bluuebunny/crossref_metadata_2025 huggingface dataset:
Myotube Morphology and Protein Metabolism are Negatively Regulated by Chemotherapy Drugs
Authors: Mora Stephen , Adegoke Olasunkanmi John
Published: June 2021

Cachexia, a condition prevalent in many cancer patients, is characterized by weight loss and fatigue resulting from decreases in muscle mass and function. Severity of cachexia negatively correlates with treatment outcomes, drug toxicity and survival. Tumor burden and cancer related‐malnutrition have been implicated in the development of the condition. Other studies have suggested a causative link between chemotherapy treatment and cachexia. To understand the mechanisms of effects of these drugs on cachexia, we investigated the effects of a common chemotherapy drug cocktail on myotube morphology, myofibrillar protein abundance, and mTORC1 signalling. On day 4 of differentiation, myotubes were treated with vehicle or a chemotherapy drug cocktail (a mixture of cisplatin (20 μg/mL), leucovorin (10 μg/mL), and 5 fluorouracil (50 μg/mL)). Compared to myotubes treated with vehicle, those treated with the drug cocktail showed dysmorphic shape and shrinkage. Drug treatment also induced a 50% decrease in the abundance of myosin heavy chain (n = 5 independent experiments, P = 0.0001), 80% reduction in troponin and tropomyosin (n=3–4, P < 0.0003) by day 6 of differentiation. To explore the reasons for the low abundance of these myofibrillar proteins, we examined treatment effects on mTORC1 (mammalian/mechanistic target of rapamycin complex 1), a signaling complex whose activity is vital for muscle anabolism. Myotubes treated with the drug cocktail showed 75% reduction in the phosphorylation of mTORC1 activator AKT (n = 3, P = 0.0061), and ≥70% reduction in phosphorylation status of mTORC1 substrates ribosomal protein S6 and its kinase, S6K1 (n = 3, P < 0.03). Drug treatment also led to 50–75% decreases in markers of mitochondria content cytochrome C oxidase (COX IV) and pyruvate dehydrogenase (P < 0.05). Because of the link between AKT activation and regulation of both protein synthesis and proteolysis, these data suggest that chemotherapy drugs decrease the abundance of myofibrillar proteins likely through the modulation of pathways that regulate protein synthesis and degradation, and mitochondrial content. To improve treatment outcomes and quality of life of patients, it is critical to identify interventions that can limit the negative effects of these drugs on muscle protein status and mitochondrial content.

Support or Funding Information

Natural Science and Engineering Research Council of Canada

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diaphragm Fiber Morphology of Ts65Dn Mice: A Model of Down Syndrome
Authors: Pascolla Jessica I. , Recceno Candace N. , Liang Chen , Greco Jacob , DeRuisseau Lara R. , DeRuisseau Keith C.
Published: June 2021

Down Syndrome (DS) is a developmental disability that results from triplication of chromosome 21. Roughly 40% of people with DS experience sleep apnea. Consistent with this finding, the Ts65Dn mouse model of DS was previously shown to display hypoxemia. Our interests center on the involvement of the nervous system and diaphragm in the etiology of hypoxemia in the Ts65Dn mouse. We hypothesized that Ts65Dn mice would exhibit a greater number of apneas and proportion of diaphragm fast twitch fibers, which may be associated with hypoxia in these mice. Male Ts65Dn mice and colony controls (WT) (n=8/group) were studied at 3 months of age using barometric plethysmography to determine the occurrence of apneas (mean±SD) over a thirty‐minute evaluation. At ten months of age the spinal cord and diaphragm tissues were harvested from mice under isoflurane anesthesia. Ts65Dn mice displayed more apneas (36.3±28.0) than WT (7.1±9.1) mice at 3 months of age (p<0.05). Diaphragm fiber type distributions and areas were determined from a subset of mice (n=2/group). Compared to WT controls, diaphragm muscles from Ts65Dn mice displayed smaller Type I/IIa and Type IIx fibers (18.8% and 5.6% smaller, respectively) and larger Type IIb fibers (17.7% larger). The distribution of Type I/IIa fibers was similar in Ts65Dn mice compared to WT (64.1% vs. 58.8%) and distribution of Type IIb fibers was less than WT (4.7% vs. 8.2%). The distribution of Type IIx fibers was similar between the groups (31.1% DS; 33.0% WT). Statistical analyses were not performed on diaphragm morphological comparisons due to the small sample size. Our preliminary analyses indicate the Type IIb fibers, in particular, of Ts65Dn diaphragms mice may be larger and less prevalent compared with control diaphragms. Our work continues to expand upon these findings by including a larger sample size to achieve appropriate statistical power and incorporating phrenic motoneuron counts in spinal cord sections.

