stimulated p‐AKT when on a chow diet. Levels of p‐AKT were the same in basal and insulin male LivARKO mice fed a chow diet. Male LivARKO mice fed a control diet displayed a 4‐fold increase in levels of p‐AKT when stimulated by insulin compared to mice not given insulin (basal). Moreover, male LivARKO mice fed a HFrD displayed no insulin stimulated p‐AKT. Foxo1 levels were not altered by insulin stimulation in male LivARKO mice fed a chow, control, or HFrD. However, it should be noted that control and HFrD both increased Foxo1 levels compared to chow diet.

Conclusion

Our results suggest that hepatic insulin action or the ability for insulin to initiate glucose uptake in liver is decreased in male LivARKO mice on HFrD. Further investigation of Research Diets Inc. data is necessary to determine why the control diet and HFrD lead to the results discovered.
Sleep Apnea and Kidney Disease: The Role of Hypoxia and Lysyl Oxidase
Authors: Thomas Ann , Jakkidi Nishka R. , Li Shiyu , Cheng Kit‐Yan , Gumz Michelle , Canales Muna T. , Mohandas Rajesh
Published: May 2022

Study Objective/Hypothesis

Chronic kidney disease (CKD) or progressive loss of kidney function, is a serious health condition that affects 1 in 7 adults or an estimated 37 million Americans. Epidemiological studies have linked CKD with Sleep Apnea (SA), a breathing disorder characterized by erratic breathing, and episodes of hypoxia. However, the mechanisms that underlie the association between CKD and SA are not well understood. Hypoxia Inducible Factor‐1α (HIF‐1α) is a transcription factor that regulates the body’s response to hypoxia. Lysyl oxidase and lysyl oxidase likes (LOX and LOX‐Ls) are a group of copper enzymes that are hypoxia responsive and pro‐fibrotic. We hypothesized that hypoxia in SA upregulates HIF‐1α to drive expression of LOX or LOX‐L enzymes, fibrosis, and CKD.

Materials & Methods

Samples from the Sleep and Nephrology and Outcomes Research Study (SNORE), a prospective observational study of sleep disorders in veterans with CKD, were used for this study. The patients were divided into three main groups: CKD with no SA (n=14), CKD with severe SA (n=12) and healthy controls (n=13). RNA from peripheral blood mononuclear cells (PBMC) was isolated using PAXGene Blood RNA tubes (Qiagen). The RNA was then used as a template for reverse transcription, converting it into cDNA and fold changes in gene expression between groups were examined using real time PCR. Differences between groups were compared using nonparametric Mann Whitney U tests and a p‐value < 0.05 was considered statistically significant.

Results

LOX expression was lower in patients with CKD compared to healthy controls, regardless of SA (CKD with SA: 0.28 ± 0.06 (p=0.04) and CKD without SA: 0.33 ± 0.07 (p=0.09)). There were no significant differences seen in any of the LOX‐L expression (LOX‐L1 (p=0.84), LOX‐L2 (p=0.35), or LOX‐L3 (p=0.93)). Similarly, HIF‐1α was lower in patients with CKD (CKD with SA: 0.69 ± 1.0 (p=0.47) and CKD without SA: 0.46 ± 0.06 (p=0.01)). There was no statistically significant correlation between LOX and HIF‐1α (r=0.04, p=0.83).

Conclusion

Our results showed that HIF‐1α and LOX expression in PBMC was decreased in patients with CKD regardless of if they had SA or not. The mechanisms underlying these observations, the functional significance, and if these changes are representative of tissue LOX expression needs further studies.
Bioluminescence resonance energy transfer‐based detection of acetyl‐CoA
Authors: Lieberman Whitney , Jhulki Isita , Meier Jordan
Published: May 2022

CoA metabolites play a critical role in the epigenetic regulation of gene expression. Of particular significance is acetyl‐CoA, a primary metabolite used by histone acetyltransferases to modify histone tails and therefore control chromatin state. In cancer cells, the dysregulation of acetyl‐CoA can lead to the aberrant expression of genes controlling cell growth. One challenge in studying these acetyl‐CoA dependent functions is the lack of optical methods to detect CoA metabolites. Towards that goal, we report a bioluminescence energy transfer (BRET)‐based approach to detect acetyl‐CoA. Our strategy is inspired by recent biophysical and chemoproteomic analyses of human acetyltransferases, which revealed that members of the N‐terminal (NAA) acetyltransferase family preferentially bind acetyl‐CoA over CoA. To explore whether we could develop an acetyl‐CoA selective ligand‐displacement assay, we first recombinantly expressed and purified a NAA‐Nanoluc fusion protein. Next, we demonstrated its ability to generate a BRET signal when incubated with CoA‐fluorophore conjugates. Finally, we evaluated whether NAA‐Nanoluc BRET was altered by the addition of CoA metabolites, which revealed a direct relationship between the affinity of the CoA and optical signal. We report the first tunable indicator displacement assay for CoA metabolites and provide a new approach for studying the engagement of acetyltransferase enzymes by small molecule inhibitors.
Genetic deletion of endothelial Caveolin‐1 is protective against metabolic disease
Authors: Luse Melissa A. , Pavelic Caitlin , Tessema Rachael , Cochran Jesse , Minshall Richard D. , Leitinger Norbert , Isakson Brant E.
Published: May 2022

