bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of clc2b chloride channel in chloride regulation of zebrafish (Danio rerio).
Authors: Yi‐Fang Wang , Chen Ruo‐Dong , Yan Jia‐Jiun , Hwang Pung‐Pung
Published: June 2021

Osmotic and ionic homeostasis in the body fluid is important for vertebrates including teleost fish, and this is mainly achieved in gill/skin ionocytes in fish. Chloride is the most abundant anion in animal tissue and plasma and participates in many physiological processes. Comparing with the regulation mechanisms of other ions that have been studied in details in zebrafish, the chloride ion regulatory pathways are less understood. In the present study, we cloned out 11 members of clc chloride channels in zebrafish. Several clc members were found to be expressed in the gills of zebrafish by RT‐PCR; however, only the mRNA of clc2b was localized by in situ hybridization in the zebrafish embryonic yolk sac and adult fish gill with an ionocyte pattern. Combination of clc2b in situ hybridization and zebrafish sodium chloride cotransporter‐like 2 (zNCC‐like 2) immunocytochemistry indicated that 2 transporters are co‐expressed on the same ionocyte. Furthermore, comparison of chloride content of clc2b morpholino knockdown and wild‐type zebrafish embryos showed significant difference. Altogether, the data suggested that clc2b plays an important role in zebrafish chloride regulation.
Response to altitude and intermittent normobaric hypoxia (INH)
Authors: Cardoso S , Samillan V , Pereira P , Werneck M , Quispe M , Amaral M , Romeo J , Rozeira C , Horowitz M
Published: June 2021

Hypoxic (HIF‐1) interventions enhance antioxidant capabilities; repair & regeneration defenses while preparing organisms for subsequent similar or even harsher stressors. More recent studies (Samillan, et. al., Hypoxia Consequences, NYAS 2009) support the clinical use of hypoxic interventions in the management of human T2D, hypertension, CV, & pulmonary disease. Reponses to altitude and INH in young control subjects (Peru) and patients with either T2D or COPD was studied. Inhalation of gas mix (pp O15 + N85) or interrupting nasal oxygen flow in pts c/COPD/Stage IV was used. OGTTs and determinations of glucose, insulin, fructosamine, HBA1c, reticulocytes (7AM‐11AM) were made. Controls (Peru, N=20, reticulocytosis) had lower levels of glucose and insulin at 2hrs post glucose administration (P,<0.0001). T2D (Brazil, N=5) had lower blood glucose (P=0.09). COPD and improved SpO2 levels were found after 5 INH/5 wks (P<0.035). These results suggest that patients will respond to INH with improved quality of life, increased tolerance to exercise, partial corrections of glucose intolerance, insulin resistance and, in general, lower dose/frequency of prescribed Rx. Support: San Pedro U., Peru. Private donations, clinical assistance by Servico Unico De Saude and Lab Araruama, Brazil.
FoxO transcription contributes to mechanical ventilation‐induced diaphragm atrophy and contractile dysfunction
Authors: Smuder Ashley J , Sollanek Kurt J , Nelson W Bradley , Min Kisuk , Powers Scott K
Published: June 2021

Mechanical ventilation (MV) is a life‐saving measure for patients in respiratory failure. However, prolonged MV results in diaphragm weakness due to both muscle fiber atrophy and contractile dysfunction. Therefore, identifying the signaling pathways responsible for MV‐induced diaphragm weakness is important. In this regard, activation of the FoxO family of transcription factors is reported to be necessary for disuse skeletal muscle atrophy and FoxO‐specific transcription can lead to the activation of major proteolytic systems responsible for muscle protein breakdown. Therefore, our goal was to determine the role that FoxO signaling plays in MV‐induced diaphragm atrophy. Cause and effect was determined by inhibiting the activation of FoxO though the use of a dominant negative FoxO (d.n.FoxO) adeno‐associated virus vector. Our results demonstrate that prolonged (12 hrs) MV results in a significant decrease in both diaphragm muscle fiber size and diaphragm specific force production. However, MV animals treated with d.n.FoxO showed a significant attenuation of both diaphragm atrophy and contractile dysfunction. In addition, inhibiting FoxO transcription attenuated the MV‐induced activation of caspase‐3, the ubiquitin‐proteasome system, and autophagy. Collectively, our findings support the hypothesis that FoxO activation plays a significant role in MV‐induced diaphragm weakness.
Matrix Metalloprotease Inhibition Negatively Affects Signaling Required for Muscle Regeneration Post‐traumatic Injury
Authors: Urso Maria L , Carrigan Christopher T , Warren Gordon L
Published: June 2021

Matrix metalloproteases (MMPs) are required for extracellular matrix turnover. We previously reported that an unfavorable ratio develops between MMPs and their inhibitors in response to traumatic muscle injury. The MMP inhibitor Batimastat (BB94) improves recovery in dystrophic muscle. We hypothesized that BB94 would promote favorable MMP and myogenic signaling, expediting functional recovery post‐injury. Injury was induced by applying a steel probe (−79ºC) to the tibialis anterior (TA) for 10s. Mice were injected with BB94 or Placebo post‐injury. TA muscles were collected after muscle function testing 48h, 5d, 10d, 18d and 30d post‐injury. In BB‐94 treated mice, protein levels of active MT‐1(−71.4%), MMP‐2 (−77.9%), MMP‐3 (−63.8%) and MMP‐9 (−86.8%) decreased (p<0.05) post‐injury compared to Placebo mice. There were no differences in TA strength loss or recovery between BB‐94 and Placebo mice (strength loss: 63.5%). mRNA for myogenic and satellite cell activation factors (Myf5, Myf6, mCadherin, SCA‐1, Notch‐1) increased in injured muscle by 2.6‐ to 4.5‐fold in Placebo mice (p<0.05), but not in BB‐94 mice. MyoD was 3.5‐fold lower in BB‐94 mice. Despite decreased MMP activity, BB‐94 did not improve functional recovery. This might be explained by the changes in myogenic gene expression. MMP‐inhibition immediately post‐injury may impair signaling required for regeneration.
Pulmonary inflammation‐induced muscle atrophy involves a GSK‐3beta‐GR signaling axis
Authors: Langen Ramon , Verhees Koen , Schols Annemie
Published: June 2021

Skeletal muscle atrophy is induced by raised glucocorticoid (GC) levels and reduced insulin/IGF‐1‐Akt signaling. Glycogen synthase kinase‐3 beta (GSK‐3b) is a downstream kinase of Akt involved in regulation of muscle protein turnover.

