bluuebunny/crossref_metadata_2025 huggingface dataset:
Restoration of delayed gastric emptying and impaired gastric compliance by anethole in an animal model of functional dyspepsia
Authors: Asano Teita , Takenaga Mitsuko
Published: June 2021

Background and Aims

Functional dyspepsia (FD) is a functional gastrointestinal disorder characterized by persistent or recurrent postprandial upper‐abdominal discomfort and epigastric pain. Although FD markedly reduces the patient's quality of life, involving elevates health‐care costs, treatment of this condition by methods other than prescribed medicines, such as natural products, could be beneficial. Delayed gastric emptying and impaired gastric accommodation play important roles in pathogenesis of FD. Anethole (1‐methoxy‐4‐((E)‐propenyl)‐benzene), a major component in essential fennel oil has been used in flavouring, alcoholic beverage production and pharmaceutical formulations. In this study, we examined the effects of anethole on delayed gastric emptying and impaired gastric compliance in animals.

Methods

Gastric emptying was monitored by phenol red marker method in mice. Induction of delayed gastric emptying was performed by the administration of clonidine, dopamine or a 5‐HT3 receptor agonist m‐CPBG. Gastric accommodation (compliance test) was examined in rats by a pressure‐volume control device, barostat. Acetylcholinesterase activity was measured using assay kit

Results

Oral administration of anethole restored delayed gastric emptying induced by clonidine administration. The dose of anethole did not affect gastric emptying in non‐stressed mice. We also found that fennel oil, which contains about 86% anethole, suppressed clonidine‐induced delayed gastric emptying. Furthermore, a Japanese herbal medicine An‐chu‐san containing anethole improved the gastric emptying in the presence of clonidine. Pharmacological and biochemical analyses suggested that anethole restores gastric emptying independently of D2, 5‐HT3, 5‐HT4 receptors and acetylcholinesterase. Finally, anethole stimulated gastric compliance in rats both with and without wrap restraint stress.

Conclusions

Taken together, our results suggest that anethole could be beneficial for treatment of postprandial distress and early satiety in patients with FD.

Support or Funding Information

This research received no specific grant from any funding agency in the public, commercial, or not‐for‐profit sectors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Function And Cardiovascular Stress Markers After A Single Dive In Aging Rats (ApoE Knockout Rats)
Authors: Ardestani simin Berenji , Eftedal Ingrid , Matchkov Vladimir , Pedersen Micahel
Published: June 2021

The compressed gas breath during diving augments partial pressure of oxygen causing the oxygen concentration of the blood to increase above normal (hyperoxia). Hyperoxia in combination with gas bubbles that develop during the decompression (ascent) phase, likely causing oxidative stress, including transient endothelial dysfunction in venous and arterial vessels. The number of aging divers is rising and aging itself is associated with a gradual impairment of endothelial function. These alterations play a central role in the pathogenesis of atherosclerosis and coronary artery disease. While diving and aging are independent modulators of cardiovascular function, little is known about their combined effect. Thus, the central question is does diving expose old divers to more oxidative stress or not?

Method

ApoE homozygous knockouts rats with impaired cardiovascular function were used as a model for aging. 10 ApoE rats (male and female) exposed to 500 kPa heliox gas (80% helium/20% oxygen) for 1 hr in a pressure chamber to simulate diving. Endothelial function examined in‐vitro by myograph in pulmonary and mesenteric artery. The oxidative stress biomarkers measured in the plasma (collected from heart) and lung tissue via TBARS assay. 10 ApoE rats served as a control group.

Results and conclusion

The results of this study demonstrated that a single dive causes endothelial dysfunction in pulmonary arteries of rats with aging cardiovascular system. This seems to be caused by reduction in NO synthesis (Fig. 1). These responses were observed just in male rats.

Support or Funding Information

This project was funded by Aarhus University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vivo Endothelial Cell‐specific Expression of AMPKα Attenuates Cold‐induced Pulmonary Vascular Dysfunction and Hypertension by Mitigating Inflammation
Authors: Wang Shirley , Xing Yu , Xu Yuechi , Chen Kai , Ali Quaisar , Ullah Mujib , Sun Zhongjie
Published: June 2021

Background and Aim

In lung biopsies from patients with pulmonary hypertension (PH), mononuclear cells are often observed in pulmonary arterial plexiform lesions, mainly consisting of leukocytes and macrophages. Endothelial AMP‐activated protein kinase α (AMPKα) is downregulated in the pulmonary arteries of patients with PH. AMPK is involved in the cellular response to metabolic stress. AMPKα expression was decreased in pulmonary arteries in a rat model of PH induced by exposure to cold. The aim of this study is to investigate whether in vivo endothelial cell‐specific expression of AMPKα attenuates cold‐induced inflammation and PH.

Methods

To investigate the role of AMPKα1 in PH, we generated Lentivirus carrying AMPKα1 full‐length cDNA driven by an endothelial‐specific promoter, Tie2 (Tie2.AMPKα1). Lentivirus with Tie2.LacZ was used as the control construct. Four groups of Sprague‐Dawley male rats (7/each group) received IV delivery of PBS, PBS, Tie2.LacZ and Tie2.AMPKα1 respectively. Following injection, one PBS group was kept at the room temperature and serve as a control. Other three groups were exposed to cold temperatures (5±1°C). The right ventricular (RV) pressure was measured by end‐point cannulation after 11 weeks of cold‐exposure.

