bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic [Ca2+]i regulation in human airway smooth muscle by STIM and Orai1 proteins
Authors: Thompson Michael A , Pabelick Christina M , Prakash Y S , Sieck Gary C 
Published: June 2021

Regulation of intracellular Ca2+ in human airway smooth muscle (ASM) involves sarcoplasmic reticulum (SR) Ca2+ release/reuptake and plasma membrane (PM) Ca2+ influx, including store‐operated Ca2+ entry (SOCE) following SR Ca2+ depletion. Studies in human ASM have shown that siRNA suppression of stromal interaction molecule 1 (STIM1) and Orai1 decrease SOCE; however, their exact role in regulating Ca2+ are still under investigation. In human ASM, STIM1 and STIM2 were found exclusively in SR membrane, and Orai1 only in PM (especially caveolae). GFP‐tagged STIM1 transfection showed intracellular/SR localization at baseline, with increased peri‐PM aggregation following store depletion with 10ƒÝM cyclopiazonic acid (CPA). Such aggregation was prevented by microtubule inhibition (nocodazole), but not actin inhibition (phalloidin). FRET analysis revealed co‐localization of STIM1 with TRPC3 and Orai1 in CPA‐stimulated cells. In fura‐2 loaded ASM cells, SOCE was significantly reduced by STIM1 and Orai1 siRNA (less so by STIM2 siRNA). However, basal Ca2+ and agonist (ACh, histamine)‐induced Ca2+ responses were substantially modulated by STIM2 suppression. These results indicate that in human ASM, STIM1 mediates SR‐PM interactions in regulating SOCE, while STIM2 modulates sub‐maximal agonist‐induced changes in Ca2+.Supported by NIH grant HL74309 (GCS), HL090595 (CMP) and HL088029 (YSP).
Impact of BDNF/TrkB signaling on recovery of phrenic activity after cervical spinal cord injury in rats
Authors: Zhan Wen‐Zhi , Mantilla Carlos B , Sieck Gary C 
Published: June 2021

Upper cervical spinal cord injury disrupts descending inspiratory‐related drive to phrenic motoneurons (PMN) paralyzing the diaphragm muscle (DIAm). There is gradual recovery of rhythmic DIAm activity ipsilateral to injury suggesting strengthened spared synaptic inputs to PMN. Neurotrophins play an important role in regulating synaptic function. Thus, we hypothesized that intrathecal brain‐derived neurotrophic factor (BDNF) enhances recovery of rhythmic phrenic activity after C2 spinal cord hemisection (C2HS), whereas quenching neurotrophins with TrkB‐IgG impairs functional recovery. An intrathecal catheter was placed at C4 to chronically infuse artificial CSF, BDNF or TrkB‐Fc using an osmotic pump. DIAm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of C2HS and 3D post‐C2HS. In the artificial CSF group, functional recovery was evident in 33% of C2HS rats at 7D and 56% of rats at 14D post‐injury. Intrathecal BDNF treatment enhanced functional recovery, with 88% of rats at 7D and 100% at 14D post‐C2HS displaying rhythmic phrenic activity. In contrast, intrathecal TrkB‐Fc treatment completely prevented functional recovery up to 14D post‐C2HS. Our results demonstrate that BDNF/TrkB signaling at the level of the PMN pool is both sufficient and necessary for functional recovery of rhythmic phrenic activity after C2HS.Supported by NIH grant AR51173.
High Frequency Spinal Cord Stimulation (HF‐SCS) to Provide Ventilatory Support in an Animal Model of Spinal Cord Injury
Authors: DiMarco Anthony F. , Kowalski Krzysztof E. 
Published: June 2021

