bluuebunny/crossref_metadata_2025 huggingface dataset:
Catalase over‐expression protects dystrophic skeletal muscle
Authors: Selsby Joshua Taylor , Tian Zouzhen , Barton Elisabeth R. , Sweeney H. Lee
Published: June 2021

Duchenne muscular dystrophy (DMD) is a fatal muscle disease caused by the absence of a functional dystrophin protein and is modeled by the mdx mouse. Oxidant stress has been previously reported in dystrophic skeletal muscle. Efforts to target this have been largely unsuccessful both in mdx mice and in early clinical trials. In an effort to reduce oxidant stress and improve muscle function, AAV 2/8 was used to over‐express the antioxidant catalase (Cat) in single hind limbs of mdx mice while the contralateral limbs served as control (Con). Animals were injected within the first five days of life with 1×1012 gc of AAV‐Cat and killed at 4 (n=6) or 6 wks of age (n=5), during and following the initial necrotic bout. At 4 and 6 wks, specific tension was increased in the Cat EDL by 15% (p<0.05), while the soleus remained unchanged. The EDL was 30% more resistant to damage caused by eccentric contractions at 6 wk (p<0.05), but not at 4 weeks and the soleus was more fatigue resistant at both time points. These data show that an effective antioxidant can protect dystrophic skeletal muscle and improve muscle function. Further, they may also serve as the ground work for future clinical trials using more powerful and/or appropriate antioxidants or antioxidant cocktails.

Supported by F32AR055005‐01 and U54AR052646‐03
Blockade of glycine receptors modifies temporal and spectral characteristics of phrenic nerve discharge in urethane‐anesthetized adult rat in vivo
Authors: Ryu Yena , Chun Hyun Hye , Poole Oneica N. , Chang Hannah , Solomon Irene C.
Published: June 2021

The neuronal mechanisms that underlie fast oscillatory rhythms in inspiratory‐related discharges are poorly understood; however, accumulating evidence suggests that fast synaptic inhibition may be amongst the mechanisms participating. In support of this idea, recent work from our laboratory in the urethane‐anesthetized adult rat preparation has shown that blockade of GABAA receptors modulates both the power and timing of spectral activity in phrenic nerve bursts. The role of glycine‐mediated synaptic inhibition requires clarification. Therefore, we examined the effects of glycine receptor antagonism, using cumulative increasing doses strychnine hydrochloride (STR; 0.2–10 mg/kg, iv), on phrenic nerve discharge in urethane‐anesthetized vagotomized adult rats (n=5). STR significantly decreased TI (from 325±43 to 208±24 ms; P=0.007) and TE (from 965±88 to 878±88 ms; P=0.031), resulting in an ~17% increase in burst frequency (P<0.004). STR also increased power of the MFO (by 123±61%) and HFO (by 83±65%) spectral peaks albeit this increase was not significant. Inspiratory network complexity (quantified by approximate entropy), however, was significantly reduced from 0.709±0.016 to 0.602±0.0198 (p=0.004). These findings suggest that glycine‐mediated synaptic inhibition may contribute to timing and spectral activity inherent in phrenic discharges in vivo. Supported by NS045321 and NS049310
A grid service‐based workflow for in‐silico screening of chemical inhibitors of phosphatases
Authors: Levesque Marshall , Chien Shu , Haga Jason
Published: June 2021

In many cases, grid computing offers the powerful alternative of sharing resources on a worldwide scale, across a number of different institutions to run computationally intensive applications without the need for a large, centralized supercomputer. This paper presents a scalable, reusable workflow assembled to choreograph large virtual screening experiments over a computational Grid using the molecular docking application DOCK. Software components were applied on multiple levels to create automated workflows that perform data delivery, job scheduling, job status query, and the collection of results to be displayed in a manageable fashion for further analysis. This was achieved using Opal OP to wrap the application as a Grid service and PERL for data manipulation purposes, alleviating the requirement for extensive knowledge of Grid infrastructure and security. With the workflow in place, a screening was performed with the ZINC 2,066,906 ligand “druglike” subset database against the catalytic site of the phosphatase SHP‐1 using the MPI version of DOCK 5.4 on the PRAGMA Grid testbed, utilizing 200 processors over 7 clusters. This is the first successful use of Grid computing to identify chemical inihibitors of enzymatic activity through virtual screening.

This project was supported by PRIME (NSF 0407508), PRIUS, PRAGMA, and Calit2.
Glucocorticoids Activate Ubiquitin Transcription in Muscle By Suppressing PI3‐Kinase: Implications for Muscle Atrophy
Authors: Price S. Russ , Zheng Bin
Published: June 2021

Muscle atrophy is a consequence of chronic systemic diseases (e.g., diabetes) that results from muscle‐specific activation of the ubiquitin‐proteasome (UbP) pathway. Glucocorticoids are required for these transcriptional responses. We previously demonstrated that glucocorticoids increased the transcription of the Ubiquitin C (UbC) gene in L6 myotubes by a mechanism involving MEK1 and the Sp1 transcription factor. Presently, we examined how glucocorticoids activate MEK1 and UbC transcription. Dexamethasone (Dex; 100nM) increased UbC promoter‐driven luciferase activity by 205±7% after 24 h and by 310±6% after 48 h (both are P<0.05 vs control). In the time course requiring ≥ 24h, Dex increased MEK/ERK signaling, induced Sp1 phosphorylation and suppressed PI3‐kinase (PI3K)/Akt signaling. To test if inhibition of the PI3K pathway reroutes cell signaling to the MEK/ERK pathway, cells were infected with a PI3K p85 subunit to suppress PI3K activity. MEK1/2 phosphorylation and UbC promoter activity were increased. Thus, we propose that glucocorticoids increase MEK signaling and UbC transcription by an indirect mechanism involving suppression of the PI3K pathway. Our findings underscore the multifaceted role that glucocorticoids play in muscle atrophy due to chronic systemic diseases associated with increased glucocorticoid production, insulin resistance and muscle wasting. Supported by NIH DK61521.
Expression of IGF‐1a and change in muscle fiber area after 12 weeks of resistance training in elderly postoperative patients
Authors: Suetta Charlotte , Clemmensen Christoffer , Andersen Jesper L , Magnusson S. Peter , Schjerling Peter , Kjaer Michael
Published: June 2021