Results

RV systolic pressure was elevated in cold‐PBS and cold‐Tie2.LacZ groups compared with the warm‐PBS controls, suggesting that continuous cold exposure causes pulmonary vascular dysfunction and pulmonary PH, namely cold‐induced PH (CIPH). By contrast, AAV delivery of Tie2.AMPKα1 prevented cold‐induced elevation of RV pressure, suggesting that downregulation of endothelial AMPK α1 plays a critical role in the pathogenesis of cold‐induced pulmonary vascular dysfunction and CIPH. RV developed hypertrophy as evidenced by a significant increase in the RV weight in cold‐exposed rats. Western blot and RT‐PCR data confirmed that Tie2.AMPKα1 effectively rescued the cold‐induced downregulation of AMPK α1 and eNOS expression in the lung. Cold exposure caused strong inflammatory responses as seen with robust infiltration of leukocytes and macrophages in cold‐exposed rats. Cold‐induced activation of macrophages also led to upregulation of TNFα and IL‐1β levels in the lung. Tie2.AMPKα1 effectively attenuated cold‐induced infiltration of leukocytes and macrophages and elevation of TNFα and IL‐1β levels.

Conclusion

Cold exposure causes pulmonary vascular dysfunction and CIPH via downregulation of endothelial AMPKα expression. In vivo endothelial cell‐specific expression of AMPK α1 attenuates CIPH partially by suppressing cold‐induced inflammatory responses.

Support or Funding Information

NIH R01 HL116863, HL122166.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Salt Diet May Stimulate Fructose Uptake in Brain Neurons and Contribute to Neuronal Apoptosis
Authors: Hahka Taija H. , Fan Yuan Yuan , Chen Qing Hui , Shan Zhiying Jenny
Published: June 2021

Clinical studies have been leaning toward the role that the central nervous system (CNS) is one of the main controls of blood pressure regulation such as the onset of fructose‐induced hypertension. The CNS is by far the most energy demanding and consuming organ in our body and it can metabolize fructose but that has consequences. However, the connection between fructose and hypertension within the CNS is still lacking in definition. Here we tested the hypothesis that a high fructose diet contributes to hypertension via increase neuronal apoptosis. Eight‐week‐old male Sprague Dawley (SD) rats were divided into 4 groups and were fed a normal salt (NS: 0.4% NaCI chow), high salt (HS: 4% NaCl chow), NS diet with fructose (F: 20% fructose drinking water), and high salt plus fructose (HS+F: 4%NaCl chow and 20% drinking water) diet, respectively, for 3 consecutive weeks. Their blood pressure was measured prior and post each diet treatment using the non‐invasive tail‐cuff technologies. All rats were then euthanized, then their blood and cerebrospinal fluid (CSF) was withdrawn for fructose measurements. HS+F rats showed an increase in mean arterial blood pressure (MAP) 126 mmHg compared to control 90 mmHg (p<0.0001). There is no significant MAP alterations in HS or F alone groups compared to NS group. Fructose measurement demonstrated that the serum fructose concentration is significantly increased in the F group (359 μM), and HS+F (409 μM) compared to control NS group (253 μM), while no significant alterations were observed in serum fructose in HS alone group (229 μM). In contrast, CSF fructose concentration is dramatically decreased in F (230 μM) and HS+F (231 μM) groups compared to NS control group (290 μM). In addition, immunostaining showed that HS+F group significantly promoted immunoreactivity of GLUT5, a fructose transporter in the brain paraventricular nucleus (PVN). Furthermore, incubation of brain neurons isolated from neonatal SD rats with 1 mM fructose for 6 hours stimulated neuronal apoptosis (27%) compared to the control (20%, P<0.05). In summary, this study demonstrated that HS+F diet decreases CSF fructose concentration, increases PVN GLUT5 expression, and results in hypertension in normal SD rats. These results suggest that HS facilitates brain neurons to uptake fructose into neuronal cells, resulting in an increase in intracellular fructose, which in turn, stimulate cell apoptosis. Increased neuronal apoptosis may contribute to hypertension.

Support or Funding Information

Funding support: R15‐HL122952, Qinghui Chen; R15‐HL129213, Zhiying Shan; Michigan Tech and portage health Foundation REF, Zhiying Shan

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Albumin induces CD44 expression in glomerular parietal epithelial cells by activating the ERK signaling pathway
Authors: Zhao Xueying , Chen Xiaoming , Cobbs Alyssa , George Jasmine , Zhang Yuanyuan , Chima Ashmeer , Emmett Nerimiah
Published: June 2021