In metabolic disease, the endothelium is exposed to a substantial increase in fatty acid concentrations creating a pathological role for endothelial cells (ECs) as lipid metabolizing cells. One mechanism whereby lipids may enter ECs is by caveolin‐1 (Cav1)‐mediated endocytosis. Because both ECs and adipocytes both have abundant Cav1 and both can readily take up lipids, we wanted to differentiate the role of Cav1 between the two cell types. For these reasons, we used an inducible EC‐specific Cav1 knockout mouse (Cav1fl/fl/Cdh5‐CreERT2+). Using these mice, we induced Cav1 genetic deletion from endothelium at 6 weeks, and began a high fat diet (HFD; 60% fat) to induce metabolic disease at 8 weeks. Normal chow fed littermates were used as controls. All mice were examined at 20 weeks (i.e., 12 weeks of HFD). Thus, we had four cohorts: Cre+ HFD, Cre‐ HFD, Cre+ normal chow, and Cre‐ normal chow all injected with tamoxifen. All mice regardless of genotype had comparable weight gain within diet groups, and gonadal fat pad mass. Food consumption was similar between groups, and electron microscopy was used to confirm presence of caveola in adipocytes, but not endothelium. Initial experiments examining plasma lipids found HFD Cre+ mice had both increased serum cholesterol (175mg/dL) and LDL (75mg/dL) compared to HFD Cre‐ controls (cholesterol, 125mg/dL; LDL, 60mg/dL). When these mice were challenged with a bolus of insulin, the blood glucose of Cre+ HFD mice dropped significantly resembling an insulin tolerance curve from normal chow fed controls. Similarly, Cre+ HFD mice were protected from hyperglycemia after bolus glucose injections, resulting in a glucose tolerance phenotype similar to that of a normal chow fed mouse (HFD Cre+ AUC 3000 vs. HFD Cre‐ AUC 60000). There was no difference in Cre+ or Cre‐ mice on normal chow diets with serum lipids or after challenge with insulin or glucose. It isn’t clear why the insulin and glucose protection correspond with significantly increased lipids in the Cre+ HFD mice. For these reasons, we began investigating the possible mechanisms with primary cultures of Human Adipose Microvascular Endothelial Cells (HAMECs) and knocked down Cav1 with siRNA. In monolayers of HAMEC without any other cell type present, we did not observe altered lipid uptake after addition of 50µM of exogenous fatty acids with a 70% knockdown of Cav1 protein. Additionally, HAMECs seahorse assays were used to determine if there were changes in metabolic function when Cav1 was knocked down. This work provides initial evidence for the unique metabolic roles of endothelial Cav1 during periods of high lipid circulation.
SIKE’s Phosphorylation Sites Alter Quaternary Structure
Authors: Etiel Daniel , Pham Alyssa , Ryu Sally , Lyons Cade , Dzotsi Marissa , Patel Diya , Nguyen Thereasa , McCool Ryan S. , Bell Jessica K.
Published: May 2022

Protein‐protein interactions (PPIs) are a crucial part of intracellular communication and function. The “rules” for specificity and/or selection of a protein‐protein interaction are not completely understood, but protein modifications, such as phosphorylation, have been implicated in this process. Suppressor of IKKepsilon (SIKE) is associated with multiple, distinct proteins including TANK‐binding kinase 1 (TBK1), STRIPAK (striatin‐interacting phosphatase and kinase), and cytoskeletal proteins tubulin and actinin. Although SIKE’s function is not fully defined in these complexes, protein modification has been observed in SIKE (phosphorylated at six serine residues: 133, 185, 187, 188, 190, and 198) that may direct PPI formation: Co‐immunoprecipitation revealed that the tubulin interaction was enhanced with the phosphomimetic SIKE (S133/185/187/188/190/198E). We hypothesize that the quaternary state of SIKE, regulated by phosphorylation, dictates SIKE PPI formation. Using the SIKE phosphomimetic mutant, size exclusion chromatography and chemical crosslinking studies showed a monomeric species, whereas native SIKE separated as a dimer. These studies support SIKE undergoing a phosphorylation‐induced change in quaternary state. Prior computational assessment of dimer interface stability with single phosphomimetic substitutions identified S187E, S190E, and S198E as significantly different from WT on a per residue basis. Phosphomimetic mutants for these sites were created and sequence confirmed. In SEC, the phosphomimetic point mutations shift the elution pattern from primarily dimer to containing more monomeric species. To assess SIKE interactions with tubulin, SIKE constructs were labeled using a maleimide‐thiol conjugation to BODIPY TMR. BODIPY‐labeled SIKE, phosphomimetic point mutations, and S6E SIKE interactions with tubulin were monitored using fluorescence polarization. These PPI assays assess the effect of phosphorylation in the selection of SIKE interaction partners. Together, this work advances our understanding of the role that phosphorylation‐induced changes in quaternary state play in regulating protein interactions.
Agreement Between MRI, Ultrasound, and Histology in Detecting Size Changes of the Vastus Lateralis Following Resistance Training
Authors: Ruple Bradley A. , Smith Morgan A. , Osburn Shelby C. , Sexton Casey L. , Godwin Josh S. , Stock Matt S. , Poole Chris N. , Roberts Michael D. , Young Kaelin C.
Published: May 2022

Hypothesis

We hypothesize that MRI‐determined vastus lateralis (VL) cross‐sectional area (CSA) will increase following resistance training and that changes in VL size determined by ultrasonography and histology methods will agree with and/or be related to MRI‐determined changes in VL size.