The mRNA splice variant of insulin‐like growth factor‐1a and the changes in muscle fiber cross sectional area after 12 weeks of rehabilitation, were studied in elderly postoperative patients. 28 subjects (age 60–86 yrs) were randomized to either resistance training (RT: 3/wk·12 wks), electrical stimulation (ES: 1 h/day·12 wks) or functional exercises (FE: 1 h/day·12 wks). Muscle biopsies were obtained from vastus lateralis (VL) of both legs at baseline, 5 and 12 wks post‐surgery to analyze for changes in mean muscle fiber area (MFA). Expression levels of IGF‐1a mRNA were determined using real‐time RT‐PCR and normalized to the Ribosomal Protein Large P0 (RPLP0) mRNA. There was no difference in MFA or resting levels of IGF‐1a between the three groups at baseline. RT resulted in a significant increase in IGF‐1a (+131 %, p<0.05) and MFA (+37%, p<0.05). In contrast, 12 weeks of ES and FE did not induce any change in MFA or in the expression of IGF‐1a. The present study is the first to demonstrate that resistance training initiated in the acute (1–2 days) postoperative phase is highly effective in increasing mean fiber area and in addition induced a marked increase in the expression of IGF‐1, supporting the idea that IGF‐1a may be involved in regulating muscle hypertrophy.

Funded by the IMK Foundation and the Maersk McKinney Moller Foundation
Effect of fasting on resting and diving metabolic rate in Steller sea lions (Eumetopias jubatus)
Authors: Svärd Caroline Elisabeth , Fahlman Andreas , Joy Ruth , Rosen Dave , Trites Andrew
Published: June 2021

The metabolic trade off between thermoregulation and fasting was assessed for foraging Steller sea lions. Pre‐dive resting (RMR) and diving (dive + surface) metabolic rates (DMR, 1 O2 · min−1) were measured for dives ranging between 20 to 50 m in 3 female Steller sea lions (mean initial body masses, Mb: 144.3, 167.4 and 219.9 kg) before and after a 9‐day fast. The sea lions lost an average of 9.8 % of their body mass (Mb) or 1.1 % per day. Dive duration and dive depth did not significantly affect DMR (p > 0.1). Average RMR and DMR before fasting ranged between 0.71 and 1.98 O2 • min−1 and was positively correlated with Mb (r2 = 0.97, p < 0.05). Both RMR (36.3 ± 5.22 %) and DMR (14.2 ± 12.3 %) decreased after fasting. The smaller decrease in DMR compared to RMR indicates that the cost of thermoregulation increased during diving compared to resting. RMR tended to increase in fasted sea lions within 1 hour of feeding, but was not observed in fed sea lions (p = 0.07). This suggests that digestion of food started while diving and had a greater energetic priority than dive performance in fasted animals.

(Support: North Pacific Marine Science Foundation and NOAA; SC supported by a graduate student grant from the Swedish Government and by travel funds from SEB).
Chronic Treatment with N‐acetylcysteine (NAC) Improves Cardiovascular Function in Syrian Cardiomyopathic Hamsters
Authors: Cruz Nildris , Escobales Nelson , Altieri Pablo I , Crespo Maria J
Published: June 2021

It has been postulated that oxidative stress is a contributing factor in the onset and development of heart failure (HF). To further evaluate this point, one‐month‐old Syrian cardiomyopathic hamsters (SCH) were treated for a 5‐month period with N‐acetylcysteine (NAC, 1g/kg/day). Vascular responses for acetylcholine (Ach), norepinephrine (NE) and Ang II were assessed in aortic rings from 6‐month‐old treated and untreated SCH. Systolic blood pressure (SBP), superoxide production in aorta and heart tissue (determined by lucigenin), and echocardiographic parameters were also evaluated. Chronic treatment with NAC improved 24% (P<0.05) the Ach‐induced relaxation (EMax value from 45.8±4% to 56.1±2% n=7) but did not modify EC50 values, NE and Ang II‐induced contraction, or SBP. Vascular superoxide production was reduced 50% by NAC treatment. Cardiac superoxide concentration, however, remained unchanged (1281±94 cpm/mg vs. 1275±82 cpm/mg, P>0.05). Ejection fraction increased from 39±4% in untreated SCH to 57±4% (n=11, P<0.05), and left ventricular end‐diastolic and end‐systolic volumes significantly decreased (P<0.05). Cardiac output index improved although did not reach statistical significance. These results suggest that antioxidant therapy alone improves cardiovascular function but does not prevent the appearance of HF in this animal model. Supported by NIH 2 SO6‐GM 08224.
Adventitial Hydrogen Peroxide (H2O2) Impairs Rat Carotid Artery Relaxation via Medial p38 MAPK
Authors: Cascino Thomas M. , Haurani Mounir J. , Pagano Patrick J.
Published: June 2021