We hypothesized that muscle GC receptor (GR) and GSK‐3b are required for muscle atrophy in response to pulmonary inflammation.

Control, muscle specific‐GR (mGRKO) or ‐GSK‐3b (mGSK‐3bKO) mice were subjected to intra‐tracheal LPS instillation to induce acute pulmonary inflammation.

Increases in circulating corticosterone levels following IT‐LPS were similar in control, mGRKO and mGSK‐3KO mice, but accompanied by substantial muscle atrophy in control mice only. Accordingly, increases in mRNA transcripts encoding effectors of the Ub 26S‐proteasome (MuRF1 and Atrogin‐1) and autophagy‐lysosomal (Bnip3, LC3b) systems were strongly attenuated in muscle of mGRKO or mGSK‐3KO mice. Interestingly, increased expression of GC‐sensitive genes (Glul, KLF15 and REDD1) and FOXO1 and ‐3a following IT‐LPS, was suppressed in skeletal muscle of GRKO and GSK‐3KO mice. This dependency of GR signaling on GSK‐3b was confirmed in cultured muscle cells.

We conclude that pulmonary inflammation‐induced muscle atrophy is dependent on an intact GSK‐3b‐GR signaling axis.

Support: Dutch Astma Foundation (3.2.07.017) and Dutch Top Institute Pharma (T1–201)
Docosahexaenoic acid antagonizes palmitate‐induced protein degradation in myotubes
Authors: Woodworth‐Hobbs Myra , Hudson Matt , Zheng Bin , Zoromsky Sara , Franch Harold , Price S. Russ
Published: June 2021

Muscle atrophy is a debilitating consequence of many chronic diseases, some of which are characterized by accumulation of lipids in skeletal muscle. High levels of saturated fatty acids (i.e. palmitic acid, PA) cause dysregulated protein metabolism in cultured myotubes, whereas the omega‐3 fatty acid docosahexaenoic acid (DHA) has beneficial metabolic effects. We hypothesized that DHA prevents PA‐induced muscle atrophy by counteracting its effects on protein degradation. To test our hypothesis, we studied C2C12 myotubes treated with 500uM PA and/or 100uM DHA. PA significantly increases the rate of protein degradation, and the response is reversed by co‐treatment with DHA. The protein kinase Akt is a key regulator of protein balance and inhibits the FoxO3 transcription factor. PA reduces the ratio of phospho‐to‐total Akt and increases the mRNA and total and nuclear protein levels of FoxO3. It also increases the levels of mRNA encoding three FoxO3 targets, the MuRF‐1 and atrogin‐1/MAFbx E3 ubiquitin ligases and the autophagy mediator Bnip3. DHA attenuates the effects of PA on Akt, FoxO3, and all three atrogenes mRNAs. These data indicate that PA induces protein degradation by inducing the ubiquitin‐proteasome and autophagic proteolytic systems and that DHA counters the catabolic effects of PA by improving Akt signaling.

Support NIH DK95610, AHA GRNT 7660020
Increased Mechano‐Growth Factor (MGF) expression in Crohn's disease mediates smooth muscle hypertrophy in intestinal strictures via activation of Erk5
Authors: Li Chao , Grider John , Kuemmerle John
Published: June 2021

Expression of the IGF‐IEc splice variant, MGF, increases in inflamed muscle and mediates cell hypertrophy. Smooth muscle cell (SMC) hypertrophy is a feature of intestinal strictures in patients with Crohn's disease.

Aims

To determine whether MGF increases in stricturing Crohn's disease and whether it regulates SMC hypertrophy.

Methods

SMC isolated from strictured and normal intestine in the same patient with Crohn's disease were used to prepare mRNA, protein lysates, or primary cell cultures. MGF transcripts were measured by qRT‐PCR and protein by ELISA. Cell hypertrophy in response to 100 nM C‐terminal MGF peptide was measured over 4d by confocal microscopy and by total protein/105 cells. Phosphorylated Erk5(Thr218/Tyr220) was measured by immunoblot analysis and immunofluorescence.

Results

MGF transcripts were increased 18 fold and MGF protein was increased 10 fold in SMC of strictured intestine compared to normal margin and was accompanied by a concomitant increase in Erk5 phosphorylation. MGF caused a time‐dependent, 2–19 fold increase, in SMC hypertrophy that was accompanied by nuclear localization of phospho‐Erk5. These effects were abolished by 30 Âμm BIX02188, a selective Erk5 inhibitor. MGF did not elicit IGF‐IR phosphorylation or SMC proliferation.

Conclusions

Increased MGF expression in Crohn's disease mediates smooth muscle hypertrophy via activation of Erk5.