Support or Funding Information

Supported by NIH 1R15HD076379‐01A1, 1R15HD076379‐01A1S1, and 1R15HD076379‐01A1S2

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Limited Fatty Acid Oxidation (FAO) in CPT2 Knockout Myocytes Associates with Insulin Resistance and Cell Stress: possible role of acylcarnitine lipotoxicity
Authors: Blackburn Michael L. , Ono‐Moore Kikumi D. , Hoppel Charles L. , Ellis Jessica M. , Adams Sean H.
Published: June 2021

Lipotoxicity (the accumulation of fat‐derived metabolites that activate cell stress pathways) has been implicated in muscle insulin resistance. We have proposed that lipotoxicity, and in particular accumulation of long‐chain acylcarnitines (LCACs), also contributes to the episodic myopathy and rhabdomyolysis that is evident in persons with inherited disorders of long‐chain FAO. Many studies in the arena of LCAC bioactivity have employed exogenous application of LCACs to cells; few studies have modified endogenously‐produced LCACs to determine myocyte outcomes. Thus, conclusions about the (patho)physiological relevance of LCAC‐associated lipotoxicity have been difficult to determine. Herein, we used CRISPR‐Cas9 technology to knock out carnitine palmitoyltransferase‐2 (CPT2) in murine C2C12 myotubes: as expected, this maneuver led to almost complete inhibition of FAO, plus significant cellular and media accumulation of palmitoylcarnitine in palmitate‐treated cells. In CPT2 KO myotubes, treatment with 500 μM palmitate led to ~2‐fold higher production of IL‐6 (a myocyte cell stress marker) and a ~1.5 to 2‐fold increased release of adenylate kinase (cell death and permeability marker) at 3 and 6 hr post‐treatment (when compared to palmitate‐treated wildtype control cells; n=4/treatment). Furthermore, CPT2 KO myotubes displayed higher sensitivity to palmitate‐induced insulin resistance: the % inhibition in terms of insulin‐induced p‐Akt abundance was 60%, 80%, and 95% in CPT2 KO cells (at 200, 500, and 750 μM palmitate, respectively), vs. 38%, 53%, and 67% in controls (p<0.01, n=8/treatment). The results indicate that limitation of long‐chain FAO by loss of CPT2 in muscle cells elicits cell stress and insulin resistance, consistent with a working model in which lipotoxicity and accumulation of LCAC trigger these outcomes. Further studies are needed to determine if endogenously‐produced LCACs contribute to the phenotypes observed in CPT2 KO myotubes, or if LCAC‐independent mechanisms are involved. It remains to be established whether these phenomena underlie the cardiac and skeletal muscle myopathies typical of inherited disorders of FAO, or contribute to the insulin resistance accompanying type 2 diabetes.

Support or Funding Information

This work was supported by USDA‐ARS Project 6026‐51000‐010‐05S.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased Fiber Excitability Does Not Contribute to Post‐Tetanic Potentiation in Mice
Authors: LE GENGYUN , Baumann Cory W , Warren Gordon L , Lowe Dawn A
Published: June 2021

Purpose

Post‐tetanic potentiation (PTP) is the enhancement of twitch force following tetanic stimulation and is an important component when studying skeletal muscle physiology and plasticity. Mechanistically, it is plausible that PTP could be associated with an increase in fiber excitability and/or any event following sarcoplasmic reticulum (SR) calcium release (i.e., at the contractile apparatus). Therefore, the goal of this study was to determine if increased fiber excitability contributes to PTP.

Methods

Adult female C57BL/6J mice were chronically implanted with stimulating electrodes on the left common peroneal nerve and electromyographic (EMG) electrodes on the left tibialis anterior (TA) muscle. Following surgical recovery (at least 2 weeks after EMG electrode implantation), in vivo PTP of the anterior crural muscles was performed with simultaneous M‐wave recording from the TA muscle. To measure PTP, baseline twitch torque production was measured using a 0.1‐ms electrical stimulus pulse. Ten seconds later, muscles were stimulated using 0.1‐ms pulses at 100 Hz for 1 s to produce a prolonged, isometric tetanic contraction. Starting 2 s later, two post‐tetanic twitches were elicited with 2 s between the two. Changes in twitch torque (i.e., PTP) and M‐wave amplitude were calculated as percent increases in twitch torque and the corresponding M‐wave from baseline to the highest values following tetanic stimulation. All data were analyzed using paired t‐tests and are reported as means ± SE.

Results

Twitch torque at baseline was 0.45 ± 0.07 mN·m and represented the unpotentiated twitch. Following the tetanic stimulation, twitch torque increased by 34.0 ± 7.3% to 0.60 ± 0.07 mN·m (p=0.01). However, no change was detected in twitch M‐wave amplitude from before to after the tetanic stimulation (0.37 ± 0.03 to 0.39 ± 0.04 mV, p=0.48).

Conclusion

On the basis of the disproportionate increase in twitch torque (34%) compared to the corresponding M‐wave amplitude increase (5%) after tetanic stimulation (p=0.02), we suggest that the PTP was not due to an increase in fiber excitability. Instead, it is more likely that PTP was attributable to an enhancement at or distal to SR calcium release such as phosphorylation of myosin regulatory light chain, which is consistent with previously reported mechanisms underlying PTP.