The vascular adventitia is a major source of H2O2. H2O2 activates smooth muscle p38 mitogen activated protein kinase (p38 MAPK). We postulated that adventitia‐derived H2O2 impairs relaxation via medial kinase activation. Isolated rat carotid arteries were bathed perivascularly (in situ) over 90 min in vehicle, angiotensin II (AngII, 500 nM), AngII + H2O2‐scavenger catalase or AngII + p38 MAPK inhibitor, then placed in myographs and preconstricted. Both acetylcholine (ACh)‐induced endothelium‐dependent and sodium nitroprusside (SNP)‐induced endothelium‐independent relaxations were impaired by AngII (p < 0.05). Catalase partially reversed the AngII effect on ACh‐induced relaxation and reversed impairment of SNP‐induced relaxations (p < 0.05). The p38 MAPK inhibitor SB203580 partially ameliorated the impairment of SNP‐induced relaxations and reversed the AngII effect on ACh‐induced relaxation (p < 0.05). In separate pretreated vessels, the media was dissected, homogenized and Western blots run for phosphorylated vs. total p38 MAPK. AngII activated medial p38 MAPK, but catalase inhibited this. These data suggest AngII elicits release of adventitial H2O2, impairing both endothelium‐dependent and –independent relaxation via the p38 MAPK pathway in the media. The data provide evidence of a significant paracrine signaling role of vascular adventitial H2O2 on vascular function.
Selenoprotein N and Oxidative Stress: Characterisation of the role of a novel selenoprotein
Authors: Arbogast Sandrine , Ferreiro Ana
Published: June 2021

Mutations of the selenoprotein N gene (SEPN1) have been identified as responsible for SEPN‐related myopathy (SEPN‐RM), an early‐onset muscle disorder. Selenoprotein N is the only selenoprotein implicated in a human genetic disorder but its function remained unknown. SelN has structural similarities with other selenoproteins which are involved in redox homeostasis. Therefore, we hypothesized that 1/ SelN depletion increase intracellular oxidant activity in cells, 2/ SelN plays a role in defence against oxidative stress. To verify this, we used human primary myoblasts and fibroblasts cultures from controls and patients carrying nonsense SEPN1 mutations. We measured intracellular oxidant activity using the DCFH assay. Furthermore, we examined the susceptibility of the cells to a wide range of H2O2 concentrations.

Myoblasts devoid of SelN show a significant increase in intracellular oxidant activity compared to controls whereas fibroblasts do not. However, fibroblasts devoid of SelN are more sensitive to H2O2 ‐induced oxidative stress than normal fibroblasts, as evidenced by a significantly greater cell death. Furthermore, pre‐treatment with the antioxidant N‐acetyl cysteine restores cell survival against H2O2 to a level comparable to controls.

This study establishes for the first time that oxidative stress is implicated in SEPN‐RM and that SelN protects human cells against oxidative stress.
Effect of weight loss on gene expression profiles and oxidative stress markers in the skeletal muscle of lactating beef cows.
Authors: Brennan Kristen M , Michal J J , Johnson K A
Published: June 2021

Elevated β‐oxidation leads to increased oxidative stress. Many genes can help protect from damage sustained during this physiological state. To investigate the effect of fat mobilization on oxidative stress and gene expression in skeletal muscle, Angus cows (n=26) were examined during weight loss and weight maintenance. At both physiological states, body weight, serum non‐esterified fatty acids (NEFA), erythrocyte superoxide dismutase (SOD) activity and mRNA levels of target genes in the Biceps femoris were measured. When cows were losing weight, serum NEFA's (P<.0001) were higher and SOD activity (P<.025) increased compared to weight maintenance. Expression of β‐oxidation genes, carnitine palmitoyltransferase 1 (2.8 fold), fatty acid‐binding protein 3 (4.6 fold), and acyl‐coenzyme A oxidase 1 (2.0 fold) were higher during weight loss compared to weight maintenance (P<.01). During weight loss cows also had higher expression (P<.01) of uncoupling proteins 2 and 3 (3.0 fold and 5.0 fold respectively), tumor protein 53 (1.9 fold), nuclear respiratory factor 1 (2.6 fold), estrogen‐related receptor α (2.4 fold), and peroxisome proliferator‐activated receptors α and γ (2.0 fold and 1.9 fold). Mobilization of body fat results in greater antioxidant activity in erythrocytes and increased expression of genes that play a key role in protecting skeletal muscle against oxidative stress.
Effect of photobiomodulation on glutathione redox status of gastrocnemius muscle in an animal model of diabetes
Authors: Lark Daniel Stephen , Gopalakrishnan Sandeep , Wilson Robert W , Sandoval Christopher L , Eells Janis T , Snyder Ann C
Published: June 2021

INTRODUCTION: Glutathione (GSH) is an inter‐ and intra‐cellular antioxidant, and is essential in reducing reactive oxygen species (ROS) to prevent cellular damage. Photobiomodulation using 670 nm light has been shown to reduce oxidative damage in several animal models of degenerative disease. PURPOSE: To investigate the effects of photobiomodulation on the GSH redox status in the white (WG) and red gastrocnemius (RG) muscles of the streptozotocin (STZ) type I diabetic rat model. METHODS: Male Wistar rats (n = 26) were used in this study. Rats (n = 16) were injected with 50mg/kg (ip) of STZ and one group of STZ rats were exposed to 670 nm light for 5 minutes/day (6 J/cm2) for seventeen days (NIR‐STZ = 8). On day 18, animals were euthanized and gastrocnemius muscles were rapidly dissected and flash frozen. Glutathione was measured using an enzymatic recycling reaction developed by Cayman Chemicals. RESULTS: In response to STZ‐induced acute hyperglycemia, muscle GSH concentrations (total, oxidized and reduced) were increased. In contrast muscle GSH concentrations (total, oxidized and reduced) in 670 nm LED treated animals were nearly identical to control values. CONCLUSIONS: Although these changes were not statistically significant, these data suggest that photobiomodulation may be beneficial in modulating redox status of skeletal muscle in diabetes.