Supported by NIH DK49691
CaMKKα regulates growth in mouse skeletal muscle
Authors: Smith Cheryl A.S. , Witczak Carol A
Published: June 2021

Intracellular Ca2+ is a key regulator of skeletal muscle growth, yet the Ca2+‐sensitive proteins that regulate this process are largely unknown. Ca2+/calmodulin‐dependent protein kinase kinase α (CaMKKα) is a Ca2+‐activated, serine/threonine kinase, and to date, no studies have examined whether CaMKKα regulates skeletal muscle growth. The goals of this study were to determine if activation of CaMKKα signaling can stimulate muscle growth, and if so whether CaMKKα plays an essential role in this process. To selectively stimulate CaMKKα signaling in muscle, mouse muscles were transfected with plasmid DNA containing empty vector or constitutively active CaMKKα. At 2 weeks, active CaMKKα increased muscle weights ~10%, and stimulated proteins that regulate protein synthesis (i.e. p70S6K, 4E‐BP1), demonstrating that activation of CaMKKα signaling can stimulate muscle growth. To determine if CaMKKα is essential for growth, plantaris muscles from CaMKKα knockout mice were stimulated to hypertrophy via unilateral functional overload. Surprisingly, muscles from knockout mice exhibited significantly greater (~15%) muscle growth compared to wild‐type, suggesting a complex role for CaMKKα in the regulation of muscle mass. Collectively, these results show that activation of CaMKKα signaling is sufficient but not necessary for growth in mouse skeletal muscle. SUPPORT: NIH R00AR056298, ECU start‐up funds.
The protective role of PGC‐1α in the recovery of muscle disuse atrophy
Authors: Kang Choung‐Hun , Goodman Craig A , Hornberger Troy A , Ji Li Li
Published: June 2021

Prolonged inactivity results in skeletal muscle atrophy including increased reactive oxygen species (ROS) generation, inflammation and protein degradation. Over‐expression of peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) has been shown to increase mitochondrial biogenesis and reduce oxidative stress.

Methods

Female FVB/N mice (N=48) were randomly divided into 4 groups: (1) control (CON) & injected with GFP (Con‐GFP); (2) CON & PGC‐1α in vivo transfection (Con‐PGC‐1α); (3) two wks of hindlimb immobilization (IM) followed by 5 days remobilization (IM‐RM) & injected with GFP (IM‐RM‐GFP); and (4) IM‐RM & PGC‐1α in vivo transfection (IM‐RM‐PGC‐1α). GFP or PGC‐1α was injected into tibialis anterior (TA) muscle of one of the hindlimbs with an electroporation system.

Results

PGC‐1α induced SIRT3 and SOD2 binding in CON and IM‐RM by enhancing SIRT3 promoter activity and protein expression. Basal SOD2 acetylation level was increased by 57% in IM‐RM, however PGC‐1α decreased SOD acetylation by 30% (P<0.05) in IM‐RM. Thus, PGC‐1α over‐expression resulted in a 15% increase in superoxide dismutase (SOD) 2 protein content and activity in IM‐RM vs CON (p<0.05). IM‐RM increased mitochondrial H2O2 generation in both GFP and PGC‐1α groups, whereas PGC‐1α attenuate IM‐RM‐induced ROS generation by 18% (P<0.05).
Skeletal muscle gp130 receptor regulation of cancer cachexiainduced muscle mass loss
Authors: Puppa Melissa , Narsale Aditi , Gao Song , Carson James
Published: June 2021

Circulating IL‐6 levels and skeletal muscle STAT3 activation are involved with muscle loss in many types of cancer cachexia. Additionally, cancer‐induced muscle loss is attenuated by muscle STAT3 signaling inhibition. STAT3 is a downstream target of gp130 receptor (gp130r) signaling, and the gp130r also is involved in the initiation of intracellular IL‐6 signaling. The purpose of this study was to determine if the skeletal muscle gp130r initiates signaling necessary for cancer‐induced muscle loss. PBS or Lewis lung carcinoma (LLC) cells were injected to 8 week old male wild type (WT) or skeletal muscle gp130r knockout mice (m‐gp130r). After ~30 days animal were sacrificed. Gastrocnemius muscle mass was decreased 27% in WT + LLC mice, but only 4% in m‐gp130r + LLC mice. Epididymal fat mass was reduced by LLC in WT (55%) and m‐gp130r mice (35%). LLC‐induced suppression of muscle mTOR signaling was not improved in m‐gp130r mice. LLC elicited a 1.7 and 1.9 fold reduction of p‐4EBP1 in WT and m‐gp130r mice, respectively. Cytochrome C protein expression was reduced 2.4 fold in WT mice by LLC, and only 1.5 fold in m‐gp130r mice. These data demonstrate that in the LLC tumor model of cancer cachexia gp130r loss attenuated cancer‐induced gastrocnemius muscle mass loss. This effect was not dependent on rescuing muscle mTOR signaling, but may involve attenuation of reduced oxidative capacity. Funded by R01CA121249
Positive association between physical activity and bone in children with Prader‐Willi Syndrome
Authors: Duran Andrea Tiana , Castner Diobel Mendoza , Tucker Jared M , Rubin Daniela A
Published: June 2021

Prader‐Willi Syndrome (PWS) presents hypotonia, congenital obesity, and low lean mass; perhaps leading to less physical activity (PA) and poor bone health.

Purpose

To explore associations between PA and bone in children with PWS, accounting for body fat. Nine boys and 9 girls with PWS participated (mean age: 11.0±2.2 y, body fat: 46.2±8.5 %). PA was assessed by accelerometry (minimum of 3 compliant weekdays and 1 compliant weekend day) and classified into moderate (MPA), vigorous (VPA), and moderate plus vigorous (MVPA). Fat mass, bone mineral content (BMC), bone mineral density (BMD), and BMD z‐score (BMDz) of the hip and full body minus the head (body) were measured by dual energy x‐ray absorptiometry. MPA was associated with hip BMD and BMDz (r=0.595, r=0.650, respectively). VPA was associated with hip BMC and BMD (r=0.589, r=0.592, respectively) and body BMC and BMD (r=0.549, r=0.572, respectively). MVPA was associated to hip BMC, BMD and BMDz (r=0.523, r=0.653, r=0.607, respectively), and body BMC, BMD and BMDz (r=0.518, r=0.555, r=0.638, respectively). Associations were significant at p<0.05. MVPA had moderate positive associations with all bone parameters. Both MPA and VPA were independently associated with bone. However, VPA showed stronger associations with BMC than MPA. As in other children, intensity of PA appears an important determinant of bone health. Supported by USAMRAA Award W81XWH‐09–1‐0682
Regulation of skeletal muscle p27 and CDK4 expression by high lipid concentrations
Authors: Dungan Cory M , Jadhav Kavita S , Willliamson David L
Published: June 2021