ObjectiveHF‐SCS is a novel technique of inspiratory muscle activation via spinal cord pathways. The purpose of this study was to evaluate the utility of HF‐SCS to provide long‐term ventilatory support utilizing intercostal/accessory muscle stimulation alone.MethodsIn 4 anesthetized dogs after bilateral phrenicotomy, electrical stimulation (300Hz, 1.2mA, 12 breath/min, 1.2s inspiratory time) was applied via a single electrode placed ventrally at the T2 spinal level over an arbitrary period of 5.5 hours.ResultsMean airway pressures and inspired volumes were 29±1cmH2O and 445±7ml during the first and 27±1cmH2O and 450±12ml during the final hour of chronic pacing, respectively (NS). Throughout the period of stimulation, airway pressure generation and inspired volume production remained unchanged. Mean end‐tidal PCO2 and SaO2 were 40±1mmHg and 95±1% and 40±1mmHg and 96±1% during the first and final hour of pacing, respectively (NS). To further assess the capacity of the system to maintain artificial ventilation, stimulus parameters were then increased to induce hyperventilation for an additional 30 min. This resulted in a sustained fall in end‐tidal PCO2 to 30±1 mmHg (p<0.05).ConclusionHF‐SCS has the potential to provide complete ventilatory support in subjects with ventilator‐dependent tetraplegia who lack adequate phrenic nerve function for diaphragm pacing.Support: NIH‐NINDS (R01NS064157)
Vagally‐mediated gastrointestinal reflexes are modulated by central glucose levels
Authors: Browning Kirsteen N 
Published: June 2021

The vagally‐mediated receptive relaxation reflex is activated upon distension of the esophagus and induces gastric relaxation and delays gastric emptying. Many gastrointestinal (GI) hormones exert dramatic control over vagally‐mediated GI functions via brainstem sites of action. The aim of this study was to determine whether glucose‐induced alterations in GI reflexes are achieved, in part, by brainstem sites of action.A balloon was used to distend the esophagus of adult rats and the resulting gastric relaxation measured via miniature strain gauges affixed to the stomach. Increasing blood glucose levels decreased the magnitude of the receptive relaxation from 0.6±0.1g to 0.4±0.1g and 0.3±0.1g at 3±0.2mM glucose, 6±0.5 and 10±1.8mM blood glucose, respectively (n=3). Similarly, altering brainstem glucose directly (via 4th ventricular application 3, 4.5 or 10mM glucose) decreased the receptive relaxation by 5±3, 15±0 and 20±3%, respectively (n=3). The glucose‐induced decreased receptive relaxation was blocked by brainstem application of the 5‐HT3 antagonist, ondansetron (30nmoles; 15±0.3% reduction to 10mM glucose vs 0±0.1% reduction in the presence of ondansetron, n=3).These results suggest that alterations in brainstem glucose levels, either directly or indirectly, exerts profound effects upon vagally‐mediated GI reflexes possibly via mechanism involving alterations in brainstem 5‐HT3 levelsSupported by DK 55530
Influence of Perfluorocarbons on Systemic Parameters of Transgenic Sickle Cell Mice
Authors: Andrade Gabriela Chagas , Pedro Flavia P. , Torres Ivo P. 
Published: June 2021

Transgenic (Tg) sickle cell disease (SCD) mice have been used to study SCD pathophysiology. Perfluorocarbon (PFC) is a synthetic oxygen (O2) carrier that can improve the blood O2 content. Thus, physiological responses to hypoxia (hyp)/reoxygenation (reox) were studied in Tg SCD mice treated with PFC. Anesthetized Tg mice were subjected to 60 min of hyp (FiO2=13%) followed by 90 min of reox (FiO2=50 %). PFC (Oxycyte, Oxygen Biotherapeutics, Inc, 6ml/kg BW) was injected at the end of the hyp. O2 saturation (SO2), heart rate (HR), and breath rate (BR) were measured each 5 min. Hemoglobin concentration (Hb), hematocrit (HCT) and venous PO2 were measured at the end of the experiment. Homozygous SCD mice (n=11) presented Hb= 6.7 ± 1.2 g/dL (mean ± SD) and HCT=31.1 ± 3.9 %, heterozygous (n=16) showed Hb=12.7 ± 1.5 g/dL and HCT=39.5 ± 3.5 %), while control mice (CB57BL6) (n=4) showed Hb=14.5 ± 1.3 g/dL and HCT=48.7 ± 3.3 %. PFC‐treated homozygous mice (n=6) showed lower SO2 (p<0.02) than the saline group (99.1 ± 0.8 % and 99.6± 0.3 %, respectively) but BR and PO2 were significantly higher in PFC‐treated mice (141 ± 8 bpm vs 134 ± 11 bpm and 51 ± 6 mm Hg vs 41 ± 9 mm Hg, respectively). HR in the PFC group was also significantly higher than in saline‐treated mice (593 ± 16 bpm vs 564 ± 16bpm). The results suggest that PFC therapy may improve cardiac and respiratory responses to hyp/reox in these severely anemic SCD mice.Support: Oxygen Biotherapeutics, Inc.
Visceral adipose tissue DNA methylation at dipeptidyl peptidase‐4 gene locus is associated with gene expression and plasma lipid levels in severe obesity
Authors: Turcot Valerie , Bouchard Luigi , Tchernof Andre , Deshaies Yves , Marceau Simon , Biron Simon , Lescelleur Odette , Berthio Laurent , Vohl Marie‐Claude 
Published: June 2021

