bluuebunny/crossref_metadata_2025_split huggingface dataset:
Myosin Quality Control Systems in Zebrafish Skeletal Muscle Development
Authors: Carlisle Casey , Prill Kendal , Pilgrim David 
Published: June 2021

In order to maintain homeostasis, defective proteins must be identified, removed, and replaced. In skeletal muscle, failures in this process can lead to a variety of incurable muscle diseases (myopathies), many of which are fatal. While aspects of protein turnover have been studied widely, the protein turnover process in skeletal muscle is still incompletely understood, especially with respect to the motor protein, myosin. By studying the assembly and maintenance of the basic unit of striated muscle, the sarcomere, we aim to elucidate the components involved and the mechanism controlling muscle myosin quality control in skeletal muscle.Zebrafish make an excellent model system to study sarcomere protein turnover due to their ability to survive with myopathies that are fatal in other organisms. Using two different zebrafish lines, steif, and herzschlag, we and others show that steif sarcomeres fail to assemble myosin thick filaments due to a loss of a critical component (Etard et al., 2007), while herzschlag sarcomeres degrade over time as a consequence of muscle contractions. Transmission electron microscopy coupled with H&E staining revealed a slow muscle atrophy specific to herzschlag embryos. To identify factors involved in response to myosin damage during assembly or maintenance, we have screened through candidate genes, myosin chaperones and muscle specific E3 enzymes, using in situ hybridization and qPCR. Our data indicates a division of function where specific E3 enzymes and myosin chaperones respond to myosin damage exclusively during assembly, while other E3 enzymes respond to myosin damage during sarcomere maintenance. Pharmaceutical inhibition of the proteasome shifts this response in steif embryos, but not herzschlag, suggesting an interconnected network of protein quality control during myosin assembly. Together, this work helps highlight potential therapeutic targets to treat myosin turnover related myopathies.Support or Funding InformationNSERCThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vitro testing of fluticasone drug delivery system for inflammatory injury and repair
Authors: Tipparaju Srinivas , Kelly Shannon , Weigel Robert , Halasz Kathleen , Tur Jared , Brotto Marco , Sutariya Vijaykumar 
Published: June 2021

Glucocorticoids such as dexamethasone and fluticasone have potent anti‐inflammatory properties, but they also have serious side effects when used for extended periods or even at higher doses. For enhanced drug delivery and to test whether glucocorticoids are able to provide benefits at lower doses we developed a new nano‐drug delivery system (called Nano‐Gluco) and tested it in vitro in a model of muscle cell injury and repair. We characterized Nano‐Gluco for its physical and chemical characteristics. We utilized zeta potential, particle size analysis, scanning electron microscopy and differential scanning calorimeter to evaluate Nano‐Gluco. To test the efficacy of the system, we used the LPS induced cellular injury model. We demonstrated that Lactate dehydrogenase (LDH) release was significantly lower in positive controls glucocorticoid treated cells compared with LPS alone. In support for the benefits Nano‐Gluco, it allowed similar protection of cells in vitro at a much lower concentration than glucocorticoids alone. We next utilized the LPS induced injury model with C2C12 (murine myoblasts) cells to identify the effects injury and repair, as well as inflammation signaling. At the molecular level, we tested key inflammatory markers such as NFKb, TNFα, IL1β along with other cell markers such as p27, FOXO for the beneficial effects of steroids in muscle cell repair and inflammation. These markers confirmed the beneficial effects of employing Nano‐Gluco. Overall, we demonstrate that our new Nano‐Gluco system provides a highly efficient method for delivery of glucocorticoids to cells for achieving anti‐inflammatory properties at a much lower concentration compared with current glucocorticoid preparation. We are now moving into animal pre‐clinical studies to advance the use Nano‐Gluco in humans.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion of p16 expressing senescent cells improves muscle mass and age‐dependent outcomes in multiple organ systems
Authors: Judge Jennifer , Shigdar Shahjahan , Davis Carol , McArdle Anne , Richardson Arlan , Brooks Susan 
Published: June 2021