Obese skeletal muscle has many of the same characteristics as aged muscle, which may suggest premature senescing of obese tissue. High lipid concentrations are a hallmark of obesity, yet little is known about its role on cell cycle. The objectives of these studies were to examine the role of high in vivo and in vitro lipid concentrations on regulators of cell cycle in skeletal muscle. Plantar flexor muscle from C57Bl/6 mice fed a low (10%) or a high (60%) fat diet for 8 wks and C2C12 myoblast cultures treated with a vehicle control or 50μM C2‐ceramide were both analyzed for protein expression of cell cycle regulators. The high fat diet increased (p<.05) body (26%) and liver mass (50%) vs low fat, along with reduced plantar flexor mass/body mass (−16%; p<.05). High fat fed mice had lower (p<.05) protein expression of p27 (−46%) and CDK4 (−25%) vs the low fat. The ceramide treated, insulin‐insensitive myoblasts had reduced CDK4 protein (−60%; p<.05), but no change in p27 vs control. Ceramide increased β‐galactosidase (85%; p<.05) and the proportion of cells in G2‐phase (28%; p<.05), and reduced proliferation (−51%; p<.05) as compared to control. Our results indicate that high lipid concentrations negatively affect muscle growth, which is associated with aberrant G1‐S cell cycle protein expression and a senescent‐like phenotype. Support: University at Buffalo Start Up Funds
Differing sympathetic response during dynamic leg exercise in heart failure and age‐matched healthy subjects
Authors: Notarius Catherine Frances , Millar Philip James , Murai Hisayoshi , Lee Morris Beverley , Floras John Stanley
Published: June 2021

In human heart failure (HF), we showed that muscle sympathetic nerve activity (MSNA) increased during dynamic handgrip exercise at a lower threshold than age‐matched healthy controls. The aim of the present study was to compare the MSNA response during contra lateral leg cycling in HF and control subjects, hypothesizing that, in contrast to the drop in MSNA reported in controls, MSNA would increase in HF. We measured heart rate (HR), blood pressure (BP) and MSNA in the fibular nerve (microneurography) in 11 HF patients (left ventricular ejection fraction= 32±2%; age 61±3 years, (mean±SE)) and 8 healthy control subjects (55±2 years), during 2 minutes of upright one‐legged unloaded cycling followed by 2 minutes at 50% of peak oxygen uptake (VO2peak). Resting HR and BP were similar between groups but MSNA was significantly greater (84.3±3.7 vs 68.8±5.5 bursts/100 heart beats, P=0.02), and VO2peak lower (18.7±2.4 vs 32.7±4.3 ml•kg/min, P=0.008) in HF. HR was increased during exercise (P=0.002) with no group difference (P=0.25). As expected, MSNA burst frequency decreased during exercise in the healthy controls yet was increased in HF (−4.8±3.3 vs +5.1±2.8 bursts/min, P=0.028). The normal reduction in MSNA is offset in HF by one or more sympathoexcitatory responses that may represent greater activation of muscle mechanoreceptor, metaboreceptor or cardiac sympathetic reflexes. (Heart and Stroke Foundation of Ontario)
Post‐exercise heart rate recovery is impaired in soccer athletes during a competitive season
Authors: Callegaro Carine Cristina , dos Santos Corrêa Ana Paula , Viecili Paulo Ricardo Nazario
Published: June 2021

Introduction

Post‐exercise heart rate recovery is mediated by the cardiac vagal reactivation. Since vagal control of the heart rate is impaired in athletes at the competition period, we hypothesized that soccer athletes engaged in a competitive season would exhibit an impaired heart rate recovery after exercise.

Methods

We studied 7 male soccer athletes (23 ± 5 years old) and 9 male sedentary individuals (26 ± 1 years old). Heart rate variability at rest was assessed by spectral analysis using the fast Fourier transform. Heart rate was measured at rest, during treadmill exercise test and recovery period.

Results

The athletes had a lower heart rate at rest than sedentary individuals. Low frequency and high frequency component of the heart rate were similar in the soccer athletes and in the sedentary individuals. During the exercise the athletes had a greater increase in heart rate (114 ± 20 bpm vs. 90 ± 18 bpm, P = 0.02) than in sedentary individuals. Soccer athletes showed a higher maximal oxygen consumption (63 ± 5 ml.kg−1.min−1) than sedentary individuals (47 ± 5 ml.kg−1.min−1; P= 0.001). Heart rate recovery in the first minute after exercise was similar in both groups.

Conclusion

Our data suggests that high‐intensity exercise performed at the competition season may impair heart rate recovery in soccer athletes.
The pressor response to activation of the central command pathway is attenuated by stimulation of peripheral sensory afferents in normotensive but not hypertensive rats
Authors: Liang Nan , Mizuno Masaki , Downey Ryan M , Iwamoto Gary A , Mitchell Jere H , Smith Scott A
Published: June 2021

Recent evidence suggests that central command (CC; a neural drive originating in higher brain centers) partially mediates the exaggerated BP response to exercise in hypertension. The mechanisms underlying this CC overactivity remain unclear. It has been suggested that CC is modulated by input from peripheral sensory afferents (PSA). A change in this modulatory relationship could potentially play a role in the generation CC overactivity in hypertension. To address this issue, the BP response to activation of the CC pathway before and during concurrent stimulation of PSA was examined in normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Rats were decerebrated and paralyzed. Activation of the CC pathway was evoked by electrical stimulation of the mesencephalic locomotor region (MLR; 20–50 μA in 10‐μA steps). Electrical stimulation of the sciatic nerve (3, 5 and 10 × motor threshold; MT) was used to activate PSA. The BP response to MLR activation was significantly attenuated by sciatic nerve stimulation at 5 and 10 × MT in WKY but not in SHR. This finding suggests that PSA input maintains the potential to inhibit CC activity in normal rats. The data further indicate that this inhibitory influence is reduced in hypertension. This change in the modulatory capacity of PSA may contribute, in part, to the development of CC overactivity in this disease. Support: NIH HL‐088422
Gastric Sonic Hedgehog Acts as a Chemoattractant for Macrophages During Tissue Regeneration
Authors: Engevik Amy Christine , Martin Jason R. , Tritschler Julie A. , Feng Rui , Schumacher Michael A. , Yang Li , Zavros Yana
Published: June 2021

Sonic Hedgehog (Shh) has been shown to regulate wound healing in various tissues. Immune cells such as macrophages (Mø) are also known to regulate repair. Despite its known function in tissue regeneration the role of Shh as a regulator of the immune system during gastric tissue repair remains unknown.