Funding Source: Biomedical Technology Alliance Award and UWM Research Growth Initiative
Inhibition of capacitative calcium entry suppresses ouabain‐induced stimulation of sodium‐hydrogen exchange in rat optic nerve astrocytes
Authors: Mandal Amritlal , Shahidullah Mohammad , Delamere Nicholas
Published: June 2021

Objective: Earlier we showed that 1 μM ouabain increased capacitative calcium entry (CCE) but did not increase cell sodium1. Here we studied cytoplasmic pH recovery after an acid load in ouabain‐treated cells and tested whether pH response were linked to altered calcium entry.

Methods: Ammonium chloride (20mM) was used to acid load the cells. BCECF and FURA‐2 were used to measure intracellular pH and calcium.

Results: Ammonium chloride removal causes the pHi to fall abruptly, followed by gradual recovery. lμM ouabain increased the rate of pHi recovery both in the presence and in the absence of bicarbonate in the bathing medium. NHE inhibitor DMA reduced the rate of pHi recovery. Ouabain did not detectably alter cytoplasmic Ca2+ (Ca2+i) but CCE inhibitors SKF96365 and 2APB reduced both the basal and ouabain‐stimulated rates of pHi recovery. Calcium chelator BAPTA‐AM also inhibited basal and ouabain‐stimulated pHi recovery. Western blot showed presence of NHE1 but not NHE2, NHE3 or NHE4.

Conclusion: Ouabain‐induced stimulation of pHi recovery was due, at least in part, to stimulation of NHE1. Although lμM ouabain failed to alter Ca2+i, the findings suggest the NHE response may be dependent on CCE.

Funding: NIH Grant EY014069
K+‐depolarization of efferent arterioles of juxtamedullary nephrons: A method for studies on Ca2+ entry and mobilization
Authors: Feng Ming‐Guo , Navar L. Gabriel
Published: June 2021

Increases in [Ca2+]i involve Ca2+entry and mobilization mechanisms. We developed a method for studies on Ca2+mobilization from intracellular stores and entry from the extracellular space. Videomicroscopic measurements of vascular dimensions were performed on the rat isolated blood‐perfused juxtamedullary nephron preparation. Single efferent arterioles (EA) which get out of glomerulus, branch to form the descending vasa recta were selected for study. We used KCl‐induced K+‐depolarization, an experimental means of activating voltage‐ activated L‐type Ca2+ channels (LCC), to exclude the possibility of the action of LCC. We found that, in contrast to afferent arterioles, superfusion with KCl (55mM) caused only a slight constriction in EA indicating a weak activity of LCC in EA. Ryanodine, a modulator of intracellular ryanodine receptor(RyR)‐induced Ca2+ release, caused constriction at low concentration (1μM) and dilation at high concentration (51μM) in K+‐depolarized EA indicating that ryanodine modulate Ca2+ release from intracellular stores via RyR. Adding NE (1μM) to the high KCl treated EA elicited strong constriction. NE‐induced vasoconstriction was markedly inhibited by pretreatment with 2‐APB (100μM), a store‐operated channel (SOC), as well as IP3 receptor blocker. However, ryanodine (51μM) failed to inhibit the NE‐induced vasoconstriction indicating that α1‐adrenergic vasoconstriction predominantly involves IP3 dependant SOC activation, rather than LCC or RyR activation in EA. Our results are consistent with the previous observations obtained using other methods. This study provides a new, simple and effective method to investigate the mechanisms of Ca2+entry and mobilization. (supported by NHLBI HL‐18426)
Effect of ET‐1 on afferent arterioles of ETB‐receptor deficient mice
Authors: Schildroth Janice , Sendeski Mauricio , Rettig Juliane , Steege Andreas , Kalk Philipp , Persson Pontus B. , Hocher Berthold , Patzak Andreas
Published: June 2021

Endothelins (ET‐1, ET‐2, ET‐3) are a family of regulatory peptides that have long‐lasting vasoconstrictor and pressor effects. Among them ET‐1 is the most active form that plays an important role in the regulation of renal function. The aim of the study was to investigate the contribution of ETA‐ and ETB‐receptors in ET‐1 induced effects on afferent arterioles. Adult ETB‐receptor deficient rescued mice and wild‐type mice were used. Cumulative dose‐response‐curves to ET‐1 (10−12 to 10−7 mol/L) were measured in isolated, perfused afferent arterioles, as well as during ETA‐receptor blockade. Further, dose‐response curves to the ETB‐receptor agonist 4‐Ala‐ET‐1 were measured. Results: 1. Afferent arterioles of wild‐type mice and ETB‐receptor deficient mice showed a concentration‐dependent constriction to ET‐1. 2. The dose‐response curves did not differ between ETB‐receptor deficient mice and wild‐type mice. 3. There was no response to ET‐1 during ETA‐receptor blockade in afferent arterioles of both groups. 4. Afferent arterioles of wild‐type mice and ETB‐receptor deficient mice did not significantly respond to 4‐Ala‐ET‐1. Conclusions: Results indicate that the ETA‐receptor mediates vasoconstriction in afferent arterioles and that there is a lack of functional ETB‐receptor activity in afferent arterioles.
Renal calcium‐sensing receptors influence renal hemodynamics via nitric oxide‐mediated vasodilation
Authors: Beierwaltes William H.
Published: June 2021