Support or Funding Information

Supported by NIH grants R01‐AG031743 and T32‐AR007612, and Metropolitan State University Professional Development Grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CRISPR/Cas9‐mediated Genome Editing in Patient‐Derived iPSC‐Cardiomyocytes Recapitulates an MYH6‐R443P Phenotype in a HLHS Family
Authors: Kim Min‐Su , Lough John , Geurts Aron , Mitchell Michael , Tomita‐Mitchell Aoy
Published: June 2021

Hypoplastic Left Heart Syndrome (HLHS) is a clinically and anatomically severe form of Congenital Heart Disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. We previously demonstrated that rare variants in the α‐myosin heavy chain (MYH6) gene are significantly enriched in HLHS (p<1×10−5, observed in 10.5% or 20/190 HLHS subjects). Analysis of clinical outcomes showed that survival without cardiac transplant was reduced in HLHS subjects with MYH6 variants (p=6×10−3) as compared to those without MYH6 variants. Transcriptome and protein expression analyses from cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β‐myosin heavy chain (MYH7) gene in subjects with MYH6 variants. Visualizing α actinin in atria showed intrinsic disrupted sarcomere structure in HLHS patients with MYH6 variants.

We observed that patient‐specific induced pluripotent stem cells (iPSCs) were defective in cardiomyogenic differentiation in vitro. Additionally, sarcomere structure in cardiomyocytes derived from iPSCs produced from a family trio (unaffected mother‐affected father‐patient) carrying a variant in MYH6 (MYH6‐R443P) was dysmorphic.

We introduced the MYH6‐R443P variant into iPSCs derived from the unaffected parent using CRISPR/cas9 technology and observed that the MYH6‐R443P phenotype in HLHS‐iPSCs was recapitulated; specifically, gene‐edited MYH6‐R443P+/− and MYH6‐R443P−/− iPSCs displayed defective cardiomyogenic differentiation, and sarcomere structure in these cardiomyocytes was dysmorphic. We conclude that the etiology of MYH6‐associated HLHS may be informed utilizing iPSCs.

Support or Funding Information

The Advancing a Healthier Wisconsin Endowment (AHW)

Wolfe Family Foundation

Great Milwaukee Foundation

Children's Research Institute

Little Heart for Life

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Auto‐antibodies Targeting Components of Sarcolemma Repair Represent a Pathogenic Mechanism in Idiopathic Immune Myopathies
Authors: McElhanon Kevin E. , Capati Ana , Young Nicholas , Hampton Jeffrey , Paleo Brian J. , Beck Eric X , Sahenk Zarife , Aggarwal Rohit , Oddis Chester V. , Jarjour Wael N. , Weisleder Noah
Published: June 2021

Idiopathic immune myopathies (IIM) represent a group of disorders causing chronic inflammation and significant damage to skeletal muscle due to an unchecked autoimmune response. The main challenge slowing study of IIM is an understanding of the pathogenic mechanism responsible for initiation and progression of skeletal muscle inflammation. Two mouse models of IIM have recently provided new insights into the pathogenesis of the disease. The synaptotagmin VII null (Syt VII−/−) mouse is characterized by defects in membrane resealing and presents with a mild form myositis at 2 months of age. A more robust model of IIM combines knock‐out of Syt VII with a FoxP3 mutation resulting in a mouse with impaired membrane resealing and regulatory T‐cell deficiency. Adoptive transfer of lymphocyte preparations isolated from this double mutant mouse model to recombination‐activating gene 1 (RAG‐1) null mice results in severe skeletal muscle inflammation. Plasma membrane resealing is a highly conserved mechanism allowing an injured cell to survive following a disruption to the lipid bilayer that compromises barrier function. Given the importance of plasma membrane barrier function in preventing exposure of intracellular antigens to the immune system, specifically in an immune‐compromised environment, it is possible that compromised sarcolemma resealing could drive pathogenesis of IIM. We show, for the first time, that a deficiency in T‐regulatory cells is not sufficient to induce sarcolemma fragility, however, purified antibodies against critical proteins facilitating the sarcolemma repair process are sufficient to reduce membrane integrity. We also demonstrate that sarcolemma integrity is reduced in distal skeletal muscle in the absence of inflammation in our novel murine model of IIM. We have established by direct ELISA that auto‐antibodies against multiple proteins involved in sarcolemma repair are elevated in IIM patient sera and find that exogenous delivery of IIM positive patient serum can compromise sarcolemma resealing in healthy skeletal muscle. These findings represent a novel mechanism that drives the progression of IIM when decreased sarcolemma integrity induces a vicious cycle of antigen presentation that directly contributes to the pathophysiology of idiopathic immune myopathies.

Support or Funding Information

NIH, National Institute of Arthritis and Musculoskeletal and Skin Diseases Award Number F31AR071745

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adaptations of Bone and Bone Vasculature to Muscular Stretch Training
Authors: Barsoum Mina‐Michael , Hotta Kazuki , Delp Michael , Ghosh Payal , Behnke Bradley , Churchill Alexandra , Boyd Brandon , Muller‐Delp Judy Marie
Published: June 2021

