bluuebunny/crossref_metadata_2025 huggingface dataset:
Organic Anion Transporting Polypeptide 1B3 can Form Homo‐ and Heterodimers
Authors: Zhang Yuchen , Hagenbuch Bruno
Published: June 2021

OATP1B3 is a 12 transmembrane domain protein expressed at the basolateral membrane of human hepatocytes where it mediates the uptake of numerous drugs and many endogenous compounds. Previous western blot results suggest the formation of OATP1B3 multimers. In order to better understand post‐translational regulation and its effects on the mechanism of OATP1B3‐mediated transport, we investigated the oligomerization status of OATP1B3. We used transient transfection of C‐terminally His‐, FLAG‐ or HA‐tagged OATP1B3 and OATP1B1 in HEK293 cells, co‐immunoprecipitation with and without cross‐linking and a Proximity Ligation Assay (Duolink PLA) to detect interactions between the different constructs. Despite similar transport rates for estradiol‐17β‐glucuronide and estrone‐3‐sulfate, immunofluorescence experiments demonstrated that in contrast to wild‐type, His‐ and FLAG‐tagged OATP1B3 where the C‐terminal end is on the cytoplasmic side of the membrane, the C‐terminal end of HA‐tagged OATP1B3 is extracellular. After cross‐linking with DTSSP anti‐FLAG antibodies were able to pull down FLAG‐tagged OATP1B3 (positive control) but also co‐transfected His‐ or HA‐tagged OATP1B3, demonstrating that OATP1B3 can form homodimer and suggesting that the C‐terminal part of the protein is not involved in the dimerization. Using the Duolink PLA technique, we could confirm co‐localization of His‐ and FLAG‐tagged OATP1B3 in transfected HEK293 cells. In addition, we were also able to detect OATP1B3‐FLAG OATP1B1‐His co‐localization suggesting that OATP1B3 not only homo‐dimerizes but also hetero‐dimerizes with OATP1B1. These findings suggest that OATP1B3 and OATP1B1 can form dimers and that the two OATPs might be co‐regulated at the post‐translational level.
Genistein Reduces Glyburide Efflux by the Human Placental BCRP Transporter: Transcriptional Regulation and Direct Inhibition
Authors: Bircsak Kristin , Lin Yixin , Aleksunes Lauren
Published: June 2021

The hypoglycemic drug glyburide is routinely used in the treatment of gestational diabetes. Fetal exposure to glyburide is typically low because the BCRP/ABCG2 transporter on placental syncytiotrophoblasts prevents its maternal‐to‐fetal transfer. The purpose of this study was to determine the effect of the dietary soy isoflavone genistein on the transcriptional regulation and direct inhibition of BCRP. For the regulation studies, human choriocarcinoma BeWo placental cells were incubated with genistein (0‐10 µM). After 48 h exposure to genistein, placental BeWo cells exhibited a 40% reduction in BCRP mRNA and protein. Down‐regulation of BCRP led to a 50% increase in the accumulation of the BCRP substrate, Hoechst 33342 in BeWo cells treated with genistein. Further, down‐regulation of BCRP by genistein was prevented by removal of serum from the media. In addition to modulating the expression of BCRP, genistein also competitively inhibited its transport in HEK cells overexpressing the wild‐type (WT) or functionally‐reduced BCRP variant (C421A). As expected, C421A‐BCRP cells had 10% greater accumulation of 3H‐glyburide than WT‐BCRP cells. Genistein increased the accumulation of 3H‐glyburide in both WT‐ and C421A‐BCRP cells by 50%. Taken together, pregnant women expressing the common loss‐of‐function C421A‐BCRP variant and/or consume genistein through a soy diet may be at an elevated risk for fetal exposure to glyburide and subsequent neonatal hypoglycemia. Support: ES020522, ES005022, ES007148, AFPE and PhRMA Predoctoral Fellowships.
Use of Vitamin A Supplementation to Enhance Neutrophil Function
Authors: Anderson Ericka , Corriden Ross , Dohrmann Simon , Nizet Victor
Published: June 2021

Vitamin A is a micronutrient essential for growth, cellular differentiation, reproduction, vision, and immunity. Retinoic acid (RA), the active metabolite of Vitamin A, affects transcriptional activity by binding to its receptors, RARs (Retinoic Acid Receptors) and RXRs (Retinoid X Receptors). Vitamin A is essential for immunity in humans, and deficiency is associated with increased morbidity and mortality from infectious diseases. Vitamin A deficient (VAD) neutrophils and macrophages exhibit impaired phagocytosis and bacterial killing, and infectious diseases can cause VAD by decreasing intake, decreasing absorption, and increasing excretion of Vitamin A. Thus, VAD and infection are thought to exist in a “vicious cycle”.

Here, we investigated whether RA treatment enhances neutrophil killing and has direct antibacterial activity. Human neutrophils were pretreated with RA and then tested for their ability to kill E. coli K1, S. aureus MRSA, and S. pyogenes. We found that RA induced production of both reactive oxygen species (ROS) and neutrophil extracellular traps (NETs), critical mediators of neutrophil bactericidal activity, and enhanced killing of all bacteria tested. RA was also found to have direct activity against S. pyogenes. These results suggest a potential for RA as a treatment for bacterial infections.