Calcium (Ca) is a cofactor in both activation of nitric oxide synthase (NOS) and vascular smooth muscle contraction. Both endothelial cells and vascular smooth muscle express Ca‐sensing receptors (CaSR), which increase intracellular Ca in response to increased extracellular Ca. Ca mimetics enhance this signaling. We hypothesized that the Ca mimetic Cinacalcet (Amgen) would cause NO‐mediated renal vasodilation. The effect of Cinacalcet on blood pressure (BP) and renal blood flow (RBF) without and with NOS inhibition by L‐NAME (10 mg/kg bw) were measured in Inaction‐anesthetized male Sprague‐Dawley rats. Without L‐NAME, an i.v. bolus of 30 mg/kg bw Cinacalcet increased BP from 102 ±5 to 112 ±4 mmHg (p<0.001) within 3 min and RBF increased 15% from 6.41 ±0.4 to 7.38 ± 0.4 ml/min/gkw (p<0.001). L‐NAME increased BP by 29 ±4 mmHg and reduced RBF by 45% (p<0.001). Then, Cinacalcet administration produced a similar pressor response as before (11 ±2 mmHg, p<0.01), but now RBF remained unchanged. Thus, we observed the systemic response to activating CaSR was pressor and NO‐independent, but the renal vasodilation was NO‐mediated. We conclude that activating the CaSR does cause NO‐mediated renal vasodilation. This dissociation of systemic and renal responses suggests that in the renal vasculature, CaSR‐activated NOS activity may be a particularly important factor in maintaining renal perfusion.
Post‐transcriptional silencing of CCR3 downregulates IL‐4 stimulated release of CCL26 in alveolar type II cells
Authors: Taka Equar , Errahali Younes J , Heiman Ann S
Published: June 2021

Trafficking and activation of cells in diseases such as asthma are, in part, modulated by eotaxin‐2 (CCL24) and eotaxin‐3 (CCL26) chemokines that signal via CCR3. Short‐interfering RNAs (siRNA) are novel tools to inhibit gene functions in human diseases. In this context, we hypothesized that alveolar type II cells can be transfected with siRNAs targeting CCR3 (CCR3‐siRNA) which will down‐regulate the receptor and decrease synthesis and release of its ligands CCL24 and CCL26. To test our hypothesis, human A549 alveolar type II epithelium‐like cells were transfected with four separate and combined CCR3‐siRNA duplexes and effects on CCR3, CCL26 and CCL24 were studied. FACS analysis showed decreased cell surface CCR3. Densitometry of lysate immunoblots indicated up to 75–84% reduction in CCR3. RT‐PCR showed an 80% decrease in CCR3 mRNA. IL‐4 stimulated CCL26 release (959 ± 201 pg/ml) was reduced up to 65% and constitutive CCL24 release (285 ± 64 pg/ml) was reduced by 80% in CCR3‐siRNA treated cells. Activation of eosinophils, assessed as superoxide anion generation, was reduced up to 80% when treated with supernatants of A549 cells pretreated with CCR3‐siRNAs. These findings suggest that CCR3‐siRNA treatment of alveolar type II cells may be useful for development of novel therapies for controlling airway inflammation. Support for this research was provided in part by NIH grants RR08111 and RR03020.
The R3/1 alveolar epithelial type I cell line ‐ Characterisation as an in vitro model for drug disposition
Authors: Ehrhardt Carsten , Endter Sibylle , Andersen Rikke , Koslowski Roland , Horalkova Lenka
Published: June 2021

The rat cell line, R3/1, has been reported to display several phenotypical features of alveolar epithelial type I cells. In order to characterise the cell line as an in vitro model for pulmonary drug disposition studies, R3/1 cells were cultured on Transwell Clear filters. The transepithelial electrical resistance was measured as a parameter for the integrity of cell layers. Presence of cell junctional proteins (i.e., E‐cadherin, occludin, ZO‐1 and ZO‐2) was studied on mRNA (by reverse transcriptase‐polymerase chain reaction) and antigen levels (by immunofluorescence microscopy), respectively. Furthermore, expression patterns of catabolic peptidases were analysed in R3/1 monolayers. These studies included carboxypeptidase M, aminopeptidases A, B, N and P, γ‐glutamyltransferase, angiotensin‐converting enzyme, dipeptidylpeptidase IV and endopeptidases 24.11 and 24.15. R3/1 cells lack the ability to form confluent monolayers, due to an absence of several structural proteins of the junctional complex (occludin, E‐cadherin). However, the level of catabolic peptidases was found to be similar to that of rat alveolar epithelial type I‐like cells in primary culture. This suggests that R3/1 cells might be a good candidate for in vitro stability assays of protein drugs intended for pulmonary administration.
Measurement and Inhibition of Airway Hyperreactivity in a Guinea Pig Model of Allergic Asthma
Authors: Riley Jason P. , Dunford Paul J
Published: June 2021

An accurate invasive technique to measure airway hyperreactivity (AHR) in a guinea pig model of asthma is described. Previous reports of the use of flexi‐Vent, a computer controlled, small animal ventilator using forced oscillation techniques have been limited to the mouse. Dexamethasone and an H4R antagonist, JNJ‐7777120, were investigated for their ability to attenuate the AHR response which was induced by systemic sensitization and aerosol challenge with ovalbumin (OVA). To attenuate the response, guinea pigs were dosed around aerosol challenge with JNJ‐7777120 or dexamethasone. Twenty‐four hours post challenge airway hyperreactivity (AHR) was measured. AHR was assessed by measuring changes in dynamic respiratory resistance (R) and compliance (C), in response to rising doses of aerosolized methacholine (Mch), in anaesthetized animals. AHR was induced by Mch in a dose dependent manner as shown by an increase in R and decrease in C. JNJ‐7777120 and dexamethasone inhibited the increase in R and decrease in C dose dependently. Measurement and pharmacological modulation of AHR responses to Mch are therefore detectable using this technique. The novel method described allows for the accurate measurement of airway hyperreactivity a major clinical hallmark of asthma and has utility in the assessment of potential therapies for lung disease.
Oxidative stress increases chloride transport in human CF nasal epithelial cell
Authors: Tessier Marie‐Claude , Boncoeur Émilie , Massé Chantal , Dagenais André , Berthiaume Yves
Published: June 2021