Hypothesis

Shh secreted from the gastric epithelium mediates the recruitment of Møs that drives tissue repair. A mouse model expressing a parietal cell‐specific deletion of Shh (PC‐ShhKO) and a mouse model expressing a myeloid cell‐specific deletion of Hedgehog receptor Smoothened (LysMCre/SmoKO) were used. Acetic acid ulcers were induced and samples collected. In controls, ulcers healed within 7 days. Tissue regeneration was accompanied by the recruitment of Møs to the stomach within 48 hours post injury. Control mice had elevated expression of cytokines and the pro‐angiogenic factor VEGF that correlated with an increased Shh tissue concentration within 3 days post injury. PC‐ShhKO mice showed complete loss of ulcer repair, the absence of Mø recruitment and reduced Shh tissue concentrations. LysMCre/SmoKO mice exhibited delayed repair of ulcerated tissue. While in vitro Møs migrated towards the Shh gradient, cells isolated from LysMCre/SmoKO mice failed to respond. Gastric Shh facilitates tissue repair by acting as a Mø chemoattractant. Funding: ACS Research Scholar Award 119072‐RSG‐10–167‐01‐MPC (Y. Zavros)
microRNA‐29b Functions as a Repressor of Intestinal Mucosal Growth by Targeting Cyclin‐dependent Kinase 2
Authors: Xiao Lan , Rao Jaladanki N. , Gorospe Myriam , Wang Jian‐Ying
Published: June 2021

Integrity of the intestinal epithelium is tightly regulated by numerous factors including microRNAs. We recently found that mucosal growth inhibition associates with an increase in microRNA‐29b (miR‐29b), but its exact role in epithelial homeostasis remains unknown. This study determined if miR‐29b inhibits intestinal mucosal growth by repressing cyclin‐dependent kinase (CDK) 2.

Methods

Studies were conducted in mice and IEC‐6 cells, and miR‐29b function was examined by its ectopic overexpression and silencing.

Results

miRNA‐29b silencing by the systemic delivery of LNA‐antimiR‐29b not only increased CDK2 but also stimulated small intestinal mucosal growth in mice. In contrast, increased levels of miRNA‐29b in IEC‐6 cells repressed CDK2 expression and caused growth arrest in G1 phase. miR‐29b repressed CDK4 translation through direct interaction with the CDK2 mRNA, and point mutation of miR‐29b binding‐site on the 3′‐untranslated region of the CDK2 mRNA prevented miR‐29b‐induced repression of CDK2 translation. CDK2 gene transcription was also inhibited by miRNA‐29b through a process involving p53. miR‐29b increased p53, whereas deletion of p53 binding‐site in the CDK2‐promoter blocked miRNA‐29b‐induced repression of CDK2 transcription.

Conclusions

These results indicate that miRNA‐29b inhibits intestinal mucosal growth by repressing CDK2 expression through distinct mechanisms. NIH Grant DK‐57819
JANUS KINASE 3 REGULATES COLONIC DIFFERENTIATION
Authors: Mishra Jayshree , Kumar Narendra
Published: June 2021

Jak3 is a non‐receptor tyrosine kinase expressed in both hematopoietic and non‐hematopoietic cells. Previously we demonstrated the molecular mechanisms of Jak3 in mucosal wound repair and homeostasis. However the role of Jak3 in intestinal differentiation is not known. In this study, we investigated the in‐vivo significance of Jak3 in intestinal differentiation. We examined the colonic morphology and the expression of different differentiation markers in the intestine. Our results showed that colonic length and colon to body‐weight ratio were significantly reduced in Jak3 KO mice compared to their wild‐type littermates that resulted in increased severity of DSS‐induced colitis. The Enterocytic differentiation marker like villin, carbonic anhydrase, chromagranin, and isoforms of mucin were also analyzed. Expression of villin, chromagranin, and carbonic anhydrase were reduced in Jak3 KO mice. Morphological analysis showed that the number of goblet cells in the crypts of large intestine and crypt lengths from the base of muscularis was reduced in Jak3 KO mice. To determine the molecular mechanism of this impairment, Jak3 interactions with barrier function protein β‐catenin were examined using in vitro model of intestinal epithelial cells. In these cells Jak3 was activated in response to IL‐2 that induced differentiation of goblet cell‐like morphology. IL‐2 also facilitated the interactions of Jak3 with β‐catenin. Knockdown of the Jak3 reduced the tight‐junction localization of β‐catenin with reduced the goblet cell characteristics. Taken together, these data indicate that Jak3 played a crucial role in colonic differentiation through its interactions with β‐catenin.
Mast cell CRF receptor subtypes differentially modulate mast cell signaling and epithelial barrier injury during stress.
Authors: Moeser Adam , Gibson Amelia , D'Costa Susan
Published: June 2021

