bluuebunny/crossref_metadata_2025_split huggingface dataset:
The significance of IL‐10 stimulation in cardiac innate responses (1095.18)
Authors: Bagchi Ashim , Akolkar Gauri , Mandal Soma , Gao Xiaoling , Yang Xi , Singal Pawan 
Published: June 2021

It is known that the cardioprotective role of IL‐10 may involve innate signaling via the activation of patterns recognition receptors, such as Toll‐like receptor 4 (TLR4). We have previously demonstrated that myeloid differentiation gene factor 88 (MyD88) plays a key role in this innate response to IL‐10. In the present study, we further characterized downstream details in IL‐10 stimulation of innate signaling using IL‐10 knockout (KO) mice. A significant increase in the expression of TLR2 was noticed in the KO mice hearts whereas TLR4 expression was not different from the wildtype (WT). There was a significant downregulation in MyD88 expression. The expression of interleukin‐1 receptor associated kinases ‐1 (IRAK‐1) was slightly but significantly less in IL‐10 KO hearts with almost no difference in IRAK‐4 expression. On the contrary, IRAK‐M (an inhibitor of MyD88‐ dependent TLR4 signaling pathway) and IRAK‐2 levels were higher in the KO mice compared to WT. Levels of TNF‐α in IL‐10 KO mice were higher both in the heart and blood. IL‐1β was elevated in the blood of IL‐10 KO mice; however, there was no change in cardiac expression. TLR2‐ mediated TNF‐α activation in IL‐10 KO mice led to an increase in apoptosis as evident from increased expression of pro‐apoptotic protein, Bax and Bax/Bcl‐xL ratio. In the KO hearts, the increased proteolytic enzyme activity of caspase3/7 as well as fibrosis in the heart suggested that TLR2‐mediated IRAK‐M/IRAK‐2 activation might enhance TGF‐β and TNF‐α. Thus an increase apoptosis as well as fibrosis in the IL‐10 KO mice suggest that this cytokine is an important player in restoring cardiac cells from damage.Grant Funding Source: Supported by the Canadian Institutes of Health Research
Long‐term atorvastatin treatment inhibited pro‐survival signaling, reduced mitochondrial function and altered ultrastructural integrity in cardiac myocytes (1095.6)
Authors: Zemljic‐Harpf Alice , Godoy Joseph , Schilling Jan , Kassan Adam , Schwarz Anna , Asfaw Elizabeth , Alvarez Erika , Dalton Nancy , Niesman Ingrid , Patel Hemal 
Published: June 2021

Introduction: Statins are widely used to lower low‐density lipoprotein cholesterol (LDL‐C) for prevention of cardiovascular disease. The FDA warns that statins may induce skeletal muscle side effects. Skeletal muscle biopsies from patients with statin myopathy showed defects in mitochondrial ultrastructure. Impaired mitochondrial function was postulated as a key cause of statin‐induced myopathy. Long‐term statin effects on cardiac muscle are currently unclear.Methods and Results: Neonatal cardiac myocytes were treated with either atorvastatin (lipophilic) or pravastatin (hydrophilic) for up to 48 hours (both at 1‐10 μM). Atorvastatin, but not pravastatin, inhibited RhoA activation, reduced mTOR/AKT signaling, and induced apoptosis. Further, atorvastatin treatment reduced protein expression of caveolin, dystrophin and epidermal growth factor receptor in cardiac myocytes. Both statins induced ER stress responses, but only atorvastatin increased CHOP initiated apoptotic cell death. Assessment of mitochondrial function after 48 hours of statin therapy revealed reduced oxygen consumption rates in atorvastatin treated cardiac myocytes, when compared to pravastatin or vehicle treated cells. The extracellular acidification rate profile was unchanged between groups at all time points. When mice underwent long‐term atorvastatin or pravastatin (5mg/kg/day for 7 months) treatment, both statins reduced LDL‐C, but only atorvastatin increased creatine kinase serum levels. Atorvastatin therapy in mice altered mitochondrial ultrastructure and induced protein aggregate formation in the heart. Atorvastatin, but not pravastatin, increased mortality in cardiomyopathic mice, when compared to vehicle treated controls.Conclusion: Atorvastatin reduced mitochondrial function, altered cardiac ultrastructure and induced apoptosis in cardiac myocytes. Our data suggest that long‐term atorvastatin treatment impaired mitochondrial integrity in cardiac muscle.
Effect of altered endogenous melatonin concentration by conditional light control on cellular organization in injured spinal cord (1096.2)
Authors: Jin Yunho , Hong Yunkyung , Park Kanghui , Hong Yonggeun 
Published: June 2021