Dipeptidyl peptidase‐4 (DPP4) is overexpressed in visceral adipose tissue of severely obese subjects with the metabolic syndrome (MS) as compared to those without MS.AimTo test whether methylation of DPP4 is associated with DPP4 mRNA levels and MS‐phenotypes.MethodsGreater omentum fat tissue DNA was extracted in 92 severely obese women undergoing biliopancreatic derivation to treat morbid obesity. Women were premenopausal, non‐diabetic and not taking medications to treat MS features. Cytosine methylation levels (%) in DPP4 CpG island (Human build 36.3; chr2: 162638045‐162639319 reverse strand) was assessed by pyrosequencing of sodium bisulfite treated DNA.ResultsMean methylation levels of combined CpG sites no.94–102 (%Meth) were calculated. The %Meth correlated negatively with DPP4 mRNA levels (r=0.25, p<0.05). Subjects with %Meth corresponding to the second tertile had lower total‐ and LDL‐cholesterol levels (PANOVA<0.05; adjusted for age, smoking, body mass index).ConclusionsIn visceral adipose tissue of severely obese women, lower methylation levels of specific DPP4 CpG sites were associated with higher mRNA levels. Methylation levels were also associated with blood lipids.Grant Funding Source: Canadian Institutes of Health Research
Restoration of fatty acid but not glucose utilization in the infarcted heart following stem cell therapy
Authors: Hughey Curtis , Ma Lianli , James Freyja , Young Pampee Paul , Wasserman David , Rottman Jeff , Shearer Jane 
Published: June 2021

AimTo determine whether stem cell therapy reduces abnormal cardiac substrate utilization following a myocardial infarction (MI).MethodsThree treatment groups, SHAM, MI and MI‐STEM receiving intramyocardial injection of mesenchymal stem cells. Echocardiography was performed at 0, 1 and 3wk. C57BL/6J mice underwent a hyperinsulinemic‐euglycemic clamp 4wk following treatment (n=8–11). To assess indices of whole body and cardiac long chain fatty acid (LFCA) and glucose utilization (R(f), R(g); umol/g wet weight/min), 2‐deoxy[(3)H]glucose and [125I]‐15‐(rho‐iodophenyl)‐3‐R,S‐methylpentadecanoic acid were administered. Both the peri‐infarct (PI) region and a non‐involved (NI) area of the left ventricle were examined.ResultsNo differences in whole body substrate utilization or insulin sensitivity, as assessed by glucose infusion rate were apparent between treatments. In the PI region, MI resulted in declines in R(f) compared to SHAM (0.25 vs 0.44). This decline was mitigated in MI‐STEM (0.33 vs 0.44). In contrast, R(g) was diminished in both the MI and MI‐STEM groups in the PI (1.77 vs 2.0 vs 3.87) compared to the SHAM. Additionally, R(f) and R(g) in the NI was not different between groups.ConclusionsStem cell therapy assists in reducing the metabolic abnormalities following a MI. Specifically, it minimizes MI‐induced declines in R(f) but not R(g).
A Pathological Role for Protease‐Activated Receptor 2 in the Heart
Authors: Pawlinski Rafal , Antoniak Silvio , Spring Denise , Verrier Edward D. , Blaxall Burns C. , Mackman Nigel 
Published: June 2021