This work was supported by the UNCF‐Merck Science Initiative Postdoctoral Fellowship (EA), and NIH grants AI096837 and AI077780 (VN).
Activation of ERBB3 Receptor Prevents TNFα Production from Human Non‐Classical Monocytes
Authors: Ryzhov Sergey , Matafonov Anton , Galindo Cristi , Zhang Qinkun , Tran Truc‐Linh , Lenihan Daniel , Geisberg Lenneman Carrie , Sawyer Douglas
Published: June 2021

Immune cells amplify the progression of chronic heart failure (CHF). Recombinant neuregulin‐1 (NRG1) represents a potential therapy for the treatment of various forms of heart failure. We hypothesized that NRG1, acting via ERBB receptors, inhibits pro‐inflammatory cell activation in CHF. Using flow cytometry, we found that peripheral blood monocytes (PBMs) represent a major subpopulation of mononuclear cells expressing both ERBB2 and ERBB3 receptors. A strong inverse correlation was found between expression of ERBB3 and TNFa mRNA in PBMs from patients with heart failure (rs=‐0.682, p=0.001) but not in control subjects (rs=‐0.042, p=0.886). To determine direct effects of ERBB3 signaling on activation of PBMs we used a model of LPS‐induced TNFa production in ex vivo short‐term culture of PBMs. We found that glial growth factor 2(GGF2; USAN ‐ cimaglermin alfa), NRG1 isoform type II, decreased LPS‐induced TNFa production in CD14lowCD16+ non‐classical monocytes. The inhibitory effect of GGF2 on LPS‐induced TNFa production in CD14lowCD16+ monocytes strongly correlated with the expression of ERBB3 (rs=0.769, p=0.013). In contrast to non‐classical PBMs, we found no effect of GGF2 in CD14highCD16‐ subset of classical monocytes irrespective of their expression of ERBB3. Taken together, our results suggest that ERBB3 signaling plays an important role in inhibition of TNFa production from CD14lowCD16+ cells. Given that non‐classical monocytes play a central role in initiation of vascular inflammation, the stimulation of ERBB3 signaling in these cells may potentially slow progression of CHF.
High Salt Diet Modulates Vascular Response in A2AAR+/+ and A2AAR‐/‐ Mice: Role of Soluble Epoxide Hydrolase, PPARγ, and KATP Channels
Authors: Pradhan Isha , Ledent Catherine , Mustafa S Jamal , Morisseau Christophe , Nayeem Mohammed
Published: June 2021

This study aims to investigate the signaling mechanism involved in high salt (HS)‐induced altered vascular response to adenosine in the presence or absence of adenosine A2A receptor (A2AAR). We hypothesized that HS‐induced enhanced vascular relaxation through A2AAR and epoxy‐eicosatrienoic acid (EETs) is dependent on peroxisome‐proliferator activated receptor‐gamma (PPARγ) and ATP‐sensitive potassium channels (KATP channels) in A2AAR+/+, while, HS‐induced contraction to adenosine is dependent on soluble epoxide hydrolase (sEH) EETs in A2AAR‐/‐ mice. Organ‐bath and Western‐blot experiments were conducted in HS (4% NaCl) and normal salt (NS, 0.18% NaCl)‐fed A2AAR+/+ and A2AAR‐/‐ mice aortae. Enhanced vasodilation to A2AAR agonist, CGS 21680, in HS‐fed A2AAR+/+ mice (+27.6 ± 4.7) was blocked by PPARγ antagonist (0.1 µM T0070907) and KATP channel blocker (10 µM Glibenclamide) to (10.6 ± 1.8% and +4.5 ± 4.9%; p<0.05). sEH inhibitor (AUDA)‐dependent vascular relaxation was mitigated by T0070907 (from +4.1 ± 2.3% to ‐7.1± 3.0 % at 10‐6 M; p<0.05). PPARγ agonist (Rosiglitazone)‐induced relaxation in HS‐A2AAR+/+ mice (+3.5 ± 2.9%) was attenuated by Glibenclamide (‐8.1 ± 1.8% at 10‐6 M; p<0.05). Conversely, HS‐induced contraction to NECA in A2AAR‐/‐ mice (‐57.29 ± 2.84%) was attenuated by AUDA (‐6.62 ± 1.24%, p<0.05). These data implicate the contribution of PPARγ and KATP channels downstream of A2AAR in vascular relaxation in HS‐fed mice while, role of sEH in vascular contraction in HS‐fed A2AAR‐/‐ mice
Glucose Metabolism is Impaired in Cultured Myotubes from Severely Obese Humans
Authors: Zou Kai , Hinkley J Matthew , Billings Sarah , Park Sanghee , Zheng Donghai , Jones Terry , Hornsby Pamela , Lenhard James , Pories Walter , Dohm G Lynis , Houmard Joseph
Published: June 2021

In vivo, severe obesity is associated with marked defects in skeletal muscle glucose metabolism. However, it is not evident if these metabolic defects are retained in human primary skeletal muscle cells (HSkMCs) raised in culture, which indicates a genetic and/or epigenetic influence. The purpose of this study was to determine whether impaired glucose metabolism is retained in differentiated myotubes established from severely obese subjects. HSkMCs obtained from lean (BMI = 23.9 ± 0.1 kg/m2) and severely obese (BMI = 47.7 ± 0.6 kg/m2) muscle biopsies were differentiated to myotubes. Radiolabeled 1‐14C glucose was used to measure glycogen synthesis (GS), glucose oxidation (GO) and non‐oxidized glycolysis (NOG) rates in the presence or absence of insulin. Insulin‐stimulated GS and GO rates were elevated to greater extents in myotubes from lean subjects (33% and 17%) in comparison to the severely obese (18% and 6%). In contrast, NOG production did not change in myotubes from lean donors in response to insulin, but largely increased (26%) in severely obese, resulting in a decreased (16%) ratio of oxidized to non‐oxidized glucose in comparison to lean controls. In addition, glycogen synthesis rate was enhanced in myotubes from lean (32%) compared to severely obese (18%). These data suggest that myotubes established from severely obese humans have impaired glucose metabolism.
Reduction of NMJ Occupancy in Dystrophic Muscle
Authors: Pratt Stephen , Shah Sameer , Lovering Richard
Published: June 2021