Methods

Twelve healthy, untrained, college‐aged males (20±1 y; BMI: 26.9±5.4 kg/m2) had VL assessments via MRI, B‐mode ultrasound (thickness and cross‐sectional area) prior to (PRE) and after 10 weeks of resistance training (POST). Muscle biopsies were obtained at the site of imaging at PRE and POST for mean fiber cross‐sectional area (fCSA) determination. Agreement between MRI mCSA (mCSAMRI) and panoramic‐based ultrasound mCSA (mCSAUS) at PRE and POST as well as change scores in these variables were examined using Bland‐Altman plots for mean bias and 95% limits of agreement (LOA) as well as Lin’s concordance correlation coefficients (CCC). The relationship between fCSA with mCSAMRI, mCSAUS, and B‐mode ultrasound thickness (VLThick), as well as VLThick and mCSAMRI were tested with Pearson’s correlations.

Results

Both mCSAMRI and VLThick significantly increased following training (28.1±6.1 to 31.1±6.9cm2 (p=0.005) and 2.35±0.35 to 2.63±0.39cm (p=0.001), respectively). mCSAUS and fCSA did not significantly increase with training (26.3±6.4 to 28.3±7.5cm2 (p=0.064), and 4896±901.3 5443.2±1449.3µm2 (p=0.12), respectively). Bland‐Altman analysis revealed a small mean bias in mCSAUS relative to MRI (‐2.29 m2, 95% LOA: ‐8.74, 4.15 cm2). Concordance between mCSAUS and mCSAMRI taken at PRE and POST time points were excellent (CCC=0.83). Bland‐Altman analysis revealed good agreement between change scores in mCSAUS and mCSAMRI (‐1.08cm2, 95% LOA: ‐5.49, 3.33 cm2). There was a moderate concordance between mCSAUS and mCSAMRI change scores (CCC=0.70). A positive, moderate correlation was found between percent change in both VLThick and mCSAMRI as well as VLThick and mCSAUS (r=0.39, p=0.071 and r=0.52, p=0.080, respectively). A negative correlation was found between percent changes in fCSA and mCSAMRI (r= ‐0.569, p=0.054). Finally, correlations were not significant between percent changes in VLThick and fCSA (r=0.053, p=0.871) or fCSA and mCSAUS (r= ‐0.271, p=0.394).

Conclusion

These data suggest that, relative to MRI, panoramic ultrasound is a reliable method to track mCSA changes of the VL. However, fCSA changes do not associate with tissue level changes, and this discordance warrants further research.
Exposure to Eosinophilic Esophagitis Limits Esophageal Carcinogenesis in a Murine Model
Authors: Fuller Anne , Kabir Mohammad F. , Karami Adam , Klochkova Alena , Mu Anbin , Klein‐Szanto Andres , Hamilton Kathryn , Whelan Kelly
Published: May 2022

Introduction

Epidemiological data suggest that eosinophilic esophagitis (EoE) patients do not develop esophageal cancer. Here, we paired murine models of EoE and esophageal squamous cell carcinoma (ESCC) to perform the first functional interrogation of the relationship between EoE and esophageal cancer.

Methods

C57B6 mice were treated with MC903/Ovalbumin (OVA) to induce EoE and the esophageal carcinogen 4‐nitroquinoline 1‐oxide (4NQO) to induce ESCC according to the experimental scheme shown in Fig 1A. Esophagi of mice treated with vehicle controls (n=6), MC903/OVA alone (n=4), 4NQO alone (n=10), and MC903 in combination with 4NQO (n=5) were dissected. Tumor frequency was recorded and tumor load (tumor size x tumor number) was determined under a dissecting microscope. H&E stained tissues were evaluated for esophageal lesions. CD45‐positive immune cells were isolated from the spleens of mice with EoE or naïve controls then co‐cultured with the murine‐derived esophageal cell line AKR. After 24‐72 hours of co‐culture, live/dead dye was used to assess AKR viability.

Results

10/10 mice treated with 4NQO alone developed ESCC as expected in this robust tumor model. In mice co‐treated with MC903/OVA and 4NQO, ESCC was detected in 4/5 mice with a significant decrease in tumor load as compared to mice treated with 4NQO alone (Fig 1B, C). 6/6 mice treated with MC903/OVA alone and 4/4 mice treated with propylene glycol (vehicle for 4NQO) failed to display tumors (Fig 1C). We further evaluated esophageal epithelium of 4NQO‐treated animals to characterize distribution of ESCC lesions. While squamous dysplasia (pre‐malignant), early ESCC and late ESCC were present in all tumor‐bearing animals; there was a decrease in early ESCC and a lack of late ESCC lesions in 4NQO‐treated mice that were exposed to MC903/OVA‐induced EoE (Fig 1D). Moreover, the total percentage of esophageal epithelium occupied by neoplastic lesions was significantly diminished in mice co‐treated with 4NQO and MC903/OVA (Fig 1E). To investigate how EoE limits esophageal carcinogenesis, we exposed AKR esophageal tumor cells to CD45‐positive cells isolated from spleens of strain‐matched donor mice with or without EoE (Fig 2A). We found a robust induction of cell death in tumor cells exposed to immune cells from EoE donor mice compared to those exposed to immune cells from vehicle‐treated controls at 48 hours (n=3, p<0.05) (Fig 2B, C).