The magnitude of bone formation and remodeling is linked to both the magnitude of strain placed on the bone and the perfusion of bone. We have previously reported an increase in bone perfusion and bone density that occurs in the femur of old rats with moderate aerobic exercise training. The purpose of this study was to determine the acute and chronic effects of static muscle stretching on bone blood flow and to determine whether chronic stretching affects bone remodeling. Old male Fischer 344 rats were randomized to either a naïve or stretch‐trained group. Static stretching of ankle flexor muscles was achieved by placement of a dorsflexion splint on the left ankle for 30 min/day, 5d/wk for 4wks. The right hindlimb served as a contralateral control (unstretched) limb. Bone blood flow was assessed during and after an acute bout of stretching in naïve rats, and at rest and during exercise in stretch‐trained rats. Following 4 weeks of daily muscle stretching, micro‐CT analysis was used to determine the volume and density of bone and the bone vasculature in the tibia. In naïve rats, placement of the splint increased blood flow to the proximal tibial metaphasis. After 4 weeks of daily stretching, blood flow to the proximal tibial metaphysis was higher in the chronically stretched limb during exercise. Chronic stretching also increased tibial bone weight, and increased total bone volume in both the proximal and distal tibial metaphyses. In trabecular bone immediately below the proximal tibial growth plate, the total volume of bone was increased, but the bone density was not changed. Total intravascular volume also increased in the region of the bone below the growth plate. These data suggest that static muscle stretching increases loading of bone and bone blood flow, leading to bone remodeling and growth which is matched by an increase in bone intravascular volume.

Support or Funding Information

NIH R15AG055029

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The FOXM1 Inhibitor RCM‐1 Decreases Carcinogenesis and Nuclear β‐catenin
Authors: Rama Nihar , Shukla Samriddhi , Milewski David , Kalin Tanya
Published: June 2021

FOXM1, a member of the Forkhead box (FOX) family of transcription factors, has previously been shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple types of cancer when abnormally upregulated. Our lab previously identified a small molecule compound, Robert Costa Memorial drug‐1 (RCM‐1), through high‐throughput screening as a FOXM1 antagonist in vitro and in an in vivo mouse model of allergen‐mediated lung inflammation. Here, we examined antitumor activities of RCM‐1 using tumor models. Confocal imaging of RCM‐1‐treated tumor cells indicated that reduced cellular proliferation was concordant with inhibition of FOXM1 nuclear localization in these cells. RCM‐1 reduced the incidence and growth of tumor cell colonies in the colony formation assay. Migration assays in vitro demonstrated the ability of RCM‐1 to inhibit migration of tumor cells. In animal models, RCM‐1 treatment inhibited growth of mouse rhabdomyosarcoma Rd76‐9 and human lung adenocarcinoma H2122. RCM‐1 decreased FOXM1 protein levels in treated tumors, reduced tumor cell proliferation and increased tumor cell apoptosis in vivo as detected by immunohistochemical staining. Western blot analysis of cultured tumor cells showed that RCM‐1 decreased protein levels and nuclear localization of β‐catenin, an important constituent of the canonical Wnt signaling pathway, and inhibited protein‐protein interaction between β‐catenin and FOXM1. Altogether, our study provides important evidence of antitumor activity of the small molecule compound RCM‐1, suggesting that RCM‐1 has promising potential as a cancer therapy in the future.

Support or Funding Information

Peter K. Lauf Award, Ohio Physiological Society; NIH funding awarded to TK.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histological Analysis of Lysophosphatidic Acid Receptor 3 Deficient Zebrafish
Authors: Chen Wei‐Min , Chang Ya‐Chi , Lin Yu‐Nung , Chiang Jui‐Chung , Lee Hsinyu
Published: June 2021

Lysophosphatidic acid (LPA) is a small bioactive phospholipid enriched in serum and exerts diverse cellular functions through activation of multiple G Protein‐coupled receptors, LPA1‐LPA6. Our previous studies demonstrated that lysophosphatidic acid receptor 3 (LPA3) is critical for megakaryopoiesis and erythropoiesis in zebrafish. However, the roles of LPA3 during the development of other organs of zebrafish remain unclear. Here, we established a zebrafish strain with targeted disruption of lysophosphatidic acid receptor 3 (Lpar3) gene by Transcription Activator‐like Effector Nuclease (TALEN). Paraffin‐section and hematoxylin‐eosin staining were performed for histological analysis to clarify the effects of LPA3 in different organs of adult zebrafish. Histological analysis results showed a shrinking of hematopoietic tissue in kidney marrow of LPA3 deficient fish, implies essential roles of LPA3 in hematopoiesis. In addition, bile duct hyperplasia and mature spermatids loss were also observed in LPA3 deficient fish. Nevertheless, no obvious morphological differences in eyes, thymus, heart, and spleen were observed. Our observation provides tissues profiles of LPA3−/− animal and possible application of LPA3 deficient zebrafish as a disease model in the future.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Function in Dopamine Receptor‐3 (D3R) Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation In Vitro
Authors: Kisling Andrew , Zakari Madaniah O , Alsahly Musaad B , Melit‐Thomas Deepthy C , Clemens Stefan , Lust Robert M , Katwa Laxmansa C
Published: June 2021