Proper development and organization at the neuromuscular junction (NMJ) is necessary for effective neuromuscular transmission. In healthy (WT) muscle, the postsynaptic acetylcholine receptors (AChRs) form a continuous, pretzel‐shaped pattern that is spatially aligned with the nerve terminal; this configuration is referred to as “NMJ occupancy”. Duchenne muscular dystrophy (DMD) is a neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Alterations of NMJ post‐synaptic structure in dystrophic (mdx) muscle are well described and we recently demonstrated further morphologic alterations and resulting NMJ transmission failure after injury. However, changes to the pre‐synaptic junction have not been well‐characterized. Here, we quantified the overlap between pre‐synaptic morphology, using antibodies against synaptic vesicles (SV), and post‐synaptic morphology, using bungarotoxin to stain the AChRs. Pre‐ and post‐synaptic structures were identified by immunofluorescence, and occupancy quantified from confocal images. The percent NMJ occupancy was calculated as SV(μm2)/AChR(μm2) * 100. In both WT and mdx mice, pre‐ and post‐synaptic structures were spatially aligned. However, there was a 2‐fold reduction in NMJ occupancy in mdx mice compared to WT (29.5 ± 4% vs 65.2 ± 13%, respectively). Portions of several mdx NMJs showed an absence of SV labeling for the corresponding AChRs, suggesting retraction of presynaptic terminals. We posit that NMJ structural abnormalities in mdx are closely associated with deficits in NMJ function and muscle contractility.
Shortening Speed Dependence of Concentric Force Potentiation in the Absence of Myosin Phosphorylation
Authors: Bunda Jordan , Gittings Bill , Vandenboom Rene
Published: June 2021

Contraction‐induced elevation of myoplasmic Ca2+ leads to sequential activation of skeletal muscle myosin light chain kinase (skMLCK), an enzyme responsible for phosphorylating the myosin regulatory light chain (RLC). In wildtype (WT) mouse skeletal muscle containing skMLCK, force potentiation correlates with RLC phosphate content; although, these muscles may contain an RLC phosphorylation‐independent component. In this study, extensor digitorum longus (EDL) muscles from knockout (KO) C57BL/6 mice, devoid of skMLCK, were activated in vitro (25°C) across a range of stimulation frequencies (10, 25, 45, 70 and 100 Hz) and subjected to shortening ramps at 0.1, 0.3 or 0.5 of maximal shortening velocity (Vmax). This was performed before and after a standard conditioning stimulus (CS) that potentiated mean concentric force at 0.3 and 0.5 Vmax to 1.20 ± 0.03 and 1.30 ± 0.02 of unpotentiated (pre‐CS) values, respectively; however, mean forces were depressed to 0.84 ± 0.03 of pre‐CS values at 0.1 Vmax (n = 3, P < 0.05). Increasing shortening speed augmented the frequency at which concentric force potentiation was maximal: 25 and 45 Hz at 0.3 and 0.5 Vmax, respectively. These results mirrored previous results from WT muscles (Gittings et al., 2012), except that concentric force potentiation was attenuated by ~50% across all frequencies at 0.3 and 0.5 Vmax, while force at 0.1 Vmax was reduced rather than unchanged. Thus, our data indicates the presence of an RLC phosphorylation‐independent mechanism for potentiation in skMLCK KO muscles that may complement the intact RLC phosphorylation mechanism in WT muscles.

Supported by NSERC (2014‐05122).
Diaphragm of Hyperkalemic Periodic Paralysis Mouse Has No Contractility Abnormality Compared to the Robust Abnormalities in EDL and Soleus
Authors: Ammar Tarek , Renaud Jean Marc
Published: June 2021

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant disease that is caused by mutations in the SCN4A gene that encodes for the Nav1.4 channel, the skeletal muscle isoform of Na+channels. HyperpKPP patients suffer from both myotonia and limb paralysis. The myotonia is the result of large membrane depolarization due to large Na+ influx through defective NaV1.4 channels, while the paralysis is caused by increases in extracellular [K+] [[K+]e). However; even though diaphragm also expresses Nav1.4 channels, HyperKPP patients rarely suffer from respiratory distress, as expected with any paralysis. Despite the fact that Na+ influxes through mutated NaV1.4 channels in diaphragm, soleus and EDL muscles from a HyperKPP mouse model are all significantly greater than in their WT counterparts, the HyperKPP diaphragm is the only muscles being spared from the K+‐induced paralysis. The lack of paralysis in diaphragm is not related to any significant increases in Na+ K+ ATPase α‐1 and α‐2 protein content, which actually only increased in HyperKPP EDL. Measurements of resting membrane potential (resting Em) and force demonstrated that the lower force generation in HyperKPP EDL and soleus compared to wild type muscles at different [K+]e was in part related to greater membrane depolarization when [K+]e was increased. However, for a given [K+]e or resting EM, HyperKPP diaphragm generated more force than WT diaphragm. It is proposed that another membrane component spares diaphragm from HyperKPP symptoms, possibly the Na+Ca2+ exchanger which can increase [Ca2+]i during contraction when [Na+]i is elevated as it does in cardiac muscle.
Impaired vasodilation due to thromboxane A2 and reactive oxygen species (ROS) production in renal hypertensive rat aorta
Authors: de Paula Tiago , Silva Bruno , Grando Marcella , Bendhack Lusiane
Published: June 2021