Conclusions

These data indicate that exposure to EoE activates an anti‐tumoral immune response that limits esophageal carcinogenesis. These studies have the potential for significant implications related to the clinical care of patients affected by EoE and ESCC.
Nicotine withdrawal after chronic exposure in adulthood blunts the ventilatory response to hypoxia in rats
Authors: Wollman Lila B. , Fregosi Ralph F.
Published: May 2022

Nicotine is one of the most addictive substances known, and it is estimated that 50.6 million US adults use nicotine in some form. It is well known that smoking, the most common form of nicotine exposure, alters lung structure and function. In contrast, the impact of chronic nicotine exposure during adulthood on neural structures involved in the control of breathing is largely unknown. In preliminary experiments in adult rats, we used plethysmography to study the effects of chronic nicotine exposure on the ventilatory response to hypoxia, which is a critical respiratory chemoreflex that enhances breathing and limits arterial O2 desaturation. Four weeks of chronic nicotine exposure through drinking water had no effect on the ventilatory response to a brief, 5‐ minute episode of hypoxia (10%) (Minute ventilation: One‐way ANOVA, Control vs Nicotine Exposure; P=0.6533). In contrast, nicotine exposed rats show a significantly blunted ventilatory response to hypoxia following 48 hours of nicotine withdrawal (Control vs Nicotine Withdrawal, P=0.0447). The reduced ventilatory response was due to a blunted increase in tidal volume (Amplitude: One‐way ANOVA, Control vs Nicotine Withdrawal; P=0.007), with a normal frequency response (Control vs Nicotine Withdrawal; P=0.2518. This has important clinical ramifications, as hypoxia is a commonly encountered medical complication, and withdrawal symptoms are often observed in hospitalized patients as it is common for them to abstain from smoking during periods of worsening health or in preparation for surgery.
Metabolomics can spot the difference: Dried Blood Spot (DBS) coming of age in a metabolomics era
Authors: Heyman Heino M. , McCulloch Scott D. , Karoly Edward D. , Mitchell Matthew W. , Goodman Kelli D. , Evans Anne M.
Published: May 2022

In the wake of the birth of personalized medical treatment, the need for validated in‐home bio‐specimen collection options from patients are required. New ways of collecting and storing bio‐specimens to retain important biological information are on the rise. For this reason, dried blood spots (DBSs) have gained increasing attention especially in the field of metabolomics. Metabolomics systematically captures the phenotype of disease, interventions, microbiome, etc. if the most relevant biological pathways are represented broadly, as has traditionally been demonstrated in clinical EDTA plasma. Here we demonstrated results from a study comparing DBS against the golden standard in blood collection, EDTA plasma. The study showed that even though DBS had a reduction of overall metabolites coverage compared to plasma (̴ 800 vs ̴1200 metabolites, respectively), all major pathways and more than 95% of the metabolic sub‐pathways that are routinely detected in plasma are conserved in DBS. Traditional biological signatures, including exercise biomarkers, dietary biomarkers, and even the cyclical trend of the female hormonal biomarkers, are equally well represented in DBS compared to plasma. This evidently showed that metabolic signatures of disease that have been well characterized in plasma are maintained in DBS. The analysis of DBS for metabolomics has come a long way and this study showed DBS to be a valuable alternative to EDTA plasma and ought be considered when plasma is impractical to collect and store i.e., at‐home collections, remote localities, resource limited settings, pediatrics, and IEM screening to name a few. DBS‐based metabolomics provides a powerful tool for personalized medicine and an effective way of bringing metabolomics into our homes.
The Impact of Long‐Duration Exposure to Microgravity and Galactic Cosmic Radiation on Corpus Cavernosum Function
Authors: R Andrade Manuella , Azeez Tooyib A. , Montgomery McLane M. , Caldwell Jacob T. , Park Hyerim , Narayanan S. Anand , Delp Michael D. , La Favor Justin D.
Published: May 2022