Dopamine is a neurotransmitter heavily involved in neural pathways regulating the reward response, body movement, mood, and cognitive function. As such, much of the research on dopamine and its five receptor subtypes has been conducted to observe the in vivo function of each receptor subtype in neurological disorders. However, recent studies by colleagues from our institution have suggested that D3Rs may play a role in cardiac‐related aging, as 2‐month old D3 receptor knock‐out (D3KO) mice show age‐related changes in cardiac function similar to 2‐year old wild‐type (WT) mice. Thus, it would be interesting to understand the role of D3R expression in LV cardiac fibroblast and its function. We isolated, cultured and characterized cardiac fibroblasts from the left ventricles (LV) of WT (23‐weeks old) and D3KO (5 and 31‐week‐old) mice. Immunohistochemistry was performed to determine the expression of D3R in LV cardiac fibroblasts, and cell culture experiments were performed to examine possible changes in WT and D3KO cell migration and proliferation. Proliferation was measured via total cell count over time points of 6, 12, 24, and 36 hours, while migration in response to scratch wound was examined as percent change in area at 3, 6, 12, 24 hours, and every 12 hours thereafter up to 86 hours. Staining with antibodies for D3R successfully showed expression of this receptor in WT fibroblasts, while cell culture experiments showed reduced proliferation and migration of D3KO fibroblasts compared to WT. Migration of D3KO fibroblasts from 5‐week old mice were significantly different (*p<0.05) in response to an artificial wound when compared to 23‐week‐old mice. Additionally, upon isolation of LV cardiac fibroblasts from 5‐week‐old and 31‐week‐old D3KO and 23‐week‐old WT mice, cell volume from D3KO fibroblast isolation was markedly reduced compared to WT isolation under identical conditions (5‐fold and 10‐fold decrease in 5‐week and 31‐week D3KO, respectively). This data suggests that D3Rs play a role in LV cardiac fibroblast migration and proliferation and support previous observations of age‐related changes in cardiac function of D3KO mice.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circulating muscle‐enriched microRNA expression correlates with myogenic gene expression in chronic alcohol administered SIV‐infected macaques
Authors: Simon Liz , Molina Patricia
Published: June 2021

Background

Skeletal muscle enriched microRNAs, myomiRs, regulate gene expression critical for myogenesis, metabolism and functional muscle mass. Previously we demonstrated that chronic alcohol and simian immunodeficiency virus infection decreased myogenic differentiation capacity of myoblasts, and this was associated with a decrease in myomiR expression. Evidence suggest that exosomes carrying myomiRs target myocytes and modulate their physiology and function. The aim of this study was to determine whether total and exosomal circulating‐myomiR expression correlates to myogenic gene expression and myoblast differentiation potential.

Methods

Gene and microRNA expression was determined in skeletal muscle tissue from simian immunodeficiency virus (SIV) infected and chronic binge alcohol (CBA) administered rhesus macaques with and without antiretroviral therapy (ART) (VEH/SIV/ART−, VEH/SIV/ART+, CBA/SIV/ART, CBA/SIV/ART+). Plasma exosomes were isolated using the total exosome isolation kit based on the principle that by sequestering water molecules, less‐soluble vesicles are forced out and is isolated by low‐speed centrifugation. Total circulating myomiR expression was determined in plasma using taqman miRNA primers and cel‐238 used as the spike‐in control.

Results

CBA decreased circulating total and exosomal miR‐206 expression. Expression of exosomal miR‐206 was positively correlated with miR‐206 expression in the muscle. Circulating and total exosomal miR‐206 also correlated with the myotube density of myoblasts and was negatively correlated with histone deacetylase 4 expression in the different treatment groups.

Conclusions

Chronic alcohol decreases circulating myomiR expression in myoblasts isolated from SIV‐infected animals. Whether circulating myomiR expression can be used as a noninvasive biomarker to predict dysfunctional muscle mass will be determined in a cohort of people living with HIV with alcohol use disorder has to be determined.

Support or Funding Information

K01AA024494 and AA09803 NIH/NIAAA

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SOX18 Variant E137K Reduces Transcriptional Activity of SOX Responsive Promoters in Cotransfected Epithelia
Authors: Sirpilla Olivia , Prokop Jeremy W. , Qutob Dinah , Freeland Thomas , Underwood Adam C.
Published: June 2021

SOX (SRY‐related box) proteins are important regulators of cellular differentiation during both embryonic development and throughout life affecting overall systemic health. Mutations within SOX genes have been found to cause many forms of cancer, cardiovascular disease, neurological abnormalities and sex reversal. Each SOX gene contains a highly conserved High‐Mobility Group‐box (HMG box) binding domain that changes transcriptional activity of target promoters by bringing distant regulatory components closer to the initiation complex. After deep sequence analysis of all 20 SOX genes, we identified a novel mutation within the SOX18 HMG‐box that exchanges glutamic acid (E) for lysine (K) at amino acid 137 (rs201931544). This mutation is present in the genome of ~0.82% of the Latino population, yet the biological implications of the E137K variant are not known. To address this, we used a luciferase‐based reporter system to assess the ability of the wildtype verses mutant form of SOX18 to activate gene transcription. First, primers were designed to amplify and clone SOX18 into the pHTCHaloTag® CMV‐neo vector (Promega). Following sequence validation, PCR was used to introduce the mutation followed by cloning of the pHTCHalo/SOX18‐E137K effector construct. Constructs were transfected into two epithelial cell lines (HeLa and A375) and activity of a luciferase reporter containing an SOX inducible synthetic promoter was measured (pGL3/AR600). Cells were transiently cotransfected with effector constructs (either Halo/SOX18, Halo/SOX18‐E137K, or empty Halo vector), the pGL3 firefly luciferase reporter as well as a control vector. Transcriptional activity facilitated by each effector was measured for luciferase activity. Results show markedly reduced luminescence (1.3 fold) in cells co‐transfected with E137K relative to native SOX18. This is a significant finding as it validates the functional implication of this mutation. Future studies focusing on additional variants found within the third alpha helix of SOX18 are currently underway.