Debilitating GI diseases, including irritable bowel syndrome (IBS) and the inflammatory bowel diseases (IBD), are initiated and exacerbated by life stress. It is known that intestinal mast cells (MC) play a critical effector role in stress‐related diseases; however, the precise signaling pathways remain poorly understood. The objective of this research was to elucidate the role of MC corticotropin releasing factor (CRF) receptors in stress‐induced MC activation and intestinal epithelial barrier injury. MC‐deficient mice (W‐sh/ W‐sh) were repleted with bone marrow‐derived MC (BMMCs) from wild type (WT), CRF1KO, or CRF2 KO mice. Following the successful repletion of BMMCs, mice were subjected to 3 hours of restraint stress (RS) and intestinal permeability, in terms of FITC 4kDa dextran (FD4) flux, was measured. Repletion of W‐sh/ W‐sh mice with CRF1 KO BMMCs ameliorated stress‐induced increases in FD4 flux. In contrast, repletion of W‐sh/ W‐sh mice with CRF2 KO BMMCS exacerbated stress‐induced barrier dysfunction compared with WT BMMC controls (p<0.05). Compared with WT BMMCs, CRF2 KO BMMCs exhibited greater intracellular Ca++ elevations and tryptase release in response to A23187. CRF2 KO BMMCs exhibited greater p44/42 activation and cAMP levels in response to CRF (0.5uM). These data show that MC CRF1 and CRF2 differentially modulate mast cell activation and intestinal barrier injury. This work was funded by NIH grant K08 DK084313 (A.J.M.).
Novel signaling role of G protein‐coupled receptor kinase‐5 in human colon cancer cell line
Authors: Raghavendra Pongali B , Parameswaran Narayanan
Published: June 2021

In this study we investigated the role and mechanisms by which G protein coupled receptor kinase‐5, a serine/threonine kinase, modulates proliferation and chemokine gene expression in human colon cancer cells in the absence and presence of Toll‐like receptor‐4 stimulation. For this, we used lentiviral based RNAi against GRK5 to knockdown GRK5 in metastatic human colon cancer cell line SW620 and examined the effects on proliferation, chemokine gene expression, and signaling in the absence and presence of lipopolysaccharide (LPS), a Toll‐like receptor‐4 ligand. Knockdown of GRK5 in SW620 cells enhanced proliferation compared to the control cells under unstimulated conditions. Compared to this, LPS treatment had very little effect on proliferation in control cells, whereas proliferation was significantly inhibited in GRK5 knockdown cells. Even though GRK5 knockdown did not affect basal mRNA expression of either CXCL8 or MIP2α, LPS‐induced CXCL8 and MIP2α were markedly inhibited in the GRK5 knockdown cells. Additionally our signaling data provide evidence that GRK5 regulates NFκB and MAPK signalling differentially under basal and TLR4 stimulated conditions. Taken together, our results provide evidence that GRK5 regulates proliferation and chemokine gene expression in the absence and presence of TLR4 stimulation via distinct signaling mechanisms in human colon cancer epithelial cells.
Pif1 helicases: helping replication forks maneuver past replication barriers
Authors: Bochman Matthew L. , Paeschke Katrin , Garcia Patricia , Zakian Virginia A.
Published: June 2021

Pif1 family 5′ to 3′ DNA helicases are found in all three domains of life. Mutation of the human PIF1 is correlated with increased propensity to breast cancer. The best studied Pif1 helicase is the multifunctional S. cerevisiae Pif1 (ScPif1). Published work shows that ScPif1 has key roles in maintenance of mitochondrial DNA, limiting telomerase processivity, processing Okazaki fragments, and suppressing DNA damage at G‐quadruplex (G4) motifs. Here we report in vitro studies that demonstrate that the budding yeast Pif1 and five bacterial Pif1 helicases are extremely proficient at unfolding G4 structures, able to do so even under single cycle conditions. In addition, ScPif1 appears to load onto G4 substrates by interacting with the G‐stacks in the G4 structure rather than the single strand loops. We used the gross chromosomal rearrangement (GCR) assay to determine if Pif1 helicases suppress DNA damage at G4 motifs. The two budding yeast Pif1 proteins, ScPif1 and ScRrm3, four bacterial Pif1 helicases, and fission yeast and human PIF1 are highly effective suppressors of G4‐induced DNA damage. The survivors of G4‐induced instability in Pif1 deficient cells have an unprecedented post‐GCR structure that involves mutation of the G4 motif and stable silencing of the marker genes used in the assay. Like ScPif1, human PIF1 (but not S. pombe or bacterial Pif1 helicases) represses telomere lengthening in S. cerevisiae.
Coordinated regulation of ZnT1, Zip6 and MT‐1 mRNA in cultured choroid plexus elicited by changes in extracellular zinc and prolactin
Authors: Aquino Mayra , Young Robin , Villalobos Alice
Published: June 2021

We are investigating regulation of zinc (Zn) exporters (ZnTs), Zn importers (ZIPs) and the Zn‐binding protein metallothionein (MT) in the choroid plexus epithelium as it adapts to changes in Zn availability or select hormones. By real‐time qRT‐PCR, we analyzed ZnT1, Zip6 and MT‐1 gene expression in cultured choroid plexus epithelial cells treated for 48 h with 10 μM DTPA an extracellular chelator or 25 μM ZnCl2 (n = 3). In Zn‐treated cells, ZnT1 mRNA increased 2‐fold at 3–48 h; MT‐1 mRNA also increased 6‐fold at 3 h, but peaked at 12 h; Zip6 mRNA increased 6‐fold at 12–48 h. Zn chelation reduced ZnT1 mRNA 60% at 3–48 h and reduced MT‐1 mRNA 90% at 6–48 h; however, Zip6 increased 6‐fold at 12–48 h. Prolactin (PRL) increases Zip6 gene expression in mammary epithelia; choroid plexus expresses PRL receptors. Choroid plexus cells were treated for 24 h with 1, 10, 100 and 1000 nM PRL (n = 1). In a concentration‐dependent manner, PRL increased ZnT1 mRNA 30–70%, but increased Zip6 mRNA 3‐ to 6‐fold and increased MT‐1 mRNA 4‐ to 10‐fold. These data indicated coordinated regulation of Zn transporters, e.g., ZnT1 and Zip6, and MT elicited by changes in Zn availability and in response to prolactin.
Mechanism of Per1 Action in Blood Pressure Control: Role of the Circadian Repressor Cry 2
Authors: Richards Jacob , All Sean , Cheng Kit‐Yan , Skopis George , Jeffers Lauren , Gumz Michelle
Published: June 2021