Cystic fibrosis is a genetic disease characterized by a defective CFTR protein, it is associated with an oxidative stress response leading to structural damage. The aim of the study was to determine the impact of oxidative stress on Cl−transport in human non‐CF and CF nasal epithelial cells. Cells were grown on a Costar filter at air/liquid interface and were treated with DMNQ 15μM for 24 hours. Cl− transport was monitored in Ussing chamber by measuring short‐circuit current (Isc) following an amphotericin B permeabilization of the basolateral membrane in presence of a Cl− gradient. Treatment with DMNQ lead to an increase of 5.6 μA/cm2 in total Isc in CF cells but no change were observed in non‐CF cells. Forskolin and genistein stimulation induced ΔIsc of 22.4 μA/cm2 inhibited by the specific CFTR inhibitor (CFTRinh 172), in both control and DMNQ treated non‐CF cells. In CF cells, these activators induced a ΔIsc of 1.19 μA/cm2 in control cells compared to 5.43 μA/cm2 in DMNQ treated cells. This increase was inhibited by NPPB, a broad range Cl− inhibitor. These result suggest that the DMNQ induced probably a non‐CFTR dependent Cl− current in CF cells. Further studies are needed to characterise the chloride channels involved in this response. Supported by the CCFF and CIHR.
Undergraduate research in neuroscience using SNNAP
Authors: Watrous James , Bortz Brandon , Cushing Daniel , Chan George , Ford Jacqueline , Podgorski Edward
Published: June 2021

There has been a growing interest in the use of SNNAP (Simulator for Neural Networks and Action Potentials) in undergraduate neurobiology courses. Most of this interest has focused on the use of SNNAP in formal laboratory sessions where students conduct short term simulations studying the properties of single neurons or slightly extended projects taking one or two laboratory sessions. This study involves the use of SNNAP during an entire semester where students are enrolled in a formal research course that requires a minimal time commitment of three hours each week and requires either a formal report or the presentation of a poster at least at the campus level. Using this approach I have had several undergraduates working individually or as part of a team investigate the behavior of more complex neural networks that include different types of synaptic connections and in some cases include the addition of more complex neurons; e.g., Morris‐Lecar, the R15 from Aplysia and the Epstein‐Marder bursting neuron. Some of these students conducted their work as part of the institution's Summer Scholars Program and the resulting work was presented at a local Sigma Xi meeting. In two instances students attended NCUR (National Conference on Undergraduate Research) to present their work. Sample simulations of these projects will be demonstrated and are available from the author. SNNAP is freely available at http://snnap.uth.tmc.edu.
The USF Undergraduate Research Experience on Women's Health: Promoting Learning that Lasts a Lifetime
Authors: Tigno Xenia T. , Vaz Kim M. , Yavneh Naomi , El‐Badri Nagwa S.
Published: June 2021

The concept of Women's Health has evolved over the past few years from a focus on disorders strictly related to female reproductive organs into a multidisciplinary construct that involves the overall well‐being of women. In the summer of 2007, the University of South Florida established an undergraduate research project on Women's Health, with a belief that a well‐planned educational activity involving both classroom instruction and direct participation of the learner in research activities can advance the cause of Women's Health, promote gender and cultural sensitivity, and emphasize the roles of sex, race, culture and class in creating health disparities in a given community. Faculty from the Colleges of Medicine, Public Health, Nursing, Arts and Sciences and Honors were engaged to expose the students to diverse disciplines and approaches to the concept, and to provide them with a variety of research mentors and projects. In addition to the 6‐week intensive mentored research project, two parallel courses were offered to provide basic information on Women's Health and the fundamentals of research. Based on the positive results from this pilot project, we expect not only to continue the program, but extend its time frame, expand its content, and open its enrolment to a greater pool of students.
Writing‐to‐learn in Investigative Physiology Laboratory Instruction
Authors: Hong Seung Mook , Laverty Gary , Baer Dee
Published: June 2021

A new investigative physiology laboratory course was developed a few years ago for biology majors. It is a 2‐credit, stand‐alone, and research‐based course. Physiological color change in lower vertebrates was the main theme of the class research and the major accomplishments of this new course were 1) students seemed more actively engaged in most experiments, and 2) ownership was tangibly exhibited in their group research projects. One of the challenges we faced, however, was the quality of the student research reports, which seemed lacking in precision in scientific analysis and writing. In the following study, we are exploring ways to enhance the critical thinking experience through low‐stakes writing assignments. First, 4 h once‐a‐week class time was divided into 1 h of planning on Day 1 and 3 h of experimentation on Day 3. Second, the students brought for discussion brief predictions of the experiment based on the assigned reading materials, which were later assessed with a minimum weight of grade. At the mid‐term reflection, students evaluated the effects of the prediction exercises both on conducting experiments and on writing a formal lab report. Most common statements were 1) it forced me to do background research on the topic and 2) it helped me with introduction write‐up. In addition, their research proposals reflected some of the critical thinking practices learned in these writing‐to‐learn exercises. These preliminary results strongly indicate the students ownership in their learning and the final research project report would show the full impact of this low‐stakes writing assignments on students critical thinking practice. Supported by Howard Hughes Medical Institute Grant
What is Dyad Pedagogy in Anatomical Education?
Authors: Sherman Lloyd R.
Published: June 2021