Spinal cord injury(SCI) causes primary and secondary damage, leading to neurologic deficits. We aimed to determine the effect of altered melatonin level in cerebrospinal fluid(CSF) by conditional light control on cellular organization and motor recovery of SCI rats. Rats were divided into 3 groups and pre‐adapted under each condition for at least 2 weeks prior to operation; L/D(12/12‐hr light/dark), LL(24‐hr constant light), DD(24‐hr constant dark). After microsurgery, we collected injured cord segments and CSF. Rats under DD condition exerted higher BBB scores than that of L/D from day 7 through 28, while LL condition induced reduction of BBB scores than L/D on day 21 and 28. Melatonin concentration in CSF was the highest in DD on day 3, which was concomitant with NeuN upregulation in injured cord lesions. Elevated melatonin concentration in DD condition was sustained day 7 only. Although NeuN expression was significantly increased in all of groups at day 28 compared to day 3, its expression was the lowest in LL condition. Olig2 was also raised in DD condition on day 28. These results suggest that higher endogenous melatonin concentration in CSF may prevents neuronal cell loss but also promotes oligodendrocyte differentiation in injured cord region, leading to motor recovery in SCI rats.Key words: Spinal cord injury, Cerebrospinal fluid, Endogenous melatonin, Light control, NeuN
Inhibition of IClswell impairs TNFα‐dependent activation of NF‐κB (1096.3)
Authors: Ettinger Nicholas , Nguyen Hong‐Ngan , Manlove Courtney , Lamb Fred 
Published: June 2021

Cytokines such as TNFα drive inflammation via transcription factors including NF‐κB. Biologic agents targeting TNFα modulate disease, but require I.V. administration and cause immune suppression. Thus there is a need to develop oral, titratable therapies. In smooth muscle cells, NF‐κB activation by TNFα depends upon intracellular, endosomal ROS production by Nox1 and deficiency of ClC‐3 disrupts ROS production at this site. TNFα also activates IClswell via endosomal ROS. We hypothesized that IClswell activity is required for NF‐κB activation by TNFα. We used a luciferase‐NF‐κB reporter to quantify responses to TNFα (10ng/ml). Activation of HEK293 cells was reduced by both Tamoxifen (10μM, 13.9±3.5 to 6.4±2.2 fold) and DCPIB (30μM, 41.5±4.9 to 8.2±0.8 fold, N=6). In addition, inhibition of this channel in HUVECs with tamoxifen resulted in a 40% decrease in NFκB activation (N=6). These data suggest that IClswell activation by TNFα may play a role in NF‐κB signaling. IClswell has also been hypothesized to allow exit of ROS from the endosome to facilitate downstream cell signaling. Thus, we tested whether inhibition of ROS production would also lead to a decrease in NFκB activation. Inhibition of ROS production by TNFα with DPI resulted in a five‐fold increase in relative NFκB activation (N=4). Identification of the specific role of this anion conductance and localization of its activity to the plasma membrane or endosomes is the subject of ongoing investigation.Grant Funding Source: NIH 5T32HD068256‐03
Role of cardiac fibroblasts in myocardial dysfunction in mice with sepsis: effect of NLRP3 inflammasome activation (1096.7)
Authors: Zhang Wenbo , Xu Xuemei , Kao Raymond , Mele Tina , Martin Claudio , Rui Tao 
Published: June 2021