Support or Funding Information

Support for this study was from NIH‐K01ES025435 (to JWP) and Walsh University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Role for ABCC4 in Regulating Pigment Granule Aggregation in Mice
Authors: Mireles Thomas , Ibarra Dora Evelyn , Wiebe Emely Reimer , Pattillo Amanda M , Rodriguez Cody J , Schuetz John D , García Dana M
Published: June 2021

ABCC4 is member of the family of ATP‐binding cassette proteins that specialize in transporting a variety of molecules out of the cell. ABCC4 exports signaling molecules present in the retina, including cyclic adenosine monophosphate (cyclic AMP). In fish, cyclic AMP regulates processes in the retinal pigment epithelium (RPE), such as dark‐adaptive pigment granule aggregation. In mammals, cyclic AMP accumulates in photoreceptors in the dark. If cyclic AMP were exported from the photoreceptors into the sub‐retinal space, it could then be available for uptake by the RPE, in which it could activate processes that drive pigment granule aggregation. We hypothesize that ABCC4 in photoreceptors mediates cAMP‐export and is needed for pigment granule migration in mice. To investigate ABCC4's involvement in pigment granule aggregation in mice, pigment granule density was measured along the basal microvilli in the RPE in both wildtype (N=3) and Abcc4 knockout mice (N=3). Pigment granule densities measured in the RPE of wild type, dark‐adapted mice differed significantly from those measured in wildtype, light‐adapted mice (p‐value = 0.02). In contrast, pigment granule density was not statistically significantly different between Abcc4 knockout mice that had been dark‐adapted or light‐adapted (p‐value = 0.13). These results support the hypothesis that ABCC4 is involved in the process of pigment granule migration in mice and are consistent with a novel role for cyclic AMP as a cell‐to cell signaling molecule in the retina.

Support or Funding Information

This project was supported by Texas State University in the form of an undergraduate research fellowship to AMP and funds from the Office of the Provost awarded to DMG in recognition of her service as Presidential Fellow. DEI received salary support from HSI STEM Program (84.031c) Award #P031C160035.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NR4A2 Inhibits ERK‐Mediated Protein Synthesis and Cell Growth in Response to Beta‐Adrenergic Stimulation in Cardiomyocytes
Authors: Ashraf Sadia , Hegazy Yassmin , Harmancey Romain
Published: June 2021

Sustained elevation of sympathetic activity is an important contributor to pathological cardiac hypertrophy, ventricular arrhythmias, and left ventricular contractile dysfunction in chronic heart failure. The orphan nuclear receptor NR4A2 is an immediate early response gene activated in the heart under beta‐adrenergic stimulation. The goal of this study was to identify the transcriptional remodeling events induced by NR4A2 expression in cardiomyocytes, and their impact on the physiological response of those cells to sustained beta‐adrenergic stimulation. Treatment of adult rat ventricular myocytes (ARVMs) with isoproterenol induced a rapid (< 4 hours) but transient (< 24 hours) increase in NR4A2 expression levels that was accompanied by increased nuclear localization of the transcription factor. Adenovirus‐mediated overexpression of NR4A2 modulated the expression of genes linked to adrenoceptor signaling, calcium signaling, cell growth and proliferation, and counteracted the increase in protein synthesis rate and cell surface area mediated by chronic isoproterenol stimulation. In consistence with those findings, NR4A2 overexpression also blocked the phosphorylative activation of ERK1/2, Akt, and of their downstream effector in protein synthesis p70S6K. Prominent among the transcriptional changes induced by NR4A2 was the > 7‐fold up‐regulation of the dual‐specificity phosphatases DUSP2 and DUSP14, two known inhibitors of ERK1/2. Pre‐treatment of NR4A2‐overexpressing cardiomyocytes with the DUSPs inhibitor BCI prevented the inhibition of ERK1/2 and p70S6K following isoproterenol stimulation. In conclusion, our results suggest that NR4A2 acts as a novel negative feedback regulator of the beta‐adrenergic receptor‐mediated growth response in cardiomyocytes, and this at least partly through DUSP‐mediated inhibition of ERK1/2 signaling.

Support or Funding Information

This work was supported by NIH Grants R01HL136438, R00HL112952, P01HL051971, and P20GM104357.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Maternal High Fructose and Offspring High Fat Intake on Programmed Hypertension in Young Adult: the Role of Nutrient Sensing Signaling in the Brain
Authors: Chao Yung‐Mei , Chan Julie Y.H.
Published: June 2021