Academic education must also be practical. Knowledge and skills acquired in the learning of anatomy should be practicable in the marketplace. Dyad Pedagogy simply avails of the natural dynamic between two individuals with mutual goals ‐ using a curriculum that is “practically”oriented. It's power comes from the very dyad unit itself to promote cognitive, affective and psycho‐motor learning all at once and at the highest levels. Learning is readily measurable by focused performance metrics akin to the taxonomies of learning devised by Benjamin Bloom et al. DP stresses anatomical projects that build knowledge and skills in problem‐solving; research; independent regular performance; quantitative analysis; oral and written presentation; decision‐making; judgment; working collaboratively; ability to self‐learn – all crucial workplace requisites. DP is a “practice‐derived” theory discovered by Sherman at Mount Sinai; it grew out of the efficacy in matching students on tasks, in a buddy‐system model. The idea took on layer upon layer of philosophical, theoretical and practical dimensions as it grew to be the single methodology practiced in Mount Sinai's Center for Excellence in Youth Education. DP fosters development of workplace skills, knowledge and attitudes, as described in the SCANS paper.
An Anatomical Comparative Study of Lower Limb Discrepancy between Inuit and Urban Population Groups
Authors: Ormond Natalie B. , Marquez Samuel , Russo Christen M , Caceres Noel
Published: June 2021

Lower limb (LL) discrepancy (LLD), frequently found within the general population (P), afflicts a wide spectrum of polytypic individuals. This condition has been linked to biomechanical abnormalities that alter posture causing kinematic changes in gait. This study compares bones of LL in two different Ps with distinct biomechanical behaviors: Inuit (I) and an Urban population (UP). Donor (n=23) & I (n=28) specimens were used from the College of Health Related Professions at SUNY Downstate anatomy program & from Museum collections. Femora & tibia were disarticulated and measured. Paired t‐test on UP revealed differences of femora with a dominance of right (46.6 cm +/− 0.4) over left (45.9 cm +/− 0.5) LL lengths from the mixed‐sex sample (n=23, p<0.05). Paired t‐test revealed femoral differences (p<0.05) with a dominance of male (48.4 cm +/− 0.7) over female (44.1 cm +/− 0.8). A mixed linear model tested for the interaction of sex on side showed males (p<0.05) having a more significant effect over females (p=0.05) but not the reverse. Femur and tibial measures were different for the I P scoring p< 0.05 for both bones. The rehabilitation perspective of PTs require a thorough knowledge of the effects of LLD that set off the different types of compensatory mechanisms employed by the patient. The appropriate therapeutic strategies can be strengthened with a more thorough knowledge of the underlying bony anatomy.
Discovery and Characterization of a novel DNA damage‐induced splice variant of Mdm2
Authors: Lents Nathan H , Dynlacht Brian David
Published: June 2021

Mdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult, and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2+108. Importantly, this insertion introduces in‐frame nonsense codons, thus encoding a profoundly truncated mdm2 protein lacking the C‐terminal RING finger domain, E3 ubiquitin ligase activity, and p53‐binding site.

A wide range of pharmacological testing revealed that Mdm2+108 is induced, in mouse and rat cells, in specific response to Adriamycin and actinomycin D, but not other modes of DNA damage. Meanwhile, antibodies against the N‐terminal region of mdm2 reveal a >90% reduction in detectable mdm2 protein upon Adriamycin treatment, while p53 accumulates to strikingly high levels. We thus conclude that this alternative spicing of Mdm2 may be an important mechanism to facilitate massive accumulation of p53 in response to genotoxic agents.
Cigarette smoke condensate inhibits dexamethasone‐induced glucocorticoid receptor nuclear translocation in lung epithelial cells
Authors: Xu Haishan , Chu Shijian
Published: June 2021

Glucocorticoids are used in the treatment of pulmonary edema, asthma and COPD. Previousinvestigations have demonstrated that the epithelial Na+ channel (ENaC) α‐subunit promoter possesses a glucocorticoid regulatory element, and is upregulated by glucocorticoids. Cigarette smoking is associated with corticosteroid resistance observed in patients with asthma and COPD. In this study, we aim to investigate effects of cigarette smoke condensate (CSC) on glucocorticoid‐dependent α‐ENaC gene expression and its possible mechanism. We found that dexamethasone (DEX) treatment resulted in significantly increased levels of α‐ENaC and nuclear glucocorticoid receptor (GR) in lung epithelial cells. Pre‐ and co‐treatment with CSC inhibited DEX‐induced α‐ENaC expression, but GR expression at both mRNA and protein levels was not affected. Furthermore, in cells pretreated with CSC, DEX was unable to increase nuclear GR to the full scale and cytoplasmic GR was proportionally increased. Our results demonstrate that (1) CSC dose‐dependently suppresses DEX‐induced α‐ENaC expression; (2) CSC does not inhibit GR expression and (3) CSC inhibits DEX‐induced GR nuclear translocation. These results suggest that reduced GR nuclear translocation may be partially responsible for cigarette smoking associated corticosteroid resistance. This work was supported by a grant from the Department of Veterans Affairs.
The distal, not the proximal promoter upregulates mitochondrial glycerophosphate acyltransferase in starved and refed rat liver cells
Authors: Shilpi Rasheda Y. , Guha Prajna , Punnette Dwayne , Haldar Dipak
Published: June 2021