Background: Inflammasomes are intracellular platforms which promote maturation and releasing of IL‐1β and IL‐18. Cardiac fibroblast is the most abundant non‐cardiomyocytes within the heart. The aim of the present study was to evaluate whether sepsis activates NLRP3 inflammasome in cardiac fibroblasts and subsequently affects function of cardiomyocytes through IL‐1β thereby results in myocardial dysfunction. Materials and Methods: NLPR3, procaspase‐1, caspase‐1 p10, pro‐IL‐1β and IL‐1β in cardiac fibroblasts were assessed with Western blot or ELISA. Cardiomyocyte intracellular cAMP was assessed with direct immunoassay kit. Mouse model of sepsis was induced by intraperitoneal injection of LPS. Mouse myocardial function was evaluated with a mouse pressure‐volume loop analysis system. Results: Treatment of cardiac fibroblasts with LPS resulted in activation of capase‐1 (increased p10) and induced maturation and releasing of IL‐1β. Genetically (siRNA) and pharmacologically (Glyburide) inhibition of NLRP3 inflammasome in cardiac fibroblasts blocked the LPS‐induced activation of the caspase‐1, as well as the maturation and releasing of the IL‐1β. Inhibition of NLRP3 inflammasome in cardiac fibroblasts prevented the decrease in intracellular cAMP in cardiomyocyte conditioned with supernatants of cardiac fibroblasts with LPS. Inhibition of the NLRP3 inflammasome attenuated the myocardial dysfunction in mice with sepsis. Conclusions: Activation of NLRP3 inflammasome in cardiac fibroblasts results in increased IL‐1β production which mediates the cardiac fibroblast‐cardiomyocyte interaction and promotes the induction of myocardial dysfunction in mice with sepsis. (HSFO GIA 2012‐000212 to TR).Grant Funding Source: HSFO
Modulation of the CX3CL1/CX3CR1 signaling pathway by acetylsalicylic acid (aspirin) in human trophoblast (1096.9)
Authors: Szukiewicz Dariusz , Alkhalayla Habib , Stangret Aleksandra 
Published: June 2021

Aspirin inhibits cyclooxygenase (COX), acting stronger on the COX‐1 than the COX‐2. COX‐independent antiinflammatory effects of aspirin were also reported. Signaling pathway related to the chemokine CX3CL1 and its receptor CX3CR1 was proposed as a new target for aspirin. CX3CL1 modulates inflammatory response within the feto‐placental unit, influencing both adhesion and chemotaxis. HT produces CX3CL1 and expresses the CX3CR1. We examined the effects of aspirin on CX3CL1 production and CX3CR1 expression in HT obtained from normal term pregnanies (N = 24). The cytotrophoblasts were isolated using Kliman’s method based on trypsin, DNase and a 5‐70% Percoll gradient centrifugation, and cultured in normoxia. Secretion of CX3CL1 was augmented by lipopolysaccharide (1μg/ml). Next, the cultures were continuously exposed to aspirin, given in 6 different concentrations (1mM to 6mM) in respective groups. CX3CL1 levels in the culture media were measured using ELISA. After 5 days the cultures were fixed and immunostained for CX3CR1. CX3CR1 expression was examined by quantitative morphometry. Aspirin administered in higher doses (4.0mM and above) produced significant (p < 0.05) inhibition of CX3CL1 production in HT. This effect was inversely proportional to significantly increased (p < 0.02) expression of CX3CR1. Thus, influencing CX3CL1/CX3CR1, aspirin interferes with placental chemokine signaling network.Grant Funding Source: Supported by WUM grant: 2M2‐W2‐13.
Protease‐activated receptor ‐3 signals independent of protease‐activated receptor‐1 to regulate endothelial nitric oxide synthase in human endothelial cells (1097.1)
Authors: Tillery Lakeisha , Motley‐Johnson Evangeline 
Published: June 2021