De novo expression of CD44 in glomerular parietal epithelial cells (PECs) leads to a pro‐sclerotic and migratory PEC phenotype in focal segmental glomerulosclerosis. However, the mechanisms underlying CD44 induction in activated PECs remain largely unknown. This study was performed to examine CD44 expression in rat PECs under proteinuric condition and its regulatory mechanisms. CD44 gene and protein levels were examined in Zucker diabetic (ZD) rat kidneys and primary cultured rat PECs exposed to exogenous albumin. Gene microarray and real‐time PCR confirmed an upregulation of CD44 mRNA in renal cortex and glomeruli of ZD rats. Immunofluorescence staining of kidney sections revealed an increase in CD44 signal in claudin‐1‐positive PECs. Enhanced CD44 staining was observed in activated PECs localized in Bowman's capsule and migrated into the glomerular tuft. Moreover, anti‐proteinuric treatment with losartan, an angiotensin II receptor blocker, significantly reduced CD44 protein level in the diabetic rat kidney. In primary cultured rat PECs, administration of rat serum albumin (RSA, 0.25 – 1 mg/ml) resulted in an activation of ERK1/2 MAPK signaling and upregulation of CD44 and claudin‐1 protein expression. This increase in claudin‐1 and CD44 protein was significantly attenuated in the presence of specific ERK1/2 inhibitor U0126. Taken together, our results demonstrate that albumin activates PECs and induces CD44 expression, at least partially via the activation of ERK signaling pathway.

Support or Funding Information

NIH SC1DK112151, NIH/NCRR/RCMI 8G12MD007602 & 8U54MD007588

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mathematical model of megalin trafficking in differentiated proximal tubule cells
Authors: Shipman Katherine E. , Long Kimberly R. , Rbaibi Youssef , Baty Catherine J. , Weisz Ora A.
Published: June 2021

The polarized epithelial cells that comprise the proximal tubule (PT) have a specialized apical endocytic pathway that allows for high‐capacity endocytosis which is necessary to recover essential nutrients and to maintain a protein‐free urine. Megalin, a multi‐ligand receptor at the apical surface of the epithelial cells, binds proteins in the ultrafiltrate and internalizes them via receptor‐mediated endocytosis. Ligands are sorted from receptors in endocytic compartments, and the receptors are recycled back to the surface. The molecular identities of the compartments involved in sorting and recycling in PT cells and the kinetics of megalin trafficking through them are unknown. When the endocytosis in the proximal tubule is dysfunctional, low molecular weight (LMW) proteinuria results. LMW proteinuria is often an early sign of kidney damage and is observed in many clinical settings, including genetic disorders, diabetes, sickle cell disease, and after renal transplantation. A significant barrier to understanding the regulation of receptor‐mediated apical endocytosis of filtered proteins has been the lack of a highly differentiated cell culture model that retains the organization and high capacity of the PT apical endocytic pathway. We previously discovered that OK cells cultured under continuous fluid shear stress develop morphological and functional features similar to that of the PT in vivo, including high apical endocytic capacity and increased megalin expression. We present an ordinary differential equation (ODE) model of megalin trafficking describing surface and internalized pools of megalin with kinetic parameters estimated from experiments using this system. Using biochemical techniques, we have estimated endocytic and recycling rates and the half‐life of surface megalin. The model is capable of achieving a steady‐state with a much larger pool of internalized megalin compared to surface within a range of realistic parameters; this is consistent with observations made by our biochemical assays and by indirect immunofluorescence of endogenous megalin. Future work includes estimated surface delivery kinetics and defining the structure and markers of the apical endocytic pathway in OK cells and mouse kidney sections using quantitative imaging. With these data, our model can be expanded and used to predict changes in megalin trafficking in response to changes in filtration rates, hormones, and in disease states.

Support or Funding Information

NIH R01 DK101484, R01 DK100357

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Training Heart Failure Patients with Reduced Ejection Fraction Attenuates their Muscle Metaboreflex and Lowers Muscle Sympathetic Nerve Activity at Rest and During Mild Dynamic Exercise
Authors: Notarius Catherine Frances , Millar Philip J. , Keir Daniel A. , Murai Hisa , Haruki Nobuhiko , O'Donnell Emma , Marzolini Susan , Oh Paul , Floras John S.
Published: June 2021