BackgroundCellular senescence is defined as permanent cell cycle arrest due to upregulation of cyclin dependent kinases such as p16. Senescent cells accumulate with age and can contribute to aging related pathologies. Recently, deletion of p16 expressing senescent cells in vivo has been shown to increase healthspan and lifespan, and ameliorate some tissue specific pathologies in mice. However, it is unknown whether cellular senescence plays a role in age associated declines in skeletal muscle function. We hypothesized that deletion of p16 expressing senescent cells will preserve age‐induced declines in muscle function and mass.MethodsTo eliminate senescent cells in vivo, we utilized p16‐3MR transgenic mice in which administration of the drug ganciclovir will induce cell death in p16 expressing cells. Mice were treated with 25 mg/kg ganciclovir or saline (control) intraperitoneally for 5 days at monthly intervals from 20–26 months of age. Animals were sacrificed at 26 months of age, muscles were weighed, and gastrocnemius muscles were tested for force production in vivo, while extensor digitorum longus and soleus muscles were tested in vitro. Muscle fiber cross sectional areas and fiber type were determined by immunohistochemistry. Additional organs were snap frozen for downstream protein, cytokine, and RNA analyses. mRNA expression was measured using qPCR.ResultsMice that were treated with ganciclovir lived longer and had a lower incidence of cancer than control mice. Muscle masses of the soleus, tibialis anterior and gastrocnemius were greater in ganciclovir‐treated mice compared with controls. The higher muscle mass in treated mice was not associated with any shift in fiber type distribution, increase in fiber cross‐sectional area, or any expansion of the volume of extracellular matrix, suggesting the higher muscle mass was due to preservation of fiber number. There was no effect of p16 deletion on muscle force production. However, upon examining muscle, spinal cord, serum and lung tissue, p16 deletion impacted age‐dependent changes in gene expression and cytokines.ConclusionsDifferential effects of senescent cell deletion on skeletal muscle mass and function suggests the role of senescent cells in skeletal muscle aging may be context specific. Further studies are needed to more thoroughly determine tissue‐specific benefits, but our work suggests that deletion of senescent cells even late in life may provide protection against global aging processes.Support or Funding InformationAG051442K12GM111725This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skeletal Muscle Function is Impaired in Mice Following Surgical Weight Loss
Authors: Stephenson Erin J , Gomes Charles K , Stayton Amanda S , Puchowicz Michelle A , Han Joan C , Pierre Joseph F 
Published: June 2021

Limb skeletal muscle mass losses in the year following bariatric surgery are far greater than the loss of lean mass experienced by inactive persons during the typical aging process. This is a clinical concern, since loss of skeletal muscle mass may have long term effects that attenuate many of the benefits associated with marked weight loss (such as sarcopenia, frailty and loss of mobility). Here, we use a mouse model of bariatric surgery (gastric sleeve) to test the hypothesis that surgical weight loss impairs skeletal muscle function. Sixteen‐week‐old male C57BL6/J mice with diet‐induced obesity (DIO; 5.21 kCal/g, 60% fat, 20% carbohydrate, 20% protein; D12492 Research Diets) were randomized into groups receiving either gastric sleeve surgery (GS; 80% resection of the greater curvature of the stomach) or sham surgery (DIO‐sham; laparotomy). A third group of age‐matched lean mice were included as a non‐surgical, non‐obese comparison group (3.82 kCal/g, 10% fat, 70% carbohydrate, 20% protein; D12450J Research Diets). Following surgery, GS mice rapidly lost 20% of their pre‐surgery weight, reducing their initial fat mass by 53% and lean mass by 20%, as measured by EchoMRI. Loss of lean mass was associated with a reduction in grip strength six‐weeks post‐surgery (−10%, p=0.04), compared to DIO‐sham, despite regain of most of the initial lean mass losses. GS mice had reduced total energy expenditure compared to DIO‐sham, as measured by indirect calorimetry at both 22°C (−7% during the light phase, p=0.0015 and −11% during the dark phase, p=0.009) and at 28°C (−7% during the light phase only, p=0.05). No differences in ambulatory activity were observed between groups. These findings, although preliminary, suggest that bariatric surgery causes molecular adaptations in the skeletal muscle that may not be easily reversed once initial lean mass losses are restored. Further work will identify whether surgical weight loss causes alterations in protein breakdown/synthesis and other metabolic adaptations that affect long‐term skeletal muscle function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Region‐Specific Metabolic Regulation of Intestinal Stem and Progenitor Cells
Authors: Stine Rachel , Seale Patrick 
Published: June 2021