We investigate the role of protease‐activated receptor 2 (PAR2) in heart pathology. PAR2 mRNA expression was analyzed by real‐time PCR in heart biopsies from patients with heart failure (HF). The role of PAR2 was investigated in mouse model of cardiac ischemia‐reperfusion (I/R) injury. Finally, we examined the effect of a cardiomyocyte‐specific overexpression of PAR2 in mice (TgPAR2). We found increased PAR2 mRNA levels in patients with ischemic HF (3.0±0.7fold) and idiopathic HF (4.3±0.6fold; P<.05 vs. controls). These levels returned to baseline after implantation of a LVAD. Interestingly, the infarct size was reduced in the hearts of PAR2−/− mice (24.3±1.7% vs. 35.8±3.4%; P<.05 vs. WT) after 2 h of reperfusion. This injury reduction was associated with less expression of pro‐inflammatory genes. PAR2−/− were protected from I/R injury‐induced heart remodeling determined by ECHO (PAR2−/− vs. WT: LV diameter: 1.43±0.06mm vs. 1.78±0.09mm; fractional shortening (FS): 49.3±1.2% vs. 40.9±1.6%; P<.05). Finally, we observed that 1 year old TgPAR2 mice developed heart hypertrophy with increased HW/BW ratio. ECHO analysis revealed that TgPAR2 mice had reduced heart function (FS: 38.3±5.1% vs. 24.5±6.7%; WT vs. TgPAR2; P<.05). These results indicate that PAR2 in the injured heart enhances inflammation and infarct size that leads to hypertrophy and HF.This study was supported by the AHA and NIH.
Integrative approach to study the molecular basis of post‐myocardial infarction remodeling in porcine heart
Authors: Kuster Diederik , Verhoeven Adrie , Merkus Daphne , Duncker Dirk 
Published: June 2021

After myocardial infarction (MI), the left ventricle (LV) undergoes remodelling in an attempt to maintain cardiac performance. Despite the apparent appropriateness, LV remodeling is an independent risk factor for heart failure. Although much is known about the role of individual proteins in cardiac remodeling, an integrative unbiased approach has been lacking, especially in large animal models, such as the pig. Therefore, we studied gene expression with microarray and transcription factor (TF) activity with Protein/DNA array and linked these to in vivo measurements of LV remodeling and function in pigs, by using network analysis. Three weeks after MI, LV remodeling and dysfunction as well as blunting of the β‐adrenergic response were noted in vivo. At gene expression level, matrix metalloproteinases were upregulated and the β‐adrenergic/cAMP signaling was disturbed. Network analysis suggested a role for inflammation and calcineurin signaling. Also several transcription factors were activated or deactivated. Linking these TFs to microarray data suggested an important role for glucocorticoid receptor (GR).The present study shows that an integrative approach, linking in vivo observations with changes in gene expression and TF activity leads to new insights in the molecular basis of post‐MI remodeling in a large animal.This study was supported by a grant from the Netherlands Heart Foundation (2005B234)
Decrease IL33 expression in cardiac fibroblasts with high concentration of glucose leads to collagen IV production: role of PKCβ
Authors: Su Zhaoliang , Xu Hu , Zhang Jingchao , Martin Claudio M. , Rui Tao 
Published: June 2021

Diabetic cardiomyopathy (DiCM) is one of major complications of diabetes mellitus. Increase in collagen production by cardiac fibroblasts has been implicated in the development of the DiCM. In the present study, we assessed the role of PKCβ/IL‐33 pathway in the high glucose (HG)‐induced collagen production in cardiac fibroblasts.MethodsCultures cardiac fibroblasts were exposed to medium containing high concentration of glucose. The PKCβ phosphorylation status, IL33 and collagen production were assessed with Western or real‐time RT‐PCR.ResultsTreatment of the cardiac fibroblasts with HG led to a decrease in IL33 mRNA and protein expression which was associated with an increase in collagen IV production. Administration of IL‐33 prevented HG‐induced collagen IV production. Expose of cardiac fibroblasts to HG resulted in PKCβ;activation as indicated by increase in phosphorylation of PKCβ;Furthermore, inhibition of PKCβ;with an inhibitor prevented the increase in collagen IV production in HG‐treated cardiac fibroblasts.ConclusionOur results suggest that PKCβ/IL33 pathway plays an important role in regulation of HG‐induced collagen IV production in cardiac fibroblasts. (CIHR MOP‐81303)
Stereologic sampling and quantitation of Mycobacterium tuberculosis (M.tb.) lesions in rhesus monkey lungs
Authors: Hyde Dallas M , Adamson Lou , Canfield Don , Tarara Ross , Hirst Linda , Christianson Miles , Wisner Eric , Ventimiglia Frank , Luciw Paul 
Published: June 2021