The circadian clock plays an integral role in blood pressure (BP) control. In the canonical model of the circadian clock, the transcription factors Clock and Bmal1 activate circadian target genes, while Period 1 (Per1) and Cryptochrome (Cry 1 or 2) repress this action. Others have shown that mice lacking Clock or Bmal1 are hypotensive, while Cry 1/2 double knockout (KO) mice exhibit salt‐sensitive hypertension. Interestingly, our data show that Per1 KO mice exhibit lower BP than wild type (WT) mice and that Per1 positively regulates expression of the alpha subunit of the renal epithelial sodium channel (αENaC). These results suggest a non‐canonical role for Per1 in the kidney and in the regulation of BP. To address this finding, we first evaluated expression of Bmal1, Clock and Cry2 in renal cortical collecting duct (CCD) cells stably transfected with Per1 shRNA and in nuclear extracts from the renal cortex of WT or Per1 KO mice. Whereas loss of Per1 had no effect on Clock or Bmal1 protein expression, Cry2 protein levels were consistently upregulated in the absence of Per1 in vitro and in vivo. Knockdown of Clock using siRNA resulted in decreased αENaC expression in CCD cells in vitro, mimicking the effect seen with Per1 knockdown. Conversely, Cry2 knockdown appeared to cause increased αENaC expression. Taken together, these data suggest that Per1 action on αENaC regulation and BP may involve the circadian repressor Cry 2.
Identifying causative genetic variants linked to reduced kidney function through congenic strain analysis and whole genome sequencing
Authors: Harmon Ashlyn C. , Johnson Ashley C. , Atanur Santosh , Maratou Klio , Aitman Tim , Garrett Michael R.
Published: June 2021

Factors such as hypertension, diabetes, and obesity contribute to chronic kidney disease (CKD), but genetic predisposition also plays a role. To investigate genetic factors involved in CKD, a novel congenic model [S.SHR(11)] was developed by transfer of a genomic segment (Chr. 11) from the spontaneously hypertensive rat (SHR) onto the Dahl salt‐sensitive (S) genetic background. We recently reported a detailed characterization of the S.SHR(11) model which demonstrated it exhibits accelerated kidney injury compared to the already highly susceptible S rat. At an advanced age, the S.SHR(11) exhibits increased BP, suggesting that kidney injury precedes changes in BP. A panel of congenic substrains [S.SHR(11)X1–9] is being used to systematically fine‐map the causative genetic variants. Whole genome sequencing has been completed, which revealed all genetic variation between the S and SHR models. Twenty‐eight missense SNP were identified within the 95% confidence interval of the genomic locus, with 15 SNP occurring within three genes. Two of these genes, Retnlg and Trat1, are immune‐related genes and are currently being investigated through in vivo and in vitro studies for their potential role in kidney injury. In summary, the congenic strain analysis and high quality sequence information will expedite identification of the causative genetic variations. Supported by RO1‐HL094446 (MRG) and T32‐HL105324 (ACH).
A SILENT POLYMORPHISM OF THE GJD2 GENE, CODING FOR Cx36 CHANNELS, IS ASSOCIATED WITH TYPE 2 DIABETES
Authors: Meda Paolo , Charollais Anne , Cigliola Valentina , Lyssenko Valeriya
Published: June 2021

Cx36 is the only connexin of the pancreatic beta cells, which produce insulin. In mice, loss of Cx36 reduces the expression of the Ins gene, the glucose‐stimulated insulin release, and the resistance of beta cells to immune aggressions. These alterations are reminiscent of those characterizing human type 2 diabetes. We investigated GJD2, the gene coding for hCx36. Using pyrosequencing, we found an exonic, synonymous rs3743123 polymorphism (SNP588) which is associated (p<0.03) with type 2 diabetes at both genotype and allele levels. A genome‐wide association study confirmed this association (p<0.002) in female type 2 diabetics, showing a minimal residual insulin secretion. To investigate the effects of SNP588, we generated clones of HeLa cells expressing either the wild type or the SNP588 form of hCx36. In cells producing the pathogenic protein, we found that the halflife of the Cx36 transcript is unchanged, and that the cognate protein allows for normal cell‐to‐cell coupling. Immunofluorescence labeling and freeze‐fracture electron microscopy indicate that the trafficking of Cx36 protein may be modified by SNP588. These observations indicate that GJD2 is a strong positional candidate for type 2 diabetes. The mechanism whereby a variation of this gene confers a susceptibility to the disease remains to be elucidated.
Identification of Btg2 as a candidate gene for hypertension in females
Authors: Hoffman Matthew J. , Xiao Bing , Nunez Lizbeth , Flister Michael , Moreno Carol
Published: June 2021

We previously found that transfer of a 3.7 Mbp region of chromosome 13 (Chr13) from the normotensive Brown Norway (BN) rat into the hypertensive Dahl salt‐sensitive (SS) rat attenuates the development of salt‐sensitive hypertension in female but not in male rats. We sought to identify the causative gene by developing and phenotyping narrow congenic strains, and by sequence and gene expression analysis of the candidate region. We created two congenic lines with BN substitutions differing by 23 Kb. The congenic regions differ by only one gene, Btg2, and have 35 sequence variants. Mean arterial pressure (MAP), measured by telemetry, was not different between strains in males after 3 weeks of 8% NaCl diet, while female with Btg2BN had significantly higher MAP (186±3.1 mmHg) than Btg2SS females (151±5.8mmHg). Btg2BN females had also significantly higher protein excretion than Btg2SS and two‐fold increase on renal medullary fibrosis. Btg2 mRNA expression was determined by qRT‐PCR in renal cortex and medulla, and BTG2 protein expression was subsequently assessed by western blot. Males had no differences in expression in renal cortex or medulla, but females had significant differences in cortex (Btg2BN: 1.8±0.2 fold change, Btg2SS: 0.4±.01). Our data suggest that Btg2 is a candidate gene salt‐sensitive hypertension in females.
A genetically based GPCR‐mediated central molecular mechanism underlying the pathophysiology of salt‐sensitive hypertension
Authors: Wainford Richard David , Carmichael Casey Yumi , Kuwabara Jill Toshiko
Published: June 2021

Aim

We have demonstrated endogenous up‐regulation of hypothalamic paraventricular nucleus (PVN) Gαi2‐proteins is required to suppress norepinephrine (NE) mediated sodium retention during high salt intake in the Sprague‐Dawley rat. In these studies we examined the role(s) of central Gαi2 proteins in sodium homeostasis and blood pressure regulation following elevated dietary salt‐intake in Brown Norway (BN), Dahl salt‐resistant (DSR), Dahl salt‐sensitive (DSS) and DSSBN8 congenic rat strains.