There is now substantial epidemiological evidence suggesting the origin of susceptibility for metabolic syndromes (MetS) and associated cardiovascular dysfunctions in adulthood can be traced back to the early life. We have previously demonstrated that maternal high fructose diet (HFD) induces programmed hypertension in adult offspring; however, the cellular and molecular signaling contributing to the developmental programming of adult hypertension is not fully understood. There are a number of nutrient sensing pathways, including silent information regulator transcript (SIRT), AMP‐activated protein kinase (AMPK), and phosphatidylinositol 3‐kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) located in the central nervous system and are responsible for the detection of sugars, amino acids, lipids and the surrogate metabolite level for the maintenance of metabolic homeostasis. Perturbation of these signaling pathways could lead to MetS and the associated cardiovascular complications. This study was designed to investigate the role of these nutrient sensing pathways in the brain on developmental programming of hypertension in offspring to maternal HFD, and the influence of high fat diet (HFatD) in offspring to these nutrient sensing pathways. Female Sprague‐Dawley rats were fed with 60% HFD or normal diet (ND) during gestation and lactation periods. After weaning both HFD and ND offspring were subjected to HFatD or ND from the age of 3 to 12 weeks. Blood pressure was monitored under conscious condition via tail‐cuff method from week 6 to 12. Expression of nutrient sensing signals in the forebrain hypothalamus was examined by Western blot analysis. Tissue oxidative stress was quantified by electron spin resonance spectroscopy. Maternal HFD induced programmed MetS and hypertension that became significant at age of 9 weeks and maintained in young adult at age of 12 weeks. Changes in both metabolic and blood pressure phenotypes were appreciably enhanced in HFD offspring with additional HFatD intake after weaning. Protein expressions of SIRT1, phosphorylated AMPK (p‐AMPK), and p‐Akt in the hypothalamus were downregulated in the HFD offspring, which were further suppressed in offspring fed with HFatD. These molecular changes were associated with tissue oxidative stress in the hypothalamus.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effects of fermented milks on physiological and neurobehavioral changes in male Sprague Dawley rats
Authors: Choi Jeonghyun , Kim Joo‐Heon , Ham Jun‐Sang , Hong Yonggeun
Published: June 2021

Melatonin is a neurohormone secreted by pineal gland and shows pluripotent functions such as anti‐inflammation, anti‐oxidation, and neuronal cell survival. The major function of melatonin is to control circadian rhythm, from awakening to sleep. Recently, many useful items emitting bright light at night time including light emitting diode (LED) and smartphone, have been emerged. They give convenience to human life, however, can cause various diseases including sleep deprivation, cancer, and obesity. Sleep disturbance by light exposure at night can lead to anxiety‐like behavior, depression, and decreased cognitive function. Therefore, this study aims to identify the effects of three types of fermented milks (FM); C, T1, T2, on sleep and neurobehaviors. Experimental animals were randomly divided into four groups; Cont (control croup), C (treatment with type C FM), T1 (treatment with type T1 FM), and T2 (treatment with type T2 FM). To determine the intestinal activation, the length from cecum to colon was measured. In T2 group, cecum/colon length was significantly increased compared to the length of other groups. To evaluate the sleep quality, electroencephalogram (EEG) was measured through intracranial electrode implantation surgery. C, T1, and T2 FM treated groups showed increased activation of δ‐wave than Cont group. Also, neurobehavioral changes were positively improved in T2 group compared to other groups. In addition, melatonin concentration containing in C, T1, and T2 was 56.0 pg/g, 77.6 pg/g, and 504.4 pg/g, respectively. Therefore, melatonin in fermented milk might have positive effects on gut‐brain bidirectional communication.

Support or Funding Information

NRF‐2017R1A2A2A01067169, KGM4611821, 2016 Creative Research Program of Inje University, Republic of Korea

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The human primordial microbiome hosts complex bacterial and fungal interkingdom ecologies that vary with gestational age
Authors: Willis Kent A , Purvis John H , Gomes Charles K , Talati Ajay J , Pierre Joseph F
Published: June 2021

Commensal microbial communities play important roles in human development. While microorganisms appear to be predominately transmitted from mother to child, the timing of transmission remains controversial. Meconium, the first stool passed after birth, was traditionally considered sterile because it originated from a sterile uterine environment. Following advances in next‐generation sequencing (NGS) techniques, more recent evidence suggests bacterial communities may exist in the gut prior to birth, however, the presence of primordial fungal communities has not been described. Here, we demonstrate the presence of multi‐kingdom microbial communities in meconium immediately following birth based on NGS. To describe the ecology of the primordial microbiome, we prospectively enrolled premature (gestational age < 32 weeks') very low birthweight (VLBW < 1500 g) and full term (FT, gestational age 37–41 weeks') newborns and collected the initial meconium. We examined the interkingdom community structure using 16S and ITS rDNA‐based amplicon sequencing followed by culture‐based techniques. We prospectively enrolled 57 VLBW and 35 FT newborns and collected a stool sample at < 48h of life from 34 infants in each group. Bacteria were found in meconium of 90% of VLBW and 82% of FT samples (χ2P=0.325) and fungi in 93% of VLBW and 62% of FT samples (χ2P=0.003). The bacterial and fungal communities differed significantly between VLBW and FT (bacteria: PERMANOVA P=0.006, fungi: PERMANOVA P=0.013). Fungal communities of VLBWs were dominated by Ascomycota, including Candida, while FT meconium contained greater Basidiomycota and taxonomic diversity. Bacterial communities in VLBW meconium were also less diverse and contained on average a third as many taxa compared with FT. These results demonstrate human meconium contains complex interkingdom microbial communities that develop with gestational age, suggesting host‐microbe interkingdom relationships may exist in the uterine environment. Intriguingly, fungi are 50% more commonly observed in premature meconium than in full term neonates which could suggest a pathological association with preterm birth.