Cyclooxygenase (COX) activity in the vascular endothelium plays a role on the vascular tone control and modulation of inflammation. Increased ROS production in hypertension can modify the relaxation induced by NO donors. This study aimed to evaluate the role of ROS on vasodilation induced by the compound NCX2121 that combines a COX inhibitor (indomethacin) and NO donor. Its effect was evaluated in normotensive (2K) and renal hypertensive (2K‐1C) rat aorta with intact endothelium. Concentration‐effect curves were constructed for NCX2121 before (control) and after incubation with the given inhibitors for soluble guanylyl‐cyclase (sGC, ODQ) or NADPH‐oxidase (apocynin). COX expression was evaluated by western blot and thromboxane A2 (TXA2) production was measured by elisa immune assay. The maximum relaxation induced by NCX2121 was similar in 2K (89.9±4.5%, n=5) and 2K‐1C aortas (78.5±4.0%, n=5). It was inhibited by ODQ only in 2K (44.2±8.3%, n=5; P<0.05), whereas it was potentiated by apocynin only in 2K‐1C rat aortas. COX expression and TXA2 production were higher in 2K‐1C than in 2K aorta. In 2K‐1C rat aorta, TXA2 production was decreased by NCX2121 from 444±72 pg/mg protein to 172±37 pg/mg protein (n=5, P<0.05). Therefore, our results indicate that in renal hypertensive rat aorta with endothelium, the NCX2121‐induced vasodilation is also dependent of COX inhibition and it is impaired by ROS production.
Cannabinoids‐Induced Vasodilation in Rat Mesenteric Artery: Possible Mechanisms of Action
Authors: Lopez‐Dyck Evelyn , Sanchez‐Pastor Enrique , Andrade‐Urzua Maria
Published: June 2021

Mesenteric artery is a resistance vessel, which has an important function in the arterial pressure maintenance. The vascular tone can be modulated through synthesis and release of vasoactive substances of the endothelium as well as through substances that can cause relaxation or contraction of the arterial smooth muscle. Some of these modulator substances are the cannabinoids. It has been shown that cannabinoids are able to produce changes in the vascular tone through different mechanisms including the activation of the cannabinoid receptors (CB1 and CB2). The aim of this study was to analyze the possible mechanisms through which cannabinoids cause rat mesenteric artery vasodilation. We have done tension experiments using synthetic cannabinoids that are selective agonists for CB1 (ACPA) and CB2 (JWH‐133) receptors, and selective antagonists (AM281 and AM630). The cannabinoids were applied in arterial rings with or without endothelium. The results show that ACPA and JWH‐133 activate cannabinoid receptors, to produce vasodilator effect in both intact‐endothelium and no‐endothelium rings, this effect was partially reverted when antagonist were used. Furthermore, involvement of big‐conductance voltage and calcium‐activated potassium (BKCa2+) channels was demonstrated by blocking with iberiotoxin. Moreover, using confocal microscopy we observed the cannabinoid receptors expression in transversal cuts, and the co‐localization of CB1 receptors and BKCa2+ channels was proved in dispersed smooth muscle cells. Therefore, cannabinoids elicit a vasodilator effect, through a mechanism dependent of the BKCa2+ channels activation.
Partial Carotid Ligation Impairs Middle Cerebral Artery Endothelial Function in Old Mice
Authors: Reihl Kelly , Walker Ashley , Donato Anthony , Lesniewski Lisa
Published: June 2021

Previous findings suggest that old mice have greater impairments in cerebral perfusion following internal carotid artery (ICA) occlusion than young mice. However, it is unknown if ICA occlusion affects endothelial function in its major downstream branch, the middle cerebral artery (MCA), in old mice. We performed partial ligation (including full ICA ligation) in the left carotid arteries of old B6D2F1 mice (29±1 mo, n=5) and then tested endothelium‐dependent dilation (EDD) of both left and right MCAs at 8 days post‐ligation. Doppler ultrasound of the left carotid artery confirmed the efficacy of partial ligation, demonstrating a 55% reduction in antegrade (p=0.02) and 110% increase in retrograde (p=0.03) blood flow that resulted in a 340% increase in oscillatory index (p=0.001) at 7 days post‐ligation compared with baseline. There were no changes in blood flow or oscillatory index in the non‐ligated right carotid artery (p>0.05). Maximal EDD was 23% lower in the left MCA compared with the right MCA (30±4% vs. 39±5%, p=0.02). After incubation with the nitric oxide (NO) synthase inhibitor, L‐NAME, maximal EDD did not differ between the left and right MCA (9±8% vs. 8±7%, p=0.47), indicating that impaired EDD in the left MCA was mediated by reduced NO bioavailability. There was no difference between the left and right MCA in the maximal response to endothelium‐independent dilation (83±2% vs. 80±4%, p=0.21). These results demonstrate that partial carotid ligation leads to impaired endothelial function of the ipsilateral MCA in old mice and may explain the impairments in cerebral perfusion following ICA occlusion.
Nandrolone Decanoate Changes The Natriuretic Peptide System in Femaile Rats
Authors: Brasil Girlandia , Lima Ewelyne , Nascimento Andrews , Caliman Izabela , dos Reis Adelina , Andrade Tadeu , Bissoli Nazaré
Published: June 2021

Introduction

The knowledge about the actions of androgenic‐anabolic steroid in females is poorly understood so the aim of the present study was evaluate the action of nandrolone decanoate (ND) and exercise on ANP system in young females.