We have shown that muscle sympathetic nerve activity (MSNA) is elevated at rest in patients with heart failure due to reduced ejection fraction (HFrEF) and, in contrast to healthy subjects, increases during moderate dynamic 1‐leg exercise. We hypothesized that exercise training in HFrEF may reduce MSNA during both mild and moderate intensity 1‐leg dynamic exercise and static handgrip; attenuate the muscle metaboreflex; and improve skin blood flow. We studied 27 stable, medicated HFrEF patients (6 women; mean age 65 ± 2 SE [range 42 to 80] years; mean left ventricular ejection fraction 30 ± 1% [range 12–40]); of either ischemic (n=18) or non‐ischemic (n=9) etiology, both before and after 6 months of cardiac rehabilitation (aerobic exercise for 45 minutes, 5 times per week at 60–70 % of VO2peak); plus 18 healthy, age and sex‐matched, medication‐free volunteers (6 women; mean age 57 ± 2 [range 48 to 72]). Before and after training, we assessed VO2peak (open‐circuit spirometry); the skin blood flow (SBF) response to reactive hyperemia (laser flowtometry); and fibular MSNA (microneurography) at rest (n=18), during 1‐leg cycling (2 min each unloaded and at 50% of VO2peak, n=16) and, to assess the muscle metaboreflex, during post‐handgrip ischemia (PHGI) after 2 min of 30% static handgrip (HG). In HFrEF, pre‐training VO2peak and skin vasodilatation were lower (P<0.001) and resting MSNA higher compared to controls (P=0.01). Exercise training improved VO2peak by 3.0±1.0 ml·kg−1·min−1 (P<0.001) and increased the SBF vasdilatory response in HFrEF to a mean value similar to controls (P=0.78). Relative to pre‐training, resting MSNA decreased by 6.0±2.0 bursts/min (P=0.01, N=18) and 7.3±3.4 burst/100 heartbeats (P=0.04). During 1‐leg cycling, MSNA decreased in control (P=0.01) but increased in HFrEF (P<0.001). Exercise training attenuated MSNA burst frequency and incidence during unloaded (P=0.04) but not loaded cycling (P=0.34). The rise in MSNA burst frequency during HG in HFrEF was unaffected by training, but its elevation during PHGI was abolished (P<0.05). We conclude that the exaggerated metaboreflex of HFrEF is attenuated by exercise training. This may contribute to a lower MSNA during mild but not moderate intensity cycling.

Support or Funding Information

Supported by Heart and Stroke Foundation of Ontario and Canadian Institutes for Health Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Sildenafil Treatment Improves Exercise Capacity in Patients with Cystic Fibrosis
Authors: Gonzalez Vincent , Siegler Nichole , Crandall Reva , Mckie Kathleen T. , Forseen Caralee , Rodriguez‐Miguelez Paula , Tucker Matthew , Harris Ryan A.
Published: June 2021

Background

Exercise capacity has been shown to predict quality of life, mortality, and risk for hospitalization in patients with Cystic Fibrosis (CF). Many patients with CF exhibit exercise intolerance, the mechanism of which has yet to be elucidated. Sildenafil, a phosphodiesterase type 5 inhibitor, increases blood flow and therefore may improve exercise capacity in patients with CF.

Purpose

The purpose of this study was to determine whether an acute dose of sildenafil improves exercise capacity in patients with CF.

Methods

13 patients with CF (mean age = 25 ± 10 years) participated in a double blind, randomized, placebo controlled study. Exercise capacity utilizing the Godfrey Protocol on a cycle ergometer was assessed at baseline and 1 hour following treatment with either sildenafil (50 mg) or placebo.

Results

A significantly greater increase (p = 0.024) in VO2 peak was observed following sildenafil treatment (4.16 ± 1.54 ml/kg/FFM) compared to placebo (1.09 ± 1.31 ml/kg/FFM). The change in maximal heart rate during exercise was statistically (p = 0.007) greater following sildenafil treatment (6.07 ± 4.04 bpm) compared to placebo (−1.54 ± 6.06 bpm). The change in chronotropic index (CRI) was significantly (p = 0.006) higher following sildenafil treatment (0.08 ± 0.02) compared with placebo (0.04 ± 0.02).

Conclusion/Interpretation

These findings suggest that an acute dose of sildenafil improves exercise capacity in patients with CF. In addition, treatment with sildenafil improved CRI. Future studies should focus on elucidating the mechanisms for improvement in exercise capacity and CRI as both are predictors of mortality.

Support or Funding Information

Supported in part by the Medical College of Georgia's Medical Scholar Program and the Laboratory of Integrative Vascular and Exercise Physiology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Low‐load Resistance Exercise with Blood Flow Restriction Changes Hypoxia‐Induced Genes Expression
Authors: Laurentino Gilberto , Aoki Marcelo , Fernandes Rodrigo , Soares Antonio , Ugrinowitsch Carlos , Hoschel Hamilton , Tricoli Valmor
Published: June 2021

PURPOSE

To investigate the effects of low‐load exercise with blood flow restriction on expression of genes related to hypoxia.

METHODS

Twenty‐four male (mean ± SD: age 21.3 ± 1.9 years, height 1.74 ± 0.8 m, body mass 73 ± 1.8 kg) were divided into three groups: low‐load exercise (LL, n=9), low‐load exercise with blood flow restriction (LL‐BFR, n=8), and high‐load exercise (HL, n=7). For LL‐BFR group a pneumatic cuff was placed on the proximal portion of the thigh and inflated at 80% of the resting arterial occlusion pressure. All subjects performed bilateral knee extension, 2x/week, during 8 weeks. LL and LL‐BFR groups performed 3–4 sets of 15 reps at 20% 1RM whereas HL performed 3–4 sets of 8–10 reps at 80% 1RM with 60s rest interval between sets. Regulated in development and DNA damage response 1 (REDD1), hypoxia‐inducible factor‐1‐alpha (HIF‐1‐alpha), vascular endothelial growth factor (VEGF), and neuronal nitric oxide synthase (nNOS) mRNA gene expression were assessed before and after training. Mixed model for repeated measures was performed for each dependent variable, and statistical significance was set at p≤ 0.05.