Support or Funding Information

The Marshall Klaus Perinatal Research Award from the American Academy of Pediatrics

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hand2 Functions to Synergistically Activate Gli Target Genes in Mandibular Neural Crest Cells
Authors: Brugmann Samantha , Elliott Kelsey H. , Chen Xiaoting , Salomone Joseph , Chaturvedi Praneet , Gebelein Brian , Weirauch Matthew T. , Peterson Kevin A.
Published: June 2021

The Hedgehog (Hh) pathway has been well‐studied and implicated in neural crest cell (NCC) survival and facial patterning; however, the mechanism(s) by which the transcription factors (TFs) of the pathway, the Gli TFs, orchestrate Hh signal transduction remains elusive. For example, Gli ChIP‐seq data from NCCs in the developing mandibular prominence (MNP) reveal that the presence of a peak is not sufficient to predict a Gli “target.” In light of these data and previous studies suggesting Gli proteins interact with co‐factors, we performed genome‐wide analyses to identify motifs that frequently appear near Gli binding regions (GBRs). These analyses revealed that E‐box motifs (bHLH TF binding sites) were significantly enriched (p=0.001) within Gli ChIP‐seq peaks compared to random sequences. Additionally, ChIP‐seq peaks for Hand2, a bHLH TF known to be necessary for MNP development, were 111‐fold enriched within Gli peaks. A comparison of Hand2f/f;Wnt1‐Cre and Gli2f/f;Gli3f/f;Wnt1‐Cre MNP phenotypes shows striking similarities, which in conjunction with the significant overlap of Gli and Hand2 co‐occupied genomic regions suggests these two proteins may work in tandem to influence Gli target gene expression. Interrogation of DNA motifs within genomic locations occupied by Gli and Hand2 together versus those occupied by Gli alone revealed the existence of two distinct types of GBRs: a ‘canonical’ GBR defined by the presence of a medially located A (GACCACCC), and a ‘deviant’ GBR defined by a T in the medial position (GACCTCCC). Interestingly, the ‘canonical’ GBR is 19‐fold enriched (p<10−136) in Gli3 peaks without a Hand2 peak, whereas it is only 3‐fold enriched (p<10−47) in Gli3 peaks that contain a Hand2 peak. To test if these distinct GBRs convey biological relevance, we identified canonical and deviant GBRs within Gli and Hand2 ChIP‐seq peaks of Gli targets important for mandibular development and linked them to reporter constructs transfected into 09–1 cells. We found that Hand2 and Gli utilize a deviant GBR to synergistically activate Gli targets including Foxd1 and Foxd2. In sum, our work establishes insight into the existence of a sequence‐dependent mechanism for Gli target gene regulation in which molecular accomplices may be necessary for optimal transcription of Gli targets in NCCs of the developing MNP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex‐Specific Alterations in LPS‐Induced Peripheral and Central Inflammation in Adult Offspring Exposed to Gestational Intermittent Hypoxia: Potential Regulation by miR‐146
Authors: Wang Tao , Kiernan Elizabeth , Ewald Andrea , Baker Tracy , Watters Jyoti J
Published: June 2021

The prevalence of obstructive sleep apnea (OSA) and concomitant intermittent hypoxia (IH) in pregnant women is rising in parallel with the obesity epidemic. IH causes profound inflammation, and it is well‐established that maternal immune activation can lead to detrimental outcomes in her offspring. Although in recent years the acute consequences of maternal OSA on the health of both the mother and the newborn have become better appreciated, little remains known about the potential long‐lasting consequences to the offspring in adulthood. Given the effect of inflammation on the neural control of breathing, we studied the consequences of gestational intermittent hypoxia (GIH) on the central and peripheral immune responses of adult male and female offspring. We exposed rat dams to IH from gestational days 10–21 (8hrs/day, 2 min cycles alternating between 10.5% O2 and 21%O2, mimicking the desaturation and resaturation kinetics of human OSA). We challenged the adult offspring with a peripheral LPS injection (1 mg/kg i.p., 3 hrs) and assessed neural (whole brain homogenates) and peripheral (spleen) cytokine gene expression. We found that GIH gene‐specifically enhanced LPS‐induced neuroinflammation in adult offspring of both sexes. Conversely, while LPS‐induced peripheral inflammation was unchanged in GIH male offspring, it was strongly attenuated in GIH females, suggesting that the central and peripheral immune responses are differentially impacted by GIH, and that these effects differ by sex. Because microRNAs (miRNAs) are critical regulators of the immune response, we tested the hypothesis that miRNAs are differentially expressed in male and female GIH offspring. In spleens from adult GIH offspring, we screened the levels of several immunoregulatory miRNAs. Interestingly, miR‐146, a strongly immunosuppressive miRNA, was highly upregulated in female spleens. We suggest that augmented miR‐146 levels suppress peripheral immune responses to an inflammatory challenge in GIH offspring, and that miR‐146 levels will be reduced in the brain where neuroinflammation is enhanced in GIH offspring. Studies are underway using lentiviral‐mediated manipulation of miR‐146 activity to more directly test this hypothesis, and to understand how central and peripheral inflammation are ultimately regulated in the context of GIH. Regulating miR‐146 expression in adults exposed to maternal OSA in utero may represent a novel therapeutic target to control the detrimental consequences of aberrant inflammatory signaling.

Support or Funding Information

Supported by NIH R01 NS085226 and HL105511, and a Wisconsin Alumni Research Foundation UW2020 grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.