Protease‐activated receptor‐3 (PAR‐3) is widely recognized as a non‐signaling elusive member of a G protein‐coupled receptor family known to regulate the endothelial nitric oxide synthase (eNOS)/nitric oxide (NO) signaling pathway via phosphorylation of its regulatory sites Thr495 and Ser1177. The eNOS/NO pathway is responsible for vascular smooth muscle cell relaxation and disruption of this pathway leads to endothelial dysfunction, laminar shear stress and vascular inflammation, a precursor to cellular induced hypertension. Using human coronary artery endothelial cells (HCAECs) as a model, we demonstrated that PAR‐3 is highly co‐expressed with PAR‐1 and ‐2 and can be activated using a synthetic peptide ligand, TFRGAP. We hypothesize that PAR‐3 can signal independently of PAR‐1 and ‐2 to regulate eNOS via site‐ specific phosphorylation and induce pro‐inflammation via the NFkB pathway. Analysis using eNOS specific antibodies showed that 500uM of TFRGAP lead to the phosphorylation of the positive site of eNOS‐Ser1177 and the negative site ‐Thr495, 30 seconds and 15 minutes respectively. Pretreatment of HCAECs with 10uM SCH79797, a selective PAR‐1 inhibitor, did not block the increase in phosphorylation at both eNOS regulatory sites stimulated by TFRGAP. Similar results where found in a stable PAR‐1 deficient cell line of HCAECs which confirmed the signaling potential of PAR‐3 using thrombin and the PAR‐3 activating peptide. Our data suggests that PAR‐3 is able to regulate eNOS/NO signaling via site‐specific phosphorylation that is independent of PAR‐1 activation. In additional studies cytokine production was measured using an ELISA after PAR‐3 activation. Data from these studies will lead to a better understanding of endothelial dysfunction and vascular inflammation while identifying potential drug targets for cardiovascular diseases.Grant Funding Source: The project [poster] described was supported by CTSA award No. UL1TR000445
Dynamic measurements of [K+]i in cardiac differentiated P19 cells with a quantum dots (QD) potassium nanobiosensor, K‐QD (1097.5)
Authors: Wong Lid , Mao Hua 
Published: June 2021

We reported the first dynamic measurements of [K+]i in intact differentiated P19 cells using a K‐QD nanobiosensor. K‐QD was synthesized by conjugating a 15‐crown‐5 K+ receptor to water soluble QD emitted at 630 nm. In Vitro spectral characterization of K‐QD confirms the sensitivity and specificity of K‐QD that is operated within the physiological K+ ranged from 4 to 130 mM. Differentiation of P19 cells was induced with 1% DMSO. A photon counting fluorescence microscope system with 405 nm excitation and 630nm emission was used to perform experiments with K‐QD loaded in the differentiated P19 cells using cation liposome. In four studies, 10‐8M to 10‐5M cumulative dose responses of [K+]i of each of the following 10 agents were determined. They were (i) quinidine, TEA and glibenclamide for the inhibition of the efflux of K+; (ii) bumetanide, ouabain, and acetylstrophanthidin for the inhibition of the influx of K+; (iii) levocromokalim and NS1619 for the activation of the K+ efflux; and (iv) nifedipine and verapamil for the inhibition of the calcium channels. Five experiments were performed per agent. The predictable and consistent responses induced by all these agents suggest that the application of K‐QD to determine the [K+]i responses in the cardiac differentiated P19 cells could be a robust platform for K+ channel compound screening, drug discovery and toxicology.Grant Funding Source: NIDDK R44‐DK084600
NHE8’s role in male fertility involves LHR protein trafficking (1097.8)
Authors: Xu Hua , Chen Huacong , Li Jing , Ghishan Fayez 
Published: June 2021