RESULTS

REDD1 expression decreased in all groups from pre‐ to post‐training (p<.0001), and significantly lower REDD1 expression was found in both LL‐BFR and HL groups when compared with LL group at post‐training (p<0.05) [Figure 1A). HIF‐1α mRNA expression increased in all groups from pre‐ to post‐training (p<.0001), and significantly greater values were observed in LL‐BFR and HL groups when compared with LL group at post‐training (p<.0001) [Figure 1B]. VEGF mRNA expression increased in both LL‐BFR and HL groups from pre‐ to post‐training (p<.0001); however, only LL‐BFR showed greater expression than LL group at post‐training (p=0.001) [Figure 1C). nNOS mRNA expression increased in all groups from preto post‐training (p<.0001), and greater expression values were observed in LL‐BFR when compared with both LL and HL groups at post‐training (p<.0001) [Figure 1D].

CONCLUSION

Low‐load resistance exercise with blood flow restriction induced changes in selected genes expression related to hypoxia following training. These results may contribute with the skeletal muscle hypertrophy responses and increased muscular angiogenesis after training period.

Support or Funding Information

This study was not supported by any funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autophagy promotes cancer chemotherapy‐induced oxidative stress and skeletal muscle dysfunction
Authors: Smuder Ashley J. , Kwon Oh Sung , Hain Brian A. , Houston Fraser E. , Talbert Erin E.
Published: June 2021

Chemotherapy‐induced cachexia is a debilitating condition characterized by fatigue and muscle wasting. The onset of muscle weakness in patients often limits treatment, resulting in reduced quality of life and increased morbidity. Our previous findings show that the chemotherapeutic agent doxorubicin (DOX) causes myotoxicity as a result of reactive oxygen species (ROS) generation during intracellular metabolism of DOX by the mitochondria, and subsequent activation of the autophagy/lysosomal system. We hypothesize that DOX‐induced accelerated autophagy results in the elimination of vital cellular organelles and proteins, resulting in functional deficits to the muscle tissue. To prevent elevated activity of the autophagy/lysosomal system, we administered a dominant negative mutation of the autophagy protein ATG5 (rAAV‐dnATG5) via intramuscular injection of the rat soleus muscle four weeks prior to treatment. Animals received either DOX (20 mg/kg ip) or saline. Two days after chemotherapy, soleus muscle specific force production and fiber cross‐sectional area were significantly reduced in control animals. These changes corresponded with deficits to mitochondrial respiration, increased mitochondrial ROS emission and the increased appearance of autophagic vacuoles within the muscle. In contrast, rAAV‐dnATG5 administration in DOX treated animals prevented the conjugation of ATG12 and ATG5 thereby reducing autophagosome formation within the soleus muscle. Interestingly, the protein expression of catalase and superoxide dismutase 2 (SOD2) was elevated in these animals, which may account for improved mitochondrial respiration and reduced mitochondrial free radical production. Finally, DOX‐induced muscle fiber atrophy and contractile dysfunction were prevented in rAAV‐dnATG5 animals. These findings indicate that accelerated autophagy is an underlying cause of chemotherapy‐induced oxidative damage and skeletal muscle weakness.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Mago Homology B (Magohb) in Skeletal Muscle
Authors: Zubair Samira , Waddell David
Published: June 2021

Skeletal muscle atrophy is a physiological condition that occurs due to immobilization, denervation, spinal cord transection and aging. Muscle wasting causes significant loss of muscle size, a measurable reduction is strength, and decreased mobility. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Mago Homology B (Magohb), an exon junction core complex component involved in splicing and mRNA surveillance, is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that Magohb is expressed in muscle cells, quantitative PCR (qPCR) was used to assess Magohb expression levels in both proliferating and differentiated muscle cells and the results demonstrate that Magohb is expressed in both proliferating myoblasts and differentiated myotubes. In order to characterize the transcriptional regulation of Magohb, fragments of the proximal promoter located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Furthermore, bioinformatic analysis of the promoter region of Magohb revealed a conserved canonical E‐box element, which is a known myogenic regulatory factor (MRF) binding site, further suggesting that Magohb may be regulated by muscle specific transcription factors. Finally, since Magohb is believed to play a role in exon‐exon junction identification and may play a role in mRNA splicing, we fused Magohb to GFP, transfected mouse myoblasts with the Magohb‐GFP expression plasmid, imaged the cells using confocal microscopy, and confirmed that Magohb is predominately localized to the nucleus. The discovery that Magohb is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of mRNA processing, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants to D.W.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Regulation and Function of Tetratricopeptide 39c (Ttc39c) in Skeletal Muscle
Authors: Hayes Caleb , Waddell David
Published: June 2021