Methods

The animals were divided in groups: S (sham surgery; vehicle), SD (sham surgery; ND, 20mg/kg/week), SE (sham surgery; resistive trainning) and SDE (sham surgery; ND, 20mg/kg/week and resistive trainning) the treatments started 21 days after surgery, and were made for 4 weeks. After that the animals were euthanized, the right atrium and left kidney were stored under refrigeration until the analysis. The ANP system evaluation were made looking for ANP gene on heart and ANP receptor on kidney (CEUA‐UFES: 031/2012). Two way ANOVA followed by bonferroni post‐test.

Results

The treatment with ND was able to increase the gene expression on atrium (S:1.142±0.102; SD: 2.802±0.488; SE:1.000±0.030; SDE: 2.962±0.385 ANP/26s mRNA expression in arbitrary units; p<0.01 when compared to S group) and also decrease the expression of ANP

image

A receptor on kidney (S:5.908±0.307, SD:1.050±0.243, SE:4.280±0.800, SDE:2.402±0.690 NPRA/26s mRNA expression in arbitrary units; p<0.01 when compared to S group). We also observed a cardiac hypertrophy and kidney hipertrophy after the treatments

image

Conclusions

So these results suggest that the ND can promote an increase on ANP release on atrium and that can be the reason of the reduction on ANP receptor on kidney.
Effect of Salt Diet on Beat‐to‐Beat Blood Pressure Dynamics in Hypertensive and Non‐hypertensive Rats
Authors: Fares S , Habib J , Engoren M , Badr K , Habib R
Published: June 2021

Objectives

Blood pressure normally exhibits substantial short and long term variability (BPV). We assessed, in salt sensitive hypertensive (SS) and non‐hypertensive (SSBN13) Dahl rats, the impact of high (HS) versus low (LS) salt diet on beat‐to‐beat BPV indices including correlation properties (detrended fluctuation analysis scale coefficient α) and complexity (Sample Entropy; SpEn)

Methods

BP data were analyzed from 9 SS [HTN] and 6 SSBN13 [No‐HTN] rats: 1) after 9 weeks of LS diet (0.4%), and 2) repeated after 2 weeks of HS diet (8%). Beat‐to‐beat systolic and diastolic BP series were derived from 2‐minute data epochs (Physionet.org). SpEn, coefficient α, standard deviation (SD) and coefficient of variation (CV, %) were compared

Results

Mean systolic and diastolic BP increased on HS – particularly in SS rats (Table). SD and CV were similar across groups irrespective of diet. On LS, HTN and No‐HTN exhibited similar systolic and diastolic complexity and correlation indices. On HS, 1) HTN showed an increased α (smoother) for systolic (p=0.015) and diastolic (p=0.021) BP; 2) No‐HTN showed lower SpEn (less complex) for systolic and diastolic BP (p=0.046), while corresponding α were unchanged; and 3) HTN showed higher systolic (p=0.003) and diastolic (p=0.001) α compared to No‐HTN. Changes in α (ΔHS‐LS) were greater in HTN versus No‐HTN rats for diastolic (p=0.034) and systolic (p=0.099) BP series

Conclusion

Hypertensive rats are more susceptible to HS with a greater rise in mean BP and reduced complexity. Yet, the comparable mean pressures in HTN and No‐HTN rats when on LS diet coupled with similar BPV dynamics suggest a protective role of low salt diet in hypertensive rats

Funding: None

Study Group
SS rats (HTN)
SSBN13 rats (No‐HTN)

Teatment

LS
HS
ΔHS‐LS
LS
HS
ΔHS‐LS

Systolic BP
mmHg
137±2ab
195±7ab
57±5b
125±2ab
155±3ab
30±2b

α
0.94±0.04a
1.15±0.04ab
0.21±0.05
0.91±0.05
0.97±0.02b
0.07±0.04

SpEn
1.43±0.06
1.26±0.06
0.16±0.10
1.51±0.09a
1.24±0.08a
‐0.28±0.09

SD
4.40±0.30
5.73±0.46
1.33±0.57
4.30±1.30
5.19±0.49
0.89±0.59

CV(%)
3.2±0.2
2.9±0.2
0.26±0.34
3.5±0.4
3.4±0.4
‐0.07±0.48

Diastolic BP
mmHg
102±3a
153±7ab
51±6b
97±1a
123±3ab
26±3b

α
0.98±0.04a
1.17±0.04ab
0.18±0.06b
0.94±0.04
0.95±0.01b
0.01±0.04b

SpEn
1.27±0.04
1.03±0.09
0.24±0.14
1.37±0.06a
1.21±0.02a
‐0.16±0.06

SD
4.03±0.31
4.70±0.48
0.67±0.59
3.80±0.50
4.65±0.56
0.85±0.58

CV(%)
3.97±0.25
3.13±0.29
0.85±0.41
3.89±0.47
3.82±0.47
‐0.08±0.48

Nbeats: 626‐953; data shown as mean±std err; a = P<0.05 LS vs HS (within‐group); b = P<0.05 SS vs SSBN13 (between‐groups; same treatment); comparisons using the rank sum and the sign rank test as appropriate
AT1 Receptors Modulate Ca2+ Transient in Adult Cardiomyocytes Treated with T3
Authors: Taniguti Ana Paula , Nocchi Eduardo , Almeida Pedro , Guatimosim Silvia , Barreto‐Chaves Maria Luiza
Published: June 2021