We reported last year that NHE8, the newest member of the sodium/hydrogen exchanger family, plays an important role in male fertility. NHE8 has abundant expression in the male reproductive organs, particularly in Leydig cells and epididymal duct cells. Male mice lacking NHE8 are infertile. Loss of NHE8 expression also resulted in reduced luteinizing hormone receptor (LHR) expression in male mice. Furthermore, luteinizing hormone (LH)‐stimulated cAMP production was reduced in isolated NHE8KO Leydig cells. After NHE8 expression was silenced by NHE8 siRNA in Leydig cells, reduced LHR expression and dramatically reduced cAMP production after LH stimulation were noted. Confocal microscopic imaging showed that LHR protein was targeted to early endosome in Leydig cells in the absence of NHE8 function instead of the plasma membrane. Therefore, male infertility in NHE8KO mice is due to the disruption of LHR protein trafficking, which results in an interruption in the precise hormone regulation that governs normal sperm development. This study is supported by NIH grant R01DK073638.Grant Funding Source: Supported by NIH grant R01DK073638
Apical NH4Cl acts to reduce intracellular Na concentration in mTAL via cellular depolarization (1098.6)
Authors: Guha Avirup , Sun Jingping , O'Connor Paul 
Published: June 2021

We have recently demonstrated that both cellular acidification and low intracellular [Na] promote NADPH oxidase activity in mTAL via activation of the voltage‐gated proton channel Hv1. NH4+ is normally present in the luminal fluid and its concentration increases during acidosis. It is known that apical uptake of NH4+ promotes intracellular acidification in mTAL, however the effect of apical NH4Cl on intracellular [Na] has not been studied. The goal of the current study was to determine the effect of increasing apical [NH4Cl] on intracellular [Na] in mTAL. We hypothesize that increasing apical NH4Cl concentration reduces intracellular [Na]. Measurements were performed using a FluoStar Omega fluorescent plate reader in cultured mTAL cells loaded with the Na sensitive dye CoroNa Green (n=6). Addition of NH4Cl to the media (HBSS pH 7.40) dose dependently reduced intracellular [Na] in cultured mTAL. Both the NKCC2 inhibitors furosemide and bumetanide lowered intracellular [Na] but did not prevent the action of NH4Cl to further reduce intracellular [Na]. Depolarization of mTAL using KCl reduced intracellular [Na] and greatly blunted the response to 8mM NH4Cl (‐62%, P<0.0002). We conclude that physiologically relevant increases in apical [NH4Cl] (4‐8mM) lower intracellular [Na] in mTAL and that these changes are mediated predominately by cellular depolarization. We speculate that during acidosis, increased NH4Cl delivery to the mTAL promotes NADPH oxidase activity in part by lowering intracellular [Na].Grant Funding Source: AHA SDG 10SDG4150061
Ion regulation and stress tolerance during oscillating salinity in two color morphs of the green crab, Carcinus maenas (1099.6)
Authors: Himes Anthony , Balschi Wingcue , Frederich Markus 
Published: June 2021