Ttc39c has been identified as a novel gene in skeletal muscle that is differentially regulated in response to neurogenic atrophy in wild‐type (WT) mice compared to Muscle RING Finger 1 (MuRF1) knockout (KO) mice. MuRF1 is a ubiquitin E3 ligase that is known to be an important mediator of muscle wasting; however, more recent research suggests that MuRF1 may also function as a regulator of atrophy‐induced gene expression. Quantitative PCR and Western blot analysis determined that Ttc39c is expressed in both proliferating and differentiated muscle cells. Furthermore, comparison of Ttc39c expression in undifferentiated and differentiated C2C12 cells demonstrated that Ttc39c levels are elevated in proliferating myoblasts, but decrease in differentiated myotubes. The transcriptional regulation of two Ttc39c isoforms with alternative transcriptional start sites were examined by cloning the unique promoter regions of each transcript. The Ttc39c reporter gene constructs were transfected into muscle cells and confirmed to have significant transcriptional activity in cultured muscle cells and are transcriptionally responsive to ectopic expression of myogenic regulatory factors (MRF). Furthermore, conserved E‐box elements in the proximal promoter regions of both Ttc39c transcripts were identified, mutated, and analyzed for their role in the transcriptional regulation of Ttc39c expression. The mutation of the conserved E‐box sequences rendered the Ttc39c reporter genes inactive, suggesting that these elements are potentially necessary for Ttc39c expression. Finally, immunoprecipitation (IP) of Ttc39c from C2C12 cells resulted in the co‐IP of numerous binding partners, which are currently being identified by proteomic analysis in the hope of elucidating a potential function for Ttc39c in skeletal muscle based on identified binding partners. Interestingly, ectopic expression of Ttc39c in C2C12 mouse myoblasts resulted in delayed cell fusion and reduction in myotube formation. The identification and characterization of novel genes that are activated during neurogenic atrophy helps improve our understanding of the molecular genetic events that result in muscle wasting and could eventually lead to new therapeutic targets in the future.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants to D.W.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Estrogen on Statin‐Induced Myotoxicity in Vitro
Authors: Roussel Jaclyn , Stambersky Ashten , Ball Karen L.
Published: June 2021

In vitro studies have suggested a greater statin‐induced myotoxicity in females. This is in contrast to what would be predicted based on estrogen (E) protection against other models of muscle damage. Therefore, the purpose of this study was to examine estrogen effects in an in vitro model of statin‐induced myotoxicity. C2C12 myoblasts were plated at a density of 0.15×105 cells in a 6‐well plate and maintained in growth media (GM) until 80%–100% confluence. Media was then replaced with differentiation media (DM) and cells allowed to differentiate for seven days. Differentiated cells were then exposed to Simvastatin (SS;10−4 mM), E alone (0.1 μM or 0.01 μM), or SS+E. After 48 hours, light microscopic images were taken to measure multinucleated cell numbers and fiber diameters. At 72 hours, immunofluorescence was performed to identify myosin positive cells and apoptotic nuclei. The SS induced toxicity was consistent with previous studies. Fiber diameters were decreased (45.7±4.1 μm vs 16.0±1.8 μm*) with significant increase in apoptotic nuclei (17%±1.4% vs 55.3%±4.3%*). Interestingly, the effects of E in combination with SS were dependent on the parameters measured. Whereas E produced a protective trend with greater fiber diameters (23.7±5.4 μm vs 16.0±1.8 μm;NS), E exacerbated the SS impact on apoptosis (74%±6.4%–87%±8.1% vs 55.3%±4.3%*). In conclusion, our data revealed contradictory influences of E on statin‐induced myotoxicity. Clarification of estrogen's effect could have significance in understanding gender specific adverse clinical responses to stain therapy. *p<0.05

Support or Funding Information

The Herbert H. and Grace A. Dow Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genetically Engineering a Sheep Model of Hypophosphatasia
Authors: Williams Diarra Kevin , Pinzon Carlos A. , Huggins Shannon , Pryor Jane H. , Georges Hanah M. , Hermann Forrest , Oldeschulte James , Westhusin Mark E. , Long Charles R. , Gaddy Dana , Suva Larry J.
Published: June 2021

Hypophosphatasia (HPP) is a rare inherited disorder that affects the development of bones and teeth. The disease is caused by mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene (ALPL) and accompanied by a highly variable clinical presentation. Although HPP patient studies have advanced our understanding of HPP, as well as documented disease severity, the rarity of the disease combined with a lack of sufficient animal models has significantly delayed mechanistic understanding and therapeutic development. Similar to other human disease models, current HPP models have been engineered virtually exclusively in rodents – specifically mice harboring null or loss‐of‐function mutations. Although useful for modeling some features of HPP, these murine models do not faithfully represent the broad spectrum of human HPP clinical bone, muscle and tooth (odonto‐HPP) phenotypes. However, in sheep both Haversian bone remodeling and tooth development are analogous to humans with sheep TNSALP amino acid sequence sharing 89% identity with human. Thus, our objective was to generate a sheep model of HPP and we posit that ALPL mutation‐specific replacements in the sheep genome using CRISPR/Cas9 will produce a model that accurately phenocopies the bone and tooth pathophysiology of HPP. To genetically engineer a sheep model of HPP, a total of 52 in vitro genetically manipulated embryos were generated targeting the Exon 10 c.1077G>A mutation using CRISPR/Cas9. Three embryos were implanted per recipient ewe and implantations were performed in a total of 17 recipient ewes. Nine ewe pregnancies were confirmed by the measurement of Pregnancy‐Associated Protein on post‐implantation day 35 and further validated by ultrasound. From 9 live newborn lambs, four were heterozygous for our specific point mutation (4/6), one compound heterozygous (1/6) and one homozygous (1/6) for a total mutation efficiency rate of 66.6% (6/9). Similar to HPP patients with the same mutation, mutant sheep have variable clinical expression accompanied with decreased bone formation and mineralization in tails observed by DXA as well as significantly and appropriately decreased serum alkaline phosphatase activity. Additionally, an apparent “shell tooth” phenotype is observed in mutant lambs, further suggesting a mutant exon 10 genotype/phenotype that more completely recapitulates human skeletal and odonto‐HPP.