Distinct metabolic programs regulate intestinal stem cell renewal and differentiation. However, the mechanisms that rewire metabolism during differentiation are poorly defined. Additionally, cells in intestinal stem cell niches have not been studied in the context of specific intestinal regions to determine if crypts along the length of the intestine are regulated by common or specialized metabolic programs. We predict that transcriptional manipulation of metabolic programming in stem cells and progenitor cells will affect the ability of these cells to properly differentiate and regenerate tissue. Here, we identify the transcription factor PRDM16 as a critical regulator of intestinal metabolism and differentiation. PRDM16 is known to affect the metabolic programming of adipocytes in thermogenic adipose tissue. Surprisingly, acute deletion of Prdm16 in adult mice causes intestinal wasting, apoptosis, and an accumulation of poorly differentiated cells in the small intestinal crypt. RNA‐seq and tracer metabolomic studies show that PRDM16 controls fatty acid oxidation (FAO) in the intestinal crypt. FAO is the process by which fatty acids are broken down to produce acetyl CoA, which can enter the TCA cycle to produce energy and other cellular building blocks. PRDM16 binds, along with its protein partners PPARγ and PPARα, to the promoter and enhancer regions of many FAO genes. The loss of Prdm16 or inhibition of FAO impaired the differentiation of stem and transit amplifying cells into mature cells. Notably, PRDM16 expression is highest in the duodenum and declines distally along the intestine. This gradient of PRDM16 levels controls region‐specific expression of the FAO program and underlies the differential reliance of region‐specific stem and progenitor cells on FAO. We also show that there is a region‐specific sensitivity to FAO inhibition in ex vivo enteroids isolated from different regions. Regional reliance of cells on FAO may have arisen due to the abundance of fats present in the proximal small intestine, where the majority of fat digestion and absorption occurs. Interestingly, this specification persists in culture where nutrients are not differentially available, raising the possibility that over time, cells have more permanently adapted to their environment. Altogether, this study establishes PRDM16 as a region‐specific regulator of intestinal metabolism and stem cell differentiation. We speculate that distinct regional metabolic profiles of stem cells could contribute to determining the character of the mature epithelial cells in different regions. Future experiments will focus on how diet affects regional metabolic profiles of intestinal stem cells and whether alterations in metabolic programming could lead to disease.Support or Funding InformationF32 DK10574303 to R. Stine, P30 DK050306 Pilot Grant to P. Seale, R01 DK10300802 to P.SealeThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rotavirus Infection Activates Intercellular Calcium Waves through Purinergic Signaling
Authors: Chang‐Graham Alexandra , Perry Jacob , Engevik Melinda , Danhof Heather , Kellen Joseph , Hyser Joseph 
Published: June 2021

Rotavirus (RV) remains the leading viral cause of diarrheal disease in children worldwide. The life‐threatening diarrhea from RV infection is potentially exacerbated by the release of signaling molecules from infected villus epithelial cells. RV increases cytosolic calcium in infected cells, which is necessary for RV replication and activation of secretory pathways. Thus, characterizing RV‐induced calcium signaling is needed to understand RV pathogenesis. However, the activation of a calcium signal from infected to uninfected cells has not been directly observed. Therefore the signaling mechanisms that facilitate widespread dysregulation of fluid secretion remain undefined. To address this, we conducted live cell fluorescence imaging in cell lines and jejunum human intestinal enteroids (jHIEs) to measure calcium signaling during RV infection.We generated African monkey kidney MA104 cells and jHIEs that stably express GFP‐based genetically encoded calcium indicators to measure calcium dynamics during RV infection. RV significantly increases the number and magnitude of calcium transients in infected wells. Further, we observed that single RV‐infected cells triggered long‐distance intercellular calcium waves that encompassed surrounding uninfected cells. These calcium waves are mediated by extracellular ATP/ADP since treatment with the ectoNTPase apyrase or the P2Y1 purinergic receptor inhibitor BPTU blocked the waves and also RV replication. We created P2Y1 receptor genetic knockouts using CRISPR/Cas9, and there were reduced calcium waves in RV‐infected knockout cells. In RV‐infected HIEs, blocking calcium waves with apyrase and BPTU decreased fluid secretion in the enteroid swelling assay and decreased serotonin secretion, which exacerbates RV diarrhea and vomiting via the enteric nervous system. Furthermore, we identified that broad anion channel blockers significantly inhibited calcium waves, supporting that ATP/ADP is released via anion channels.Our results demonstrate that RV‐infected cells release extracellular purines to cause calcium signaling in uninfected cells important for RV replication and secretory pathways. Our data points to purinergic signaling as a therapeutic target for developing anti‐diarrheal drugs. Furthermore, we are the first to show that viruses can exploit purinergic signaling and calcium waves to potentially amplify pathophysiological signaling important for diarrhea.Support or Funding InformationNIH F30 DK112563, R01 DK115507, R03 DK110270, K01 DK093657, BCM New Faculty Seed Grant and Funding, Augustana College TMC Summer Research Internship Program, TMC DDC Enteroid Core (P30DK056338)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Circadian Clock Components PER2 and BMAL1 are Altered During Infection of Helicobacter pylori
Authors: Teal Emma L , Li Jing , Chakrabarti Jayati L , Broda Taylor , Steele Nina , Rosselot Drew , Sundaram Nambirajan , Hawkins Jennifer , Hong Christian , Helmrath Michael A , Wells James , Diwan Tayyab L , Zavros Yana L 
Published: June 2021