Carcinus maenas occurs in two color morphs: green after molting and red after prolonged intermolt. The ability to ion regulate in C. maenas is well documented in green morphs at low salinity, but no studies have focused on fluctuating salinity levels, like that of a tidal environment. We investigated 1) how the ability to ion regulate differ between the two morphs, 2) how oscillating salinity changes affect both morphs, and 3) if these changes vary with sex.Red and green morphs of both sexes were exposed to constantly low salinity (12 ppt) or oscillating salinity, changing from 12 to 32 ppt every 6 h. We assessed hemolymph osmolarity, running endurance on a treadmill, reaction time, and mRNA and protein expression of key ion transporters in the gills.Green morphs are better ion regulators than red morphs, with higher mRNA expression of cytosolic carbonic anhydrase (cCA), Na+K+ATPase (NKX), and Na+H+ antiporter (NHX). Greater mRNA expression of cCA, NHX, and NKX in crabs exposed to constantly low salinity shows that low salinity is more stressful than oscillating salinity. Little difference was noted between sexes.Our data show that the well‐documented ion regulatory capabilities need to be viewed in a color morph specific context. Oscillating salinity being less strenuous than constantly low salinity is in agreement with the conditions in the crabs’ natural habitat as they are rarely exposed to constantly low salinity.Grant Funding Source: NSF‐IOB0640478 to M.F., APS undergraduate summer research fellowship to A.H.
The (pro)renin receptor blocker peptide effects on regulation of body weight and glucose homeostasis in mice with diet‐induced obesity (1101.10)
Authors: Shamansurova Zulaykho , Tan Paul , Bisotto Sandra , Michel Catherine , Nguyen Thi , Schiller Peter , Lavoie Julie 
Published: June 2021

Introduction. The Prorenin receptor [(P)RR] is a recently discovered member of the renin‐angiotensin system (RAS). Adipose tissue (AT) is a 2nd major source of circulating angiotensinogen during obesity and (P)RR effects in adipose tissue have not been clearly identified yet. In this study, we investigated the relationship between (P)RR and body weight (BW) and expression of gene and proteins in the insulin signaling pathway in AT.Material and methods. 12 week‐old C57BL/6 male mice were placed on a high‐fat/high‐carbohydrate (HF/HC) or normal diet (ND) and treated with a (P)RR blocker [(P)RR‐B] or saline for 10 weeks. BW and food intake was assessed weekly. At the end of treatment, mice were euthanized, plasma was collected for the measurement of glucose (G) and insulin (I); AT were separated, weighed and flash frozen. The GLUTs gene expressions were measured by q‐PCR; PKB proteins detected by Western‐Blot.Results.(P)RR‐B administration lowered BW in mice on HF/HC as a result of lower body fat mass. Circulating levels of G,I and the G/I ratio were lowered by the (P)RR‐B on both diet. The GLUTs gene expression in AT was affected by HF/HC and improved with (P)RR‐B administration. Moreover, the PKB phosphorylation in AT was affected with HF/HC, where (P)RR‐B administration improve PKB phosphorylation in both diet.Conclusion. The pharmacologic blockade of the (P)RR lowered BW in mice on a HF/HC, improved glucose homeostasis potentially by improving GLUTs gene expression profile and PKB phosphorylation in AT.Grant Funding Source: Supported by CDA, MDRC
Post‐mortal CO2 release by insects at high temperatures (1101.14)
Authors: Heinrich Erica , Gray Emilie , Vorhees Ashley , Bradley Timothy 
Published: June 2021

Researchers utilizing thermolimit respirometry to study insect thermal tolerance have previously reported an unexplained surge of carbon dioxide release by insects following death at high temperatures. This phenomenon has been referred to as the “post‐mortal peak” (PMP). In some insects, the CO2 release rate during the PMP may be up to 50% higher than the maximal rate achieved in the live insect. We have observed the PMP in fruit flies, mosquitoes, crickets, cockroaches, and beetles. While it has been verified that the PMP occurs after death, the cause of death does play a role in the appearance of the PMP. We have observed the PMP only when an insect dies due to high temperature stress. Furthermore, the PMP does not occur in the absence of atmospheric oxygen, or if the dead insect is subjected to cyanide prior to the initiation of the peak. On the basis of those results, we hypothesize that the CO2 released in the PMP derives from the insect’s mitochondria. We are currently simultaneously measuring CO2 release and oxygen uptake during thermal ramping in order to clarify the source and function of the PMP. Understanding this event may provide insights into the physiological implications of heat stress in insects and the timeline of biochemical events before and after death at high temperatures.Grant Funding Source: Supported by The National Science Foundation