TB is a global infectious killer. Detection systems for TB are not efficient, TB drugs are over 40 years old, and the BCG vaccine dates back over 80 years; hence, there is an urgent need to develop methods to find faster, easier and more effective diagnostics, drugs and vaccines to control TB. Research in nonhuman primate models addresses these needs. Six healthy macaques were intratracheally inoculated with 500 CFU of the clinical M.tb. Erdman strain and followed for disease progression. M.tb. infection was confirmed in all 6 monkeys. At 3 months after inoculation, 2 animals developed significant TB symptoms and were euthanized and necropsied. The remaining 4 animals were necropsied at 6 months. Lungs slices (5 mm thick), overlaid with a Plexiglas template with 5 mm holes, were used to cut cores for the volume of TB lesions and bacterial burden. Lung cores were fixed, embedded, sectioned and stained with H and E. We estimated the volume of TB lesion by stereologic point counting, and homogenates of lung cores were serially diluted and plated to measure CFU. The volume TB lesions and the number of M.tb. CFU were estimated from lung core samples. Our results show that a Plexiglas overlay and circular knives enables a stratified random sampling of TB infected lung tissue in a BSL‐3 environment that produces accurate and efficient estimation of the amount of TB lesions and the number of TB bacteria.NCRR RR000169 and Aeras Foundation
Chemical‐genetic activation of the Unfolded Protein Response reduces protein misfolding
Authors: Chiang Wei‐Chieh , Joseph Victory , Messah Carissa , Lin Jonathan H 
Published: June 2021

Unfolded Protein Response (UPR) comprises a set of intracellular signaling pathways ‐ controlled by Ire1, Perk, and Atf6 genes ‐ that detect protein misfolding in the endoplasmic reticulum (ER) and activate a broad transcriptional program that restores cellular proteostasis. UPR transcriptional targets include chaperones, ER‐associated protein degradation (ERAD) and the ubiquitin‐proteasome system components, and ER structural and transport proteins. Many human diseases arise from protein misfolding. We hypothesized that UPR signaling could be harnessed to prevent protein misfolding. To test this, we developed chemical‐genetic tools that allowed us to selectively activate individual UPR pathways. We then assessed how individual UPR pathways affected rhodopsin protein bearing a proline to histidine missense mutation at residue 23 (P23H). P23H rhodopsin was previously demonstrated to form insoluble aggregates that are retained in the ER and ultimately cause cell death and hereditary Retinitis Pigmentosa. We found that artificial UPR signaling selectively removed misfolded rhodopsin from the ER and enhanced its degradation. This suggests that UPR signaling pathways may be used to treat protein misfolding diseases.(Supported by NIH EY018313, Fight for Sight Foundation, Cystic Fibrosis Foundation, and American Federation for Aging Research)
Campylobacter jejuni disrupts polarized TLR9 signaling in colonic epithelial cells
Authors: O'Hara Jennifer , Friis Lorna , Feener Troy , Szymanski Christine , Beck Paul 
Published: June 2021