Background/HypothesisSeveral gastrointestinal functions have been demonstrated to be under circadian clock control and colorectal cancer tissues have been found to have a disruption in core circadian clock components BMAL1 and PER2. Those suffering from chronic Helicobacter pylori (H. pylori) infection are at a heightened risk of developing gastric adenocarcinoma. Compared to young individuals, the aged population suffer from an increased susceptibility to H. pylori infection as well as diminished expression of circadian clock components which could further predispose them to the development of gastric cancer. We therefore hypothesize that chronic H. pylori infection induces a hyperproliferative response in the gastric epithelium via BMAL1 activation resulting in a susceptibility to gastric cancer.MethodsGastric organoids (hFGOs) derived from primary gastric tissue of either aged (> 55 years of age) or young (14 – 20 years) individuals were generated and subsequently infected with H. pylori. Analysis for proliferation and expression of circadian clock components BMAL1 and PER2 was performed for a period of 24 hours post‐infection. Organoids infected with H. pylori were analyzed for gastric cancer markers by qRT‐PCR.ResultsGastric fundic tissue obtained from individuals infected with H. pylori were found to have a significantly upregulated expression of cytoplasmic BMAL1. hFGOs exhibited a robust circadian expression of Bmal1, Per2, and phosphorylated (inactive) GSK‐3β. In comparison to uninfected controls, hFGOs infected with H. pylori displayed a heightened expression of BMAL1, phosphorylated GSK‐3β, and epithelial proliferation. Gastric organoids derived from of elderly patients was found to have a diminished rhythmic expression of clock genes Per2 and Bmal1. This dampening in expression of circadian clock components correlates with an increased expression of markers for intestinal metaplasia.ConclusionsPersistent infection by H. pylori inactivates GSK‐3β resulting in the activation of the BMAL1:CLOCK complex leading to a hyperproliferation in gastric epithelial cells. This mechanism could heighten the risk of developing gastric cancer in the aged population and in individuals with chronic H. pylori infection. Per2 knockout mice have been generated and infected with H. pylori in order to further understand this mechanism in vivo. Results are ongoing and pending.Support or Funding InformationNIH 1U19AI116491‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of FADS Variants on Responses to Diet in Patients With ALI
Authors: Dosso Beverly , Waits Charlotte Mae , Simms Kelli , Sergeant Susan , Files D. Clark , Chilton Floyd , Langefeld Carl , Rahbar Elaheh 
Published: June 2021

Nutritional support in the intensive care unit (ICU) is a vital element of patient care. Polyunsaturated fatty acids (PUFAs) like γ‐linolenic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been recommended in immunomodulatory diets because of their anti‐inflammatory effects. However, clinical trials have failed to show unanimous benefit with PUFA‐rich diets. Metabolic conversion of these PUFAs are dependent on fatty acid desaturase (FADS) and elongase (ELOVL) enzymes, which are unequal across humans. In fact, several genetic variants within FADS and ELOVL genes have been shown to impact PUFA metabolism. Select variants are also differentially expressed in African Americans. We hypothesize that these genetic variants may help explain the differential responses to PUFA‐rich diets. In this study, we aimed to investigate gene‐diet interactions in patients with acute lung injury (ALI).Banked DNA and plasma samples from the OMEGA randomized clinical trial (NCT00609180) were used to conduct a secondary analysis on genetic variants within FADS and ELOVL regions; 43 SNPs were genotyped. Plasma PUFA levels were quantified using gas chromatography. Of the 272 enrolled, 143 received omega‐rich vs. 129 placebo diets. All statistical analyses were stratified by race and adjusted for age and gender.Several SNPs were associated with PUFA levels. Notably, SNP rs174537 had a significant impact on arachidonic acid (ARA). Caucasian T allele carriers on the omega‐rich diet had lower ARA levels (p=0.022, Fig. 1). Interestingly, this SNP had a major impact on PUFAs within African Americans on both placebo and omega‐rich diets. ARA levels were significantly higher and DHA levels were low (p=0.0007) in African Americans. Genotype at rs174537 was associated with patient outcomes; Caucasians with the T allele had significantly more VENT‐free and ICU‐free days (~>12%) than GGs, but this was not observed within African Americans. Instead, a significant interaction between gender and diet was seen, with African American women on placebo diet having 40% fewer VENT‐free (p=0.009) and ICU‐free days (p=0.022) than African American men. Mortality was highest in GGs receiving omega‐rich diets in both races, however this was not statistically significant. These data suggest strong ethnic, gender and genetic factors influencing the response to omega‐rich diets. Further investigation on other gene‐gene interactions (e.g. between FADS and ELOVL variants), and epigenetic modifications are needed to understand the underlying mechanisms regulating PUFA metabolism and biosynthesis in critically injured and ill patients.Support or Funding InformationNIH K25 HL133611This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.