During spaceflight missions, astronauts are exposed to an extreme environment with high levels of radiation and microgravity, which negatively influence the cardiovascular system. With an increased development in space exploration and growing interest in manned missions to mars, it is of high importance to evaluate the health risks associated with long‐duration spaceflights. The increased risk of oxidative stress and inflammatory damage post‐spaceflight has been associated with cardiovascular dysfunction, but little is known about the influence of space exposure on erectile function, which is a critical component for quality of life in men. The purpose of this investigation was to determine the influence of long‐duration spaceflight on corpus cavernosum (CC) function. 86 adult male Sprague‐Dawley rats were randomized into 6 different groups with half of them enduring 4‐weeks of hindlimb unloading (HLU), and exposed to sham, 0.75Gy or 1.5Gy of simulated galactic cosmic radiation at the Ground‐based GCR Simulator at the NASA Space Radiation Laboratory (NSRL). Following a 6–9‐month recovery the rats were sacrificed, and CC tissue segments were harvested and mounted into a muscle strip myograph system for ex vivo functional assessment. CC reactivity to six 10‐s,30V‐electric field stimulations with progressive frequencies in the range of 1‐32 Hz targeting the non‐adrenergic non‐cholinergic (NANC) stimulation was assessed. The effects of HLU and radiation were determined by two‐way repeated measures ANOVA. High levels of radiation significantly decreased NANC‐mediated relaxation of the CC. Treatment with nitric oxide synthase (NOS) inhibitor L‐NAME completely inhibited tissue response to electric stimulation, showing that the relaxation was mainly driven by nitric oxide. Incubation of the CC tissue with the xanthine oxidase inhibitor allopurinol, the mitochondria‐targeted antioxidant mito‐TEMPO, and the superoxide dismutase (SOD) mimetic TEMPOL increased the relaxation response of the erectile tissue for the groups exposed to radiation, with little or opposite effect on the groups under no radiation exposure. Upon treatment with the Arginase inhibitor S‐(2‐boronoethyl)‐l‐cysteine (BEC), CC tissue for the groups under radiation exposure showed a significant improvement in relaxation under NANC electric field stimulation. Comparably, western blot analysis showed increased levels of oxidative stress and lipid peroxidation in the corpus cavernosum on the groups under radiation exposure by assessment of 4‐hydroxynonenal (HNE), as well as higher levels of arginase 1 and 2. This information suggests that increased oxidative stress from radiation exposure in long spaceflight trips impairs corpus cavernosum vasoreactivity and exposes new factors to consider with space exploration to ensure that astronauts can return to normal life after long trips in space.
Pulmonary Hypertension Induces Sex Differences in Control of Contracting Rat Skeletal Muscle Oxygen Pressures
Authors: Schulze Kiana M. , Weber Ramona E. , Horn Andrew G. , Colburn Trenton D. , Ade Carl J. , Poole David C. , Musch Timothy I.
Published: May 2022

Pulmonary hypertension (PH) is a disease characterized by pulmonary vascular remodeling, cardiac impairments, and skeletal muscle dysfunction. Males and females display different features of the disease, including a higher incidence in females, while males present with greater mortality. Exercise intolerance is a cardinal manifestation of PH, but any potential differences regarding skeletal muscle function during contractions between males and females have not been studied. This investigation tested the hypothesis that sexual dimorphism exists in the oxygen delivery (Q̇O2)‐to‐oxygen utilization (V̇O2) matching with PH as represented by oxygen partial pressures (PO2) at rest and following the onset of contractions. Considering that females display greater reliance upon nitric oxide (NO) for vascular function and PH decreases NO bioavailability, we hypothesized that PH would attenuate the skeletal muscle PO2 response to a greater extent in females than males.

Methods

Thirty‐three Sprague‐Dawley rats were randomly assigned to healthy (HC) or PH groups. PH rats (male: n=7; female: n=9) were administered monocrotaline (MCT; 50 mg/kg, i.p.) while HC rats (male: n=9; female: n=8) were given saline. Disease progression was monitored via echocardiography. Phosphorescence quenching determined the O2 partial pressure in the interstitial space (PO2is) in the spinotrapezius muscle at rest and during contractions.

Results

All PH rats displayed criteria for disease including right ventricular (RV) hypertrophy, elevated RV systolic pressure, and pulmonary congestion compared to HC. Indicators of PH severity did not differ between males and females (P>0.05). Females displayed a greater reduction in baseline PO2is in PH versus HC when compared to male counterparts (‐29 ± 3% vs. ‐18 ± 2%; P<0.05). No sex differences were present in PO2is kinetics (i.e., tau, amplitude) throughout contractions. However, females with PH reached a lower nadir in PO2is than PH males (4.2 ± 0.3 vs. 7.4 ± 0.4 mmHg; P<0.05).

Conclusions

These findings support that Q̇O2‐to‐V̇O2 matching is impaired in a sex specific manner in skeletal muscle. Individual contributions of O2 delivery and utilization, as well as biochemical analysis of NO in skeletal muscle will be invaluable to elucidating the mechanistic bases for the effects seen herein.
Extracellular Matrix Content and Remodeling Does Not Differ Between Higher‐Responders and Lower‐Responders to Resistance Training
Authors: Godwin Joshua S. , Ruple Bradley , Sexton Casey , Smith Morgan , Fruge Andrew , Young Kaelin , Mobley Christopher , Roberts Michael
Published: May 2022

Objective

To determine if markers of skeletal muscle extracellular matrix (ECM) content and ECM remodeling are indicators of hypertrophy in previously untrained collage aged males.

Hypotheses

Higher‐responders (HR) to resistance training would demonstrate more malleable changes in assayed ECM markers than lower‐responders (LR).

Methods

Untrained, college‐aged males (n=38, 21±3 years old) participated in 10 weeks of full‐body progressive resistance training (2x weekly) which included the leg press, bench press, leg extension, deadlift, and lat pulldown exercises. Participants completed a pre‐testing battery (DXA, ultrasound, pQCT and strength testing) and donated a vastus lateralis (VL) biopsy approximately one week prior to training. Participants completed post‐testing 72 hours following their last training bout, and this mimicked pre‐testing. Participants were then sorted into HR (n=10) and LR (n=10) based on changes in lean body mass, mid‐thigh cross‐sectional area (CSA), VL thickness, and deadlift strength changes. Muscle tissue from these 20 participants were processed for western blotting targeting MMP‐2, MMP‐9, MMP‐14, TIMP‐1, TIMP‐2, collagen‐1, and collagen‐4 protein levels. Global MMP activity was also performed on whole tissue lysates, and fascial thickness was measured from ultrasound images. All dependent variables were analyzed in SPSS (v22.0) using two‐way (group*time) repeated measures ANOVAs.