Campylobacter jejuni infection is associated with an increased risk of developing inflammatory bowel disease (IBD) via mechanisms that remain obscure. TLR9 signaling in response to bacterial DNA is essential to intestinal homeostasis, possibly by regulating the T helper (Th) 17‐cell line. IL‐25 is a known inhibitor of the proinflammatory Th17 pathway.AimTo assess whether C. jejuni contributes to the development of IBD by disrupting TLR9 signaling using a human colonic (T84) cell line and a DSS model of colitis.ResultsApical application of a TLR9 agonist (ISS‐ODN, 5μg/mL) elicited a significant increase in transepithelial resistance across control T84 monolayers, indicating a tightening of the epithelial barrier. This response was lost in C. jejuni‐infected monolayers. Infected cells secreted significantly more IL‐8 in response to a basolaterally applied TLR9 agonist when compared to control cells. Real‐time PCR revealed a greater than 2 fold increase in TLR9 expression in human colonic biopsies exposed to C.jejuni. C57Bl/6 mice inoculated with 1 × 108 C.jejuni demonstrated an increase in occult blood score and a significant reduction in colonic IL‐25 levels following treatment with DSS compared to mice treated with C. jejuni or DSS alone.ConclusionsC. jejuni may increase the risk of developing IBD by disrupting epithelial TLR9 signaling and reducing IL‐25 levels. Funded by CAG‐CIHR‐CCFC.
Decreased expression of Paneth cell antimicrobial peptides coincide with bacterial translocation after starvation
Authors: Hodin Caroline M , Lenaerts Kaatje , de Haan Jacco‐Juri , Grootjans Joep , Hadfoune M'hamed , Hooper Lora V , Heineman Eric , Buurman Wim A 
Published: June 2021

The lack of enteral feeding, with or without parenteral nutritional support has been reported to result in increased translocation of bacteria, which is considered to play an important role in the high morbidity and mortality rates of critical illnesses such as acute pancreatitis. Recently, Paneth cells, important constituents of the innate intestinal immunity, were shown to be crucial in host protection against invasion of both commensal and pathogenic bacteria.This study investigates the influence of food deprivation on Paneth cell function and small intestinal integrity. Significant decreases in RNA expression of lysozyme (p< 0.01), cryptdin (p< 0.05) and RegIIIγ (p<0.05) were seen after 48 hours of food deprivation in C57BL/6 mice (n=15/group). Accordingly, protein expression of lysozyme and the precursor form of RegIIIγ were also shown to decrease significantly (p<0.01, p<0.05 respectively). Furthermore, increased bacterial translocation to mesenteric lymph nodes occurred (p<0.01), while HE staining showed normal intestinal architecture, distribution and expression of respectively tight junction proteins zonula occludens‐1 and claudin‐3 were unchanged.In conclusion, the current study demonstrates that enteral starvation causes Paneth cell dysfunction paralleled by increased bacterial translocation without disturbances of the small intestinal physical barrier.
Intravascular flow fields shape intussusceptive pillars in the chick chorioallantoic membrane
Authors: Lee Grace S. , Filipovic Nenad , Milosevic Zarko , Miele Lino F. , Lin Miao , Simpson Dinee C. , Konerding Moritz A. , Tsuda Akira , Mentzer Steven J. 
Published: June 2021

Intussusceptive (nonsprouting) angiogenesis is an essential process in the development of the chick chorioallantoic membrane (CAM). The influence of intravascular flow fields on intussusceptive pillar formation in the CAM, however, remains largely unknown. Using fluorescent vascular tracers and digital time‐series video reconstructions, intussusceptive pillars and the surrounding flow fields were identified during the development days 12–16. The mechanical forces within the CAM vessels, including wall shear stress, velocity, and blood pressure, were mapped using 3D finite element models and computational flow simulations. The pillars demonstrated a quasi‐circular circumference (shape factor 0.41+0.13) and comprised 1.7+1.2% of the bifurcation cross‐sectional area within 2nd and 3rd order vessels. The presence of pillars was independent of vessel diameter (R=0.32) or bifurcation angle (R=0.48) (p>0.4), but blood flow surrounding the pillars demonstrated a narrow range of velocities. More importantly, the location and geometry of the pillar correlated closely with the streamlines of the dominant vessel flow (p=0.02). We conclude that these spatial dynamics reflect a responsiveness of blood vessels to intravascular flow fields and indicate the importance of mechanical forces in shaping both intussusceptive angiogenesis and vessel structure in the microcirculation.Grant Funding Source: NIH Grant HL47078, HL75426 and HL94567