Increased thyroid hormone (TH) levels promote cardiac hypertrophy and arrhythmia by direct actions on myocardium and by interaction with other systems as local renin‐angiotensin system (RAS). Previous studies have confirmed that T3 rapidly increases AT1R and angiotensin II protein levels in cultured cardiomyocytes. In the present study, we evaluated the contribution of AT1R on Ca2+ transient cardiomyocytes treated with T3. Adult ventricular cardiomyocytes (n=07) were isolated and treated for 30 minutes with T3 (15nM, Sigma), T3 plus losartan (LOS, 1µM, Sigma) or LOS alone. Intracellular Ca2+ (Ca2+ i) imaging experiments were performed in Fluo‐4 AM (10µM; Invitrogen) loaded‐cardiomyocytes and electrically stimulated at 1 Hz.The confocal imaging was performed with a Zeiss LSM 510META microscope. The Ca2+ level was reported as F/F0, where F0 is the resting Ca2+ fluorescence. T3 treatment induced a significant increase on Ca2+i transient (3,53±0,88 in T3 vs. 2,89±0,69 in Control, ANOVA, p蠄0.05) in adult cardimyocytes, which was attenuated by concomitant LOS treatment (3,08±0,79 in T3+LOS vs. T3, ANOVA, p蠄0,05) demonstrating a possible contribution of AT1R in the action of T3 in inducing Ca2+i transient in adult cardiomyocytes.

Supported by FAPESP #2012/18515‐4
Cerebrolysin effects on cardiovascular hemodynamic parameters in Sprague Dawley rats with chronic hyperglycemia induced by streptozotocin
Authors: Zurita Enedina , De Jesús Luis , Suarez Jorge , Gomez Ma. de Jesus
Published: June 2021

Autonomic denervation is a late complication of diabetes mellitus and is considered a cause of different cardiovascular disorders. In this study we evaluated the effect of Cerebrolysin (CBL) a neurotrophic factor, on cardiovascular hemodynamic parameters in rats with chronic hyperglycemia. Male Sprague Dawley rats were administered with streptozotocin (single dose, 60mg/Kg,ip). Hyperglycemic and normoglycemic rats were administered during 8 weeks with vehicle (saline 0.9 %) or Cerebrolysin (2ml/Kg/day, ip), at 60 days, after the induction of hyperglycemia. At the end, different parameters were measured in conscious rats: body weight (BW), heart rate (HR), heart rate variability (HRV), systolic arterial blood pressure (SABP), and blood glucose (BG). Heart weight (HW) and the ratio HW/BW were obtained after euthanized rats. The results shown that in the hyperglycemic rats the administration of CBL produce an increase in BW and HW, HR and HRV and a decreases in SABP as compared with hyperglycemic vehicle rats (BW 315±1 vs. 258±4), HW 1.4±0.07 vs. 1.1±0.05), ratio HW/BW 0.47±0.03 vs. 0.31±0.05), HR 322±8 vs. 278±1 beat/min), SABP 168±12 vs. 145±9 mmHg) (p< 0.001 for all comparison). The results suggest that the neurotrophic factor CBL, could improve the cardiovascular parameters in rats with chronic hyperglycemia and could be considered and adjacent treatment to patients with diabetes mellitus.
Rapid Change in Afterload After Gastric By‐Pass Surgery
Authors: DeVallance Evan , Lee Kyuwan , Grogg Kristen , LeMaster Kent , Brader Alan , Tabone Lawrence , Rosati Kristen , Quinlin Robert , Frisbee Jefferson , Chantler Paul
Published: June 2021

Obesity is associated with increased arterial dysfunction, an independent risk factor linking obesity to an elevated risk of cardiovascular (CV) mortality. Interventions are needed to alleviate the burden of obesity on the CV system. Gastric bypass surgery (GBS) is an intervention associated with rapid weight loss and a reduction in future CV events. We aim to show the immediate effects of GBS on CV function assessed by pulse wave analysis on 12 GBS patients. As expected weight decreased (pre: 132±6 to post: 125±5 Kg, p<0.01) 1‐week post GBS this coincided with a reduction in waist circumference (pre: 122±5 to post: 112±3 cm, p<0.05). Central systolic blood pressure (pre: 112±4 to post: 105±2 mmHg, p=0.08), augmentation index (pre:16±3 to post:8±3 ,p=0.06), an indirect measure of stiffness, and central augmentation pressure (pre: 5±1 to post: 3±1 mmHg, p=0.06) tended to decrease 1‐week post GBS. These changes were independent of a change in brachial BP. These measures suggest GBS and postoperative care reduce arterial load leading to a decrease in cardiac afterload. The reduction in afterload allows for shorter ejection duration (pre: 326±8 post: 302±11 ms, p<0.05) and higher contractile rates (dP/dt pre: 672±54 post: 737±46, p<0.01) leading to a decrease of the oxygen demand in the myocardium (PTIs pre: 2346±120 post: 2194±107 area/min, p<0.05) and more efficient cardiac work. GBS quickly improves central arterial function alleviating some of the cardiac workload.
Endocardial Prevalence of Coronary Collaterals Becomes Transmurally More Uniform in a Human Heart with Left Ventricular Hypertrophy
Authors: van Lier Monique , Oost Elco , van der Wal Allard , Spaan Jos , Siebes Maria , van den Wijngaard Jeroen
Published: June 2021

Aim

To quantify and characterize coronary collaterals (CC) in human hearts.