Results

No significant group*time interactions or main effects of time were detected for MMP‐2, MMP‐9, TIMP‐1, TIMP‐2, Collagen‐1, or Collagen‐4 protein levels (p > 0.05). MMP activity and VL fascial thickness also revealed no significant group*time interactions or main effects of time (p > 0.05). A significant main effect of time (p = 0.009) and group*time interaction (p = 0.007) was detected for MMP‐14. Further analysis revealed a significant difference between HR and LR prior to training (p = 0.026) along with a significant decrease from pre to post in HR (p = 0.002)

Conclusions

Our data suggest that skeletal muscle ECM markers did not differentiate higher‐responders versus lower‐responders to resistance training. However, MMP‐14 was responsive to resistance training and significantly different between response clusters prior to resistance training, and this warrants further investigation.
To ChIP, or to CUT, that is the question: Comparative Evaluation of NextGen Methodologies for Studying the genome‐wide distribution of Histone H3 Lysine 9 di‐methyl mark in pancreatic cells
Authors: Urrutia Guillermo , Abrudan Jenica L. , Du Meijun , de Assuncao Thiago Milech , Mathison Angela J. , Zimmermann Michael T. , Lomberk Gwen , Urrutia Raul
Published: May 2022

Our laboratories study the role of the G9a‐H3K9me2 pathway in PDAC initiation and development in response to oncogenic KRASG12D mutation. Understanding how the epigenomic landscape responds to this mutation remains fundamental for the identification of potential mechanisms and targets for pancreatic oncogenesis. Unfortunately, because of its low levels in human cells and its discrete “block‐like” genome‐wide deposition, the H3K9me2 mark is more difficult to study than more abundant, sharp peak‐forming marks (e.g. H3K27ac). The aim of this study was to perform a comparison among multiple Next Generation Sequencing (NGS) technologies to identify the methodology with the best technical performance for detection of H3K9me2 at a genome‐wide scale. To do so, we performed Chromatin Immunoprecipitation (ChIP), CUT&RUN and CUT&TAG assays for the Histone H3 Lysine 9 di‐methyl histone mark (H3K9me2) followed by NGS on two pancreatic ductal epithelial‐like cells: hTERT‐HPNE E6/E7 and the KRASG12D mutant‐expressing hTERT‐HPNE E6/E7 KRASG12D cells. As expected, ChIP‐Seq required the largest number of total reads for peak detection. Both ChIP‐Seq and CUT&TAG had the highest percentage of high‐quality reads (>75%), while CUT&RUN performed below the 50% mark. No significant differences in library size were observed among all three techniques. Overall, CUT&TAG allowed identification of the largest number of H3K9me2‐marked genes in parental HPNE and KRASG12D‐expressing HPNE cells. Spearman correlation values above 0.8 for inter‐sample comparison indicated a high degree of repeatability for CUT&TAG replicates. In addition, CUT&TAG showed the largest occupation of the H3K9me2 mark near the transcription start site (TSS) of identified genes. Therefore, we have compared three methodologies for the evaluation of H3K9me2 occupation at the genome‐wide scale and identified CUT&TAG as the method with the highest quality, lowest noise‐to‐signal ratio, and highest repeatability. In conclusion, CUT&TAG represents the best option for NGS of the H3K9me2 histone mark in pancreatic cells. Notably, because this methodology is the best among the three tested for miniaturization of epigenomic studies, this information bears relevance to translational research in human, where sample availability is limited.
KLF8 and OGT/O‐GlcNAcylation regulate breast cancer stem‐like cells
Authors: Le Minh Giang , Dhamelia Tejsi , Akella Neha , Reginato Mauricio
Published: May 2022

One of the main challenges in treating breast cancer is the intra‐tumor heterogeneity, which, partly, is maintained and promoted by a sub‐population of breast cancer cells called breast cancer stem‐like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self‐renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O‐GlcNAc transferase (OGT). OGT is responsible to adding O‐GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP‐GlcNAc – final product of the hexosamine biosynthetic pathway. This modification is termed O‐GlcNAcylation and both OGT and O‐GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O‐GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA‐seq analysis of BCSC‐enriched mammospheres with OGT overexpression revealed Kruppel‐like‐factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression and ALDH activity in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and ALDH+BCSC population. Knockdown of KLF8 also reduced breast tumor growth in vivo. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O‐GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O‐GlcNAcylation, suggesting a possible feed‐forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple‐negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem‐like cells population.
Resolving the Maturation of Elastomeric Protein Condensates
Authors: Vidal Ceballos Alfredo , Diaz A. Jairo A. , Preston Jonathan , Vairamon Christo , Shen Christopher , Koder Ronald , Elbaum‐Garfinkle Shana
Published: May 2022