The effect of starvation and refeeding of liver cells on the activity of mitochondrial glycerophosphate acyltransferase (mtGPAT) was investigated. Rat liver cells were cultured in media lacking glucose, pyruvate, and fetal bovine serum for different periods of time, then placed in media, which contained those compounds, for additional time. After 24, 48 and 72 hrs of starvation and 24 hrs of refeeding of the cells, mtGPAT activity increased up to 5 fold. The viability of the cells was between 85% and 90%. Western blot results confirmed the presence of a larger amount of mtGPAT protein in the group of cells that was starved for 24, 48, and 72 hrs and refed for 24 hrs. The results of RT‐PCR showed enhancement of mRNA levels in the cells after starvation and refeeding. These results suggest that refeeding of liver cells induce an increase in mtGPAT. Additional experiments were done with liver cells transfected with pGL3‐ basic vector containing distal or proximal promoter of mtGPAT and luciferase as the reporter gene. Starvation and refeeding caused 2 to 2.5 fold increase in the luciferase activity in cells transfected with the distal promoter over control cells. No change in the luciferase activity was observed in the cells transfected with the proximal promoter. The results are in keeping with the idea that the increase in mtGPAT is regulated via the distal and not the proximal promoter. Supported by NIH grant GM‐57643.
EID‐1 interacts with the orphan nuclear receptor SF‐1 and represses its transactivation
Authors: Kim Don‐Kyu , Park Yun‐Yong , Choi Hueng‐Sik
Published: June 2021

The orphan nuclear receptor, SF‐1, plays a pivotal role on the development and differentiation of the endocrine and reproductive systems, and also regulates the transcription of a host of genes, including several steroidogenic enzymes and gonadotropin genes. Here, we demonstrate that a previously unidentified repressor, EID‐1, is a novel SF‐1 interacting protein, which represses the transactivation of SF‐1. A transient transfection assay revealed that EID‐1 specifically inhibited SF‐1, but not LRH‐1, ERRγ, or mCAR. Using yeast two hybrid and GST pull‐down assays, we determined that EID‐1 exhibited strong interaction with SF‐1. In addition, EID‐1 was determined to be colocalized with SF‐1 in mammalian living cells. EID‐1 was found to interact specifically with the AF‐2 domain of SF‐1, and competed with SRC‐1 to inhibit the transactivation of SF‐1. Physiologically, EID‐1 was expressed in the mouse testis, and EID‐1 expression in the testis was determined to decrease during the development of the mouse testis. The results of the present study suggest that EID‐1 can act as a repressor, regulating the function of SF‐1.
Development of Resorufin Fluorescence Assays for monitoring the ATP‐ase Activity of Human Chaperone Hsp‐70
Authors: Clement Cristina Corina
Published: June 2021

In response to environmental stress, there is an increase in the expression of heat shock proteins. Heat shock proteins are molecular chaperones that maintain cell homeostasis through various cellular mechanisms. Heat shock protein 70 is a specific family of heat shock proteins that uses an ATP‐driven mechanism to bind its client proteins. The overexpression of Hsp70 has been shown to prevent apoptosis and lead to tumorigenesis, but the inhibition of Hsp70 has been linked to an increase in apoptosis and tumor cell death. These anti‐apoptotic functions of Hsp‐70 prompted many groups of researchers toward development of sensitive assays for monitoring the ATP‐ase activity of this chaperone which can further be used for discovery of new potential competitive inhibitors of its enzymatic activity. This paper presents the development of resorufin fluorescence based assays for monitoring the ATP‐ase activity of Hsp‐70 by measuring the inorganic phosphate (Pi) or the adenosine diphosphate (ADP) production during the multi turn‐over enzymatic reactions. Enzyme‐coupled reactions were developed to convert the Pi or ADP in hydrogen peroxide which in turn was used to oxidize the reagent amplex red into resorufin which emits fluorescence at 590 nm upon excitation at 530 nm. The non‐hydrolysable ATP analog AMP‐PNP showed inhibition of human Hsp‐70 ATP‐ase activity in the low mM range.
Adiponectin Repressed Proliferation of Colon Cancer Cells via AMPK Activation
Authors: Kim A Young , Lee Yun Sok , Choi Jin Woo , Kim Jae Bum
Published: June 2021

Adiponectin is a member of adipocytockines and regulates whole body homeostasis. Recently, it has been shown that the level of plasma adiponectin is negatively correlated with the risk for certain cancers including breast, prostate, gastric, and colon cancers. However, it is largely unknown the role of adiponectin on colon cancers. In this work, we investigated the effects of adiponectin on the growth of colon cancer cells. Adiponectin specifically attenuated the growth of colon cancer cell lines, including HCT 116, HT‐29, and LoVo while it did not reduce the growth of normal cell lines such as HEK 293 and RWPE1. Consistently, adiponectin treatment into colon cancer cells enhanced the expression of CDK inhibitors including p21 and p27, indicating that adiponectin attenuates rapid growth of colon cancer cells by regulating of cell cycle. Moreover, adiponectin in colon cancer cells stimulated the activity of AMPK, which appears to be crucial to mediate suppression of cell growth. Collectively, our data suggest that adiponectin is a negative regulator of colon cancer growth, and that the growth inhibition effects of adiponectin on colon cancer cells might be dependent on AMPK activation.

This work was supported in part by grants from the Molecular and Cellular BioDiscovery Research Program (M10748000258‐07N4800‐25810) and the National Research Laboratory Program (R0A‐2004‐000‐10359‐0) of Korea Science and Engineering Foundation (KOSEF). J.B.K. and A.Y.K. was supported by a BK21 Research Fellowship from the Ministry of Education and Human Resources Development.