Support or Funding Information

1. William Townsend Porter Pre‐doctoral Fellowship

2. TAMU/Texas Heart Institute Center for Cell and Organ Biotechnology Innovation Kitchen Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Status Of Protein Translation In A Metabolically Flexible Mammal, The Common Tenrec, Tenrec ecaudatus
Authors: Wilson Jared Nicholas , van Breukelen Frank
Published: June 2021

Common tenrecs, Tenrec ecaudatus, are small tropical hibernating mammals endemic to Madagascar. Tenrecs possess many phenotypic features associated to ancestral mammals, suggesting they may act as a model of an ancestral form of hibernation. Common tenrecs are able to hibernate at ambient temperatures ranging from 12 to 28 °C and do not undergo brief periodic returns to euthermy during the hibernation season, a shared feature among all other known hibernators. Tenrecs display extreme metabolic flexibility. For instance, they can spontaneously enter torpor during the active season, termed facultative torpor (FT). During both hibernation and FT, oxygen consumption rates have been observed to be 1–14% of active rates depending on ambient temperatures. Protein metabolism may account for 30% or more of standard oxygen consumption rates for most animals. Polysome profiles were used to determine the status of translation in tenrecs as a function of core body temperature (Tb) and activity state. Our findings will provide mechanistic data that will be used to elucidate how common tenrecs are able to regulate metabolic flux exclusive of Tb.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Friend or Family? Social Recognition and Physiological Stress in the Self‐Fertilizing Mangrove Rivulus Fish (Kryptolebias marmoratus)
Authors: Trueman Justin D. , Currie Suzie
Published: June 2021

Social behavior has the capacity to influence how animals respond to environmental stress. Living in social groups may be beneficial to animals as it can reduce predation risk and provide foraging benefits. Advantages may be further enhanced if animals associate with related individuals (kin), with individuals of the same phenotype and/or with familiar conspecifics, regardless of relatedness. On the other hand, grouping may incur costs if there is competition for resources or increased aggression. Given the potential influence of sociality on physiological responses to stress, it is important to understand an animal's social behavior and if certain social interactions result in physiological stress. The mangrove rivulus is a tropical, self‐fertilizing fish that forms isogenic lineages, providing a model fish with a natural and extreme form of kin. Using distinct strains of rivulus originating from Belize and Honduras, we predicted that rivulus would use both relatedness and familiarity as cues to recognize conspecifics. We used a choice chamber and in two separate experiments, gave focal fish of each strain the choice between related or familiar stimulus fish. We took water samples to determine cortisol concentrations, as we predicted that association with non‐kin and/or unfamiliar fish would be stressful and result in increases is the stress hormone. We found no significant differences in the proportion of time either lineage spent with familiar or related conspecifics. We further analysed association time for aggressive behaviour as we predicted that rivulus would be less aggressive to both familiar and related conspecifics. Interestingly, we determined that the Belize lineage is not aggressive towards related or unrelated individuals whereas the Honduran lineage is aggressive to unrelated conspecifics only. Collectively, our data suggest that rivulus detect conspecifics using both familiarity and relatedness; kin discrimination is not universal and appears to have a genetic basis. My further analyses will determine whether or not association with non‐kin and/or unfamiliar individuals is physiologically stressful.

Support or Funding Information

Research supported by APS UGSRF and Mount Allison University. Cortisol analysis is in collaboration with Dr. Ryan Earley, University of Alabama. Research supervised by Dr. Suzie Currie.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Do Body Shape/Proportions predict injury? Novel insights from a 3D body scan imaging.
Authors: Talty Kevin , Thomas Diana M. , Kuiper Patrick , Morse Steven , Bigelman Kevin , Heymsfield Steven , Scioletti Michael
Published: June 2021

Background

Posture, obesity, and body shape are well‐established predictors of injury and athletic performance. However, due to the manual burden in collecting numerous anthropometric measures, to date, no large database and systematic data analysis determining body shape effects on performance and injury have gone beyond using standard measurements like BMI, waist circumference and hip circumference.

Objective

To use machine learning based Artificial Neural Network (ANN) and decision tree analysis on a large nationally representative database of automatically captured body anthropometrics to predict basic training‐related injury.

Methods

Over N=20,896soldiers (28% female) recruited for US Army basic training at Fort Jackson, SC were scanned for uniform fitting using the Human Solutions Kinect based 3D imaging technology. Each subject image consisted of 161 body shape measurements. After removing subjects with missing measurements, the remaining subjects were split into injured (91 recruits) and non‐injured (13,296 recruits) groups. 75% of each group was placed into a training group and the other 25% into a testing group. An ANN and Decision Tree model evaluated using the area under the curve(AUC) was developed to predict severe physical injury occurrence during 10 weeks of US Army basic training. Body length measurement was compared against height to evaluate whether certain body proportions were more at risk for injury.