Tropoelastin is a key protein in the formation of connective tissue such as lungs, arteries, and cartilage. The assembly and further cross‐linking process of tropoelastin culminates in the formation of elastin fibers, a resilient biomaterial capable of withstanding numerous cycles of stress and strain. Like other intrinsically disordered proteins, tropoelastin can undergo liquid‐liquid phase separation in vitro and in the extracellular space. This event is thought to aid in the self‐assembly and subsequent maturation of elastin fibers. Although the mechanical properties and morphology of mature elastin fibers have been extensively studied, the properties of elastin liquid droplets and their subsequent maturation into a solid remains poorly understood. Here, we use a model mini‐elastin polypeptide that mimics the domain architecture of naturally occurring tropoelastin to characterize this transition. We use fluorescence recovery after photobleaching (FRAP) and microrheology to capture the transition of elastin droplets from a liquid to a solid‐like state. We find that elastin droplets behave as viscous fluids at early incubation times, however, a rapid liquid‐to‐solid transition is observed in a timeframe of 80 minutes when held at constant temperature, even in the absence of cross‐linker. We further resolve the changes in dynamics, diffusion, and material properties of elastin condensates over the course of this transition. This work, which reveals the material transition from within elastin condensates, lends new insight into the early steps of the self‐assembly process of elastin while also contributing to the expanding repertoire of condensate maturation models in biological systems.
Skeletal Muscle LINE‐1 Gene Regulation and Inflammatory Response to Chronic, Voluntary Endurance Training in Rodents
Authors: Osburn Shelby , Mesquita Paulo , Neal Frances , Ruple Bradley , Holmes Matthew , Roberts Michael
Published: May 2022

Objective

We sought to determine the effects of chronic, voluntary wheel running on Long Interspersed Nuclear Element‐1 (LINE‐1) mRNA expression and promoter methylation, as well as markers of the cGAS‐STING inflammatory pathway, in skeletal muscle.

Hypotheses

LINE‐1 mRNA expression will increase with age, and exercise will mitigate this increase. This difference in expression will be driven through changes in promoter methylation. The cGAS‐STING inflammatory pathway will be upregulated with age and downregulated with rats that exercise‐trained.

Methods

Plantaris and soleus skeletal muscles were collected from female Lewis rats that belonged to one of three groups: a young control group (CTL; n=10; 6 mos old), an aged sedentary group (SED; n=12; 15 mos old), and an aged exercise group with access to a running wheel for 9 months (EX; n=12; 15 mos old). RNA, DNA, and protein were isolated from the tissues for analysis. Analysis included RT‐qPCR for LINE‐1 mRNA expression, methylated DNA Immunoprecipitation (MeDIP) to assess the methylation status of the LINE‐1 promoter via RT‐qPCR, and Western blotting for markers of the cGAS‐STING pathway and 4Hydroxynonenal (4HNE). Two primer sets were used for RT‐qPCR: L1‐3 probed for the most active form of LINE‐1 and L1‐Tot probed for all full‐length LINE‐1 elements.

Results

There was no significant difference in plantaris LINE‐1 mRNA expression or for L1‐3 or L1‐Tot. Plantaris LINE‐1 promoter methylation was undetectable in most samples, so the results are not presented herein. Soleus L1‐3 mRNA was significantly higher in EX compared to CTL (p=0.036) and there was a trend between SED and EX (p=0.053). L1‐Tot also was trending (p=0.059), where EX was higher than CTL (p=0.025). Methylation of the LINE‐1 promoter was significantly different for both L1‐3 and L1‐Tot (p=0.021 and p=0.028, respectively). In both instances, LINE‐1 DNA in SED and EX were significantly more methylated compared to CTL. There were no differences in cGAS protein expression, p‐/pan STING protein expression, or 4HNE expression.

Conclusions

Contrary to the original hypothesis, LINE‐1 mRNA expression and DNA methylation were higher in the exercise group. Additionally, chronic exercise did not affect the cGAS‐STING inflammatory pathway, highlighting this pathway may be more relevant in extreme age and disease states. Previous skeletal muscle research has focused on using mixed muscle types to look at LINE‐1 expression and regulation, so the differences seen between type I (soleus) and type II (plantaris) fibers is a novel finding that warrants further investigation to understand the underlying mechanisms.
Time course of blood level of alpha‐1 antitrypsin over 96 hours in smokers and hypertensive patients after acute myocardial infarction
Authors: Khatib Said Y. , AlJinabi Mukhallad M.
Published: May 2022

Introduction

Alpha ‐ 1 antitrypsin (A1AT), released from the liver, is known to protect lung tissue from damage. Some evidence suggests such protection is extended to myocardial tissues during myocardial ischemia/infarction.

Objective

The aim of the study was to determine the time course of A1AT levels in the plasma of myocardial infarction patients over 96 hours and compare it with healthy individuals.

Methods

Blood samples were collected from patients (n=40, age 58 + 11 yrs, males 29 and females 11), at 1, 4, 24, 48 and 96 hrs after confirmed diagnosis and admission to King Salman Cardiac Center in King Fahad Medical City, Riyadh, Saudi Arabia. IRB approval and consent was signed by all patients. The patients were divided into smokers (n=10) and non‐smokers (27), Hypertensive (23) and non‐hypertensive (14). Blood samples were collected in purple top tubes containing EDTA to prevent blood coagulation and immediately centrifuged at 6000 rpm at 4 C for 20 min. Plasma was decanted and stored at – 20 C until analyzed. AlAT was determined in plasma using Eliza kits (abcam, USA).