Methods

BN, DSR, DSS or DSSBN8 rats were maintained on a normal (0.4% NaCl) or high (8% NaCl) diet for 21‐days Additional groups of DSR and DSS rats received a 21‐day i.c.v. infusion of a Gαi2 targeted oligodeoxynucleotide during high salt‐intake. On day‐21 MAP, 24h metabolic balance, plasma NE and PVN Gαi2 protein levels were determined (N=5/group).

Results

HS‐intake did not alter MAP, suppressed plasma NE (P<0.05), and evoked a site‐specific increase in PVN Gαi2 protein levels in salt‐resistant BN and DSR (4.8 and 4.2‐fold respectively, P<0.05), but not hypertensive DSS, rats. Brain Gαi2 protein downregulation evoked hypertension, sodium retention and elevated plasma NE content in DSR rats (P<0.05) and profoundly exacerbated DSS hypertension (P<0.05). In DSSBN8 rats BN chromosome 8 substitution attenuated hypertension, sodium retention and global sympathoexcitation vs. DSS rats (P<0.05) and restored PVN specific Gαi2 protein up‐regulation.

Conclusion

PVN Gαi2 protein‐gated pathways represent a genetically conserved central molecular mechanism that suppresses NE mediated renal sodium reabsorption to maintain a salt‐resistant phenotype. Owing to SNP's in the GNAI2 gene correlating with hypertension in human subjects our findings have translational implications for human hypertension ‐ HL107330.
Epigenetic inhibition by 5 Aza 2′ deoxycytidine mitigates hypertension in hyperhomocysteinemia
Authors: Narayanan Nithya , Pushpakumar Sathnur Basappa , Qipshidze Natia , Bratcher Adrienne P , Tyagi Neetu , Tyagi Suresh C
Published: June 2021

Hyperhomocysteinemia (HHcy) is a risk factor for hypertension which is associated with aortic and cardiovascular pathologies. Although the exact underlying mechanisms are still not clear, emerging evidence implicates a role of epigenetic regulation. We hypothesize that 5 Aza 2 deoxycytidine (5 Aza dC), DNMT inhibitor, lowers blood pressure by regulating methylation levels in HHCy. Wild type (WT) and CBS +/− (HHcy) mice were administered with 5 Aza dC (0.5mg/kg body weight, DNA methyl transferase (DNMT) inhibitor), for four weeks. Blood pressure measured using tail cuff and radio telemetry methods showed a significant decrease in CBS +/− mice following treatment. mRNA expression levels of DNMTs were found to be lower in CBS+/− treatment group when compared to controls. Aortic vessel reactivity was increased in myobath studies following treatment with 5 Aza dC. Ultrasound revealed an increase in abdominal and ascending aorta diameter in treated CBS +/− mice. We conclude that inhibition of DNMT, 5 Aza dC, lowers blood pressure by regulating methylation levels in HHcy and has therapeutic potential in hypertension.
Head to Head: The Role of Competition in Undergraduate Education
Authors: Van Nuland Sonya Elizabeth , Roach Victoria A , Wilson Timothy D , Belliveau Daniel J
Published: June 2021

Opponents argue that academic competition increases student anxiety and divides their attention. Yet, little evidence concerning the application of academic game style competition exists. Could game‐like competition in the classroom be a viable and beneficial method of engaging students? This study aims to sample the effects of anonymous peer competition and performance using an e‐classroom response system (ERS). Students (n= 136) were recruited from an undergraduate anatomy course. Using a crossover design, students were exposed to two competitive treatments, a tournament with many competitive elements and a quiz with few competitive elements. Students were assessed using qualitative surveys, a baseline anatomy knowledge quiz and their course grades. Preliminary data indicates a positive student response toward the use of competitive ERS in education. Approximately 77% of students found the competitive tournament to be engaging. Additionally, 62% reported enjoying the incorporation of competitive elements into their undergraduate anatomy course. We hypothesize that academic competition among peers in an online tournament setting encourages increased familiarization with lecture material, resulting in improved exam performance. Additionally, we believe that knowledge of personal tournament rank will influence a student's scholarly motivation and study habits prior to examination.

Grant Funding Source: Ontario Graduate Scholarship
Exploring the learning environment in a medical school anatomy course: does group emotional intelligence affect team performance?
Authors: Porter Samuel Grant , Holman Michelle , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

Emotional intelligence (EI), the ability to perceive and respond to emotions, has been shown to increase academic achievement and team performance. Studies to support this association exist largely in the business world, but its impact in medical education is relatively unexplored. The purpose of this study was to determine whether emotional intelligence correlates with individual or team academic performance in the anatomy course. This study hypothesizes that individuals or teams with higher emotional intelligence will score higher in the course and that team‐based learning will increase emotional intelligence scores. Medical students were asked to complete the Self‐report Emotions Intelligence Test (SSEIT) at the beginning and the end of the 7‐ week anatomy course. Average EI scores for each team were compared to final course grades as well as cumulative daily quiz grades. Individual EI scores were also compared to individual grades, and change in EI scores over the course was assessed. Preliminary analysis of initial EI scores vs. averaged quiz scores at the midpoint of the course does not appear to show significant correlations. Final results, to be analyzed at the end of the course, may hold stronger predictive value in determining association between EI and team performance. This research is supported by the Department of Anatomy at the Mayo Clinic, Rochester, MN.

Grant Funding Source: Department of Anatomy, Mayo Clinic