Results

The AUC for the ANN was 0.79 and the AUC for the Decision Tree model was 0.70. It was determined that body proportions of shorter legs and longer torso more than doubled the risk of injury during US Army basic training

Conclusions

Machine learning models that leverage data sources like those from the newly emerging 3D body image scanners may be used to predict severe injury during vigorous physical activity. These results can be used to develop personalized prevention strategies reducing injury and retaining individuals within their activity protocols.

Support or Funding Information

None

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the Roles of Novel Endogenous Ligand of Aryl Hydrocarbon Receptor in Neural Development
Authors: Chuang Pei‐Yun , Chan Ya‐Yun , Wu Pei‐yi , Chen Pei‐Jen , Lee Hsinyu
Published: June 2021

Activation of aryl hydrocarbon receptor (AHR) by xenobiotic toxic chemicals such as 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin regulates a variety of cellular processes including embryogenesis, tumorigenesis and inflammation. However, the identity of endogenous AHR ligands remains elusive. A potential AHR ligand, compound X, was identified from zebrafish embryos using previous developed cell free bioassay for dioxin‐like compounds. In this study, we aim to investigate the effect of compound X in AHR activation and further explore its physiological effects. Compound X induces AHR translocation into nucleus and upregulates downstream cytochrome P450 1A1 (CYP1A1) in vitro. It also promotes neuronal differentiation in neuroblastoma (NB) cells, which is consistent with our previous studies that overexpression of AHR leads to NB cells differentiation. Furthermore, compound X enhances zCyp1a, myelin‐associated myelin basic protein (zMbp) and SRY (sex determining region Y)‐box 10 (zSox10) expression and improves the mobility of zebrafish larvae via Ahr2 pathway. Therefore, our results suggest that compound X is an endogenous AHR ligand, and plays a critical role during neural differentiation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myosin Light Chain Kinase‐210 Induces ER‐PM Junctions and STIM1 Puncta Formation to Augment Store‐Operated Ca2+ Entry
Authors: Srivastava Nityanand , Mohammad Tauseef , Amin Ruhul , Joshi Jagdish Chandra , Klomp Jennifer , Li Wennan , Kini Vidisha , Knezevic Nebojsa , Obukhov Alexander G. , Karginov Andrei , Komarova Yulia , Mehta Dolly
Published: June 2021

Myosin light chain kinase‐Long (MYLK‐L or MYLK‐210), a Ca2+‐calmodulin dependent kinase containing a unique 922‐amino acid N‐terminal domain is a predominant isoform of MYLK expressed in endothelial cells (ECs). N‐terminus of MYLK‐210 has been shown to contain several single nucleotide polymorphisms (SNPs) in an actin binding region which is associated with severity of acute lung injury (ALI). However, significance of SNPs in regulating MYLK‐210 function remains unclear. The classical function of MYLK‐210 is to catalyze the phosphorylation of myosin light chain to induce EC contraction, leading to inter‐endothelial gap formation and increase permeability, a hall mark of ALI. Intriguingly, we showed that MYLK‐210, through its actin‐binding motif, regulates store‐operated Ca2+ entry (SOCE) but not receptor‐operated Ca2+ entry (ROCE). We showed that MYLK‐210 null endothelial cells (ECs) or MYLK‐210 depleted ECs displayed diminished Ca2+ entry in response to SOCE‐activator thapsigargin and thrombin. We generated MYLK‐210 constructs lacking C‐terminus (N‐MYLK) or N‐terminus (C‐MYLK) to investigate the role of specific domains of MYLK‐210 in inducing SOCE activity. We found that N‐MYLK but not C‐MYLK rescued SOCE activity in MYLK‐210 null ECs, indicating thereby that MYLK regulates SOCE in a kinase independent manner. Additionally, SOCE activation failed to increase lung vascular hyper‐permeability in MYLK‐210 null mice. We identified unique actin binding motif, DVRGLL within N terminus of MYLK‐L which enables MYLK‐210 to induce SOCE. Stromal‐interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta which translocate to plasma membrane that trigger SOCE via plasmalemmal Ca2+selective channels such as transient receptor potential channel 1 (TRPC1) and Orai1. Thus, we addressed if MYLK‐L controlled STIM1 function by regulating puncta formation. We showed that STIM1 formed puncta in control ECs but these puncta were markedly reduced in MYLK depleted ECs. Formation of endoplasmic reticulum (ER)‐plasma membrane (PM) junction is required for STIM1 activation of SOCE. Using MAPPER (membrane‐attached peripheral ER) that selectively labels ER–PM junctions, we found that MYLK induced ER‐PM junction, but these junctions were markedly reduced in the density in MYLK‐knockdown cells. Our study thereby defines for the first time the clinical validity of SNPs within actin binding region of MYLK in inducing ALI through organizing ER‐PM junctions, STIM1 activity, SOCE and lung vascular hyperpermeability.

Support or Funding Information

National Institute of Health (NIH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.