Results

A1AT level were significantly less in smokers after an MI (1158+177 ug/ml at 96 hrs, P<0.05, n=10) compared to (1547+128 ug/ml, n=27, mean +SEM) in non‐smokers. A1AT after an MI attack was comparable in hypertensive (1179+213 ug/ml) with that in non‐hypertensive patients(1122+190ug/ml).

Conclusion

The plasma levels of A1AT seem to be significantly less during an MI attack in smokers compared to non‐smoker but not in hypertensive patients where the level is compared to non‐hypertensive. From other data from this study in those patients in whom the level of A1AT dropped to more than 50 % of normal values (< 600 ug/ml) they passed away and did not survive the MI attack. It is therefore suggested that A1AT could have some protective effect on ischemic myocardium. More studies are needed to confirm this suggestion.
Using Synthetic Biology methods to construct a functional estrogen biosensor based on the dimerization‐dependent Red Fluorescent Protein
Authors: Case Emma , Desharnais Brianna , Kragh Caroline , Neka Ganad , Roecklein‐Canfield Jennifer
Published: May 2022

The presence of estrogenic compounds (endocrine‐disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time‐consuming techniques such as liquid chromatography‐mass spectrometry and high performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Synthetic biology allows for the construction of genetic devises composed of DNA sequences modified to be interchangeable and provide novel functions. New tools and devices are constantly needed to enhance the already extensive list of novel genetic parts. Our approach to the design of an estrogen responsive element uses rational engineering to express dimerization dependent Red Fluorescent Proteins (ddRFP) fused to the Estrogen Receptor alpha protein (ERalpha) in bacteria cells. These two monomeric weakly fluorescent RFP proteins derived from mutant dTomato (Campbell et al,2021) are strongly fluorescent upon dimerization. These RFP constructs are brought together in the cell when ERalpha dimerizes in the presence of estrogen. We present here the construction strategy and proof of concept data demonstrating the efficiency of this dual‐functional biosensor and it effectiveness for detection of estrogenic compounds in contaminated water.
Ovarian Hormone‐Dependent miR‐146b‐5p Regulation of Plasminogen Activator Inhibitor‐1 in Renal Epithelial Cells
Authors: Adam Ryan J. , Paterson Mark R. , Grzybowski Michael N. , Geurts Aron M. , Kriegel Alison J.
Published: May 2022

Chronic kidney disease (CKD) progression differs in males and females and the influence of gonadal hormones on pathology are incompletely understood. We utilized the 5/6 nephrectomy (5/6Nx) rat model, which recapitulates hallmarks of human CKD, to study differences in regulation of CKD pathology between sexes. We previously found that genomic deletion of microRNA 146b‐5p (miR‐146b) results in a sex‐dependent phenotype in 5/6Nx rats. Male miR‐146b‐/‐ rats develop renal pathology similar to their WT counterparts. However, female miR‐146b‐/‐ rats experience markedly exacerbated renal pathology relative to WT, 5/6Nx, females as well as WT and miR‐146b‐/‐, male, 5/6Nx rats. These sex differences are abolished by ovary removal, suggesting that renoprotective effects of female gonadal hormones are miR‐146b dependent. Our objective is to identify a molecular mechanism behind these effects. PAI‐1 has previously been shown to be regulated by miR‐146b‐5p isomiR, miR‐146a‐5p, which shares a common seed sequence. PAI‐1 expression is also known to be ovarian hormone dependent, its renal expression is low in health, but elevated in many disease settings, where it may contribute to remodeling. We hypothesized that PAI‐1 expression in rat proximal tubular epithelial cells (NRK‐52E) will be dependent upon (1) the presence of miR‐146b, (2) ovarian hormones, and (3) transforming growth factor beta (TGFβ), which is elevated in our 5/6Nx rats and known to contribute to renal pathology. To aid in testing our hypothesis we used CRISPR‐Cas9 technology to generate miR‐146b knockout rat proximal tubule epithelial (NRK‐52E) cells. Results. PAI‐1 mRNA was highly elevated in untreated miR‐146b KO rat NRK‐52E cells relative to WT NRK‐52E cell controls (11.25 ± 5.3 vs. 1.00 ± 0.68, P<0.05). Conversely, WT NRK‐52E cells treated with pre‐miR‐146b had significantly reduced PAI‐1 mRNA expression (0.137 ± 0.14 vs. 1.00 ± 0.45, P<0.05). Treatment of WT NRK‐52E cells with β‐estradiol did not significantly alter PAI‐1 mRNA expression relative to untreated controls, whereas treatment with progesterone (P4) significantly increased it. Treatment of WT NRK‐52E cells with TGFβ resulted in a significant increase of PAI‐1 mRNA expression. Lastly, treatment of WT NRK‐52E cells with P4 and TGFβ combined resulted in a significant increase in PAI‐1 mRNA expression, and this expression was significantly reduced by transfection with pre‐miR‐146b. Collectively, these observations demonstrate that miR‐146b, P4, and TGFβ regulate PAI‐1 expression in NRK‐52E cells and thereby suggest PAI‐1 as a mechanistic candidate behind the sex‐specific effects of miR‐146b in CKD.