Methods

We obtained one heart with left ventricular hypertrophy (LVH), and an old inferior posterolateral infarction (64 years, 595 g) and one normal heart without prior coronary artery disease (84 years, 330 g). The coronary arteries were filled with fluorescent replica material. Hearts were frozen, alternately cut and block‐face imaged using an imaging cryomicrotome (voxel size 32 μm). Collateral transmural distribution, connectivity and diameter were determined.

Results

Three times more collaterals were detected in the normal (n=4308) compared to the LVH heart (n=1272). In the normal heart, collaterals were mainly in the LV free wall (endo: 78%, mid: 14%, epi: 5%), whereas a more uniform distribution over the LV free wall (endo: 25%, mid: 12%, epi: 21%) and septum (endo: 27%, mid: 10%, epi: 2%) was found in the LVH heart. More intraCC (within major branches) than interCC (between major branches) were detected in the normal (4191 vs. 117) and LVH (1187 vs. 85) heart. InterCC were most prevalent between LCX‐LAD (99%) in the normal and between LCX‐LAD (62%) and LAD‐RCA (33%) in the LVH heart. Most intraCC were within the LAD and LCX territories in both hearts. The median CC diameter was larger in the LVH compared to the normal heart for both interCC (92.3 vs. 71.6 μm, p<0.001) and intraCC (81.88 vs. 60.4 μm, p<0.001).

Conclusions

As in healthy canine hearts, human hearts have abundant intra‐ and interCC collaterals. Transmural distribution was more homogeneous in LVH, with fewer but larger collaterals.
Variability in IV Adenosine‐Induced Coronary Microvascular Resistance and Systemic Pressure Compromises FFR Determination despite Stable Stenosis Hemodynamics
Authors: Casadonte Lorena , Marques Koen , Spaan Jos , Siebes Maria
Published: June 2021

Aim

To investigate the effect of intracoronary (IC) and intravenous (IV) adenosine administration on variability in coronary and stenosis hemodynamics.

Methods

In 12 diseased coronary vessels (25‐56% DS), aortic pressure (Pa), distal pressure (Pd) and flow velocity (v) were simultaneously measured at baseline and hyperemia induced by IC (40 μg bolus) and IV adenosine administration (140 μg/kg/min). At baseline and throughout the hyperemic response we obtained cycle‐averaged pressure and velocity signals to derive microvascular resistance (MR = Pd/v) and FFR= Pd/Pa. From fitted stenosis pressure gradient‐velocity (ΔP‐v) relations, we determined ΔP at v= 30 cm/s (dPv30).

Results

Maximal v was similar for IC and IV adenosine (50 ± 4 vs. 54 ± 5 cm/s) with no difference in MR (1.73 ± 0.10 vs. 1.74 ± 0.13 mmHg/cm/s). However, MR during IV hyperemia rose between 9 and 135% in the post‐peak period, with concomitant fluctuations in v (13 to 61% decline) and pressures (up to 34% decline). Minimal FFR coincided with peak v for IC adenosine, but occurred 21 ± 8 s after peak v for IV adenosine, when Pa had decreased by 13% and HR increased by 25% vs. baseline. IV and IC derived stenosis ΔP‐v relations followed a stable common quadratic relationship, with no difference in dPv30.

Conclusion

IV adenosine caused persistent systemic and coronary hemodynamic variability. IC adenosine yielded comparable hyperemia with minor effects on systemic hemodynamics. Consistent ΔP‐v relations and dPv30 provide reliable means for stenosis evaluation free from maximal vasodilation.
Prdx6PLA2‐derived Lysophosphatidic Acid Signaling is required for Nox2 Activation in Pulmonary Microvascular Endothelial Cells and Alveolar Macrophages
Authors: Vazquez‐Medina Jose Pablo , Dodia Chandra , Chatterjee Shampa , Fisher Aron
Published: June 2021

The phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) is required for agonist‐induced NADPH oxidase (Nox2) activation in pulmonary microvascular endothelial cells (PMVEC) and alveolar macrophages (AM). Lysophosphatidylcholine (LPC) is a major product of the PLA2 activity of Prdx6 that it is readily metabolized to lysophosphatidic acid (LPA) by the enzyme autoaxin/lysophospholipase D (ATX/LysoPLD). LPA is a bioactive lipid that signals through G protein‐coupled receptors. LPA receptor activation stimulates pathways regulated by small GTPases, such as the Nox2 cytosolic activator, rac1. We propose that Prdx6PLA2‐derived LPA receptor signaling activates Nox2 in PMVEC and AM. Angiotensin II (Ang II) or phorbol ester (PMA) treatment increased oxidant generation in isolated perfused lungs, PMVEC and AM from WT mice but not in lungs or cells from Nox2 null, Prdx6 null or Prdx6D140A knock‐in mice (which lack Prdx6PLA2 activity). Exogenous LPA restored oxidant generation in Prdx6 null and Prdx6D140A cells. Treatment with the LPA receptor blocker Ki16425 significantly inhibited agonist‐induced oxidant generation in WT lungs and cells and also blocked agonist‐induced rac1 translocation to the plasma membrane in PMVEC. Inhibition of ATX/LysoPLD with HA130 decreased PMA‐ and LPC‐induced oxidant generation in PMVEC; inhibition was reversed by concurrent treatment with LPA. These results suggest that LPA signaling is required for agonist‐induced Nox2 activation and that Prdx6PLA2 likely generates LPC that needs conversion to LPA by ATX/LysoPLD for Nox2 activation. Funded by HL105509.
Induction of miR‐21 Expression by Freshly Fractured Silica Involves ROS‐Mediated ERK Pathway
Authors: Ding Min , Hou Lichao , Bowman Linda , Meighan Terence , Settle Tabatha , Liu Jun
Published: June 2021

Silica particles are considered to be fibrogenic agents and established carcinogens, but the mechanisms for disease initiation and progression are not well understood. Earlier studies demonstrated that the tumor suppressor gene, PDCD4, and its upstream regulator, miR‐21, may be considered as oncogenes for novel cancer prevention or anti‐cancer therapies. The present study examined the alterations of miR‐21‐PDCD4 signaling in JB6 cells after exposure to freshly fractured silica particles. The results showed that (1) silica caused PDCD4 inhibition in JB6 cells; (2) exposure of cells to silica caused a significant increase of miR‐21 expression and decrease of PDCD4 expression; (3) inhibition of ERKs or p38 with U0126 or SB 203580 reversed silica‐induced PDCD4 inhibition; and (4) ROS scavengers, N‐acetyl‐L‐cysteine, reversed the inhibitory effect of silica on PDCD4 expression; (5) chronically exposed human lung epithelial BEAS‐2B or JB6 cells to low‐dose silica resulted in neoplastic transformation assayed by soft agar. These findings demonstrate that freshly fractured silica particles induce miR‐21 expression and PDCD4 inhibition, which may be mediated through ROS and ERK pathways. Unraveling the complex mechanisms associated with these events may provide insights into the initiation and progression of silica‐induced carcinogenesis.
Select Rab GTPases Mediate Pulmonary Endothelial Barrier
Authors: Chichger Havovi , Braza Julie , Boni Geraldine , Duong Huetran , Harrington Elizabeth
Published: June 2021

Vascular permeability, a major hallmark of acute lung Injury (ALI), is regulated in part by VE‐cadherin (VE‐cad) forming adherens junctions at the interendothelial junctions (IEJ). We have recently demonstrated that the endosomal protein, p18, binds to the early endosome to enhance recycling of VE‐cad to IEJ, improve endothelial (EC) barrier function and attenuate pulmonary edema formation. The endosome system is a dynamic process which sorts cargo and directs it either back to the plasma membrane, through Rab4‐mediated recycling or to the lysosome through Rab7 and Rab9. We thus hypothesize that Rab GTPases regulates EC barrier function through modulation of VE‐cad trafficking to and from the IEJ. Lung EC were transfected with wild type Rab4 (Rab4wt), GTP‐locked endosome‐anchored Rab4 (Rab4Q67L), GDP‐locked non‐endosome binding Rabs, Rab4S22N, Rab7T22N and Rab9S21N or GFP as control. The effects of Rab4, Rab7 and Rab9 on EC endocytosis of VE‐cad, in vitro EC barrier function and in vivo pulmonary edema formation were examined. We show that activation of pro‐recycling Rab4wt and Rab4Q67L or inhibition of pro‐degradative Rab9S21N protects EC barrier function in settings of ALI in vitro and in vivo in settings of LPS‐induced injury. In contrast, Rab4S22N or pro‐lysosome forming Rab7T22N play no protective role on EC barrier integrity. We further show enhanced levels of VE‐cad at the plasma membrane concomitant with improved EC barrier function. We conclude that Rab4 and Rab9 regulate pulmonary EC barrier function by enhancing recycling of VE‐cad+ endosomes to the IEJ. We propose that targeting Rab GTPase‐mediated endocytic trafficking represents a novel therapy in the treatment of ALI.
Prolonged High Glucose Treatment Increased Orai1 Protein Expression through Inhibition of Lysosomal Pathway in Human Mesangial Cells
Authors: Chaudhari Sarika , Wang Yanxia , Ma Rong
Published: June 2021

The Orai1‐mediated store operated calcium entry (SOCE) is associated with many physiological and pathological processes in a variety of cells, including glomerular mesangial cells (MCs). We have previously demonstrated that prolonged treatment of MCs with high glucose (HG) significantly increased Orai1 expression at protein level, but not at mRNA level. These findings suggest that a post‐transcriptional mechanism(s) contributes to the HG effect. The aim of the present study was to identify if proteosomal and/or lysosomal pathways were involved in the increased abundance of Orai1 protein in response to HG. Cultured human MCs were with and without treatment with 10 μM MG132 (an inhibitor of proteosomal pathway) or 10 mM ammonium chloride (an inhibitor of lysosomal pathway) in the presence of normal glucose (NG, 5.6 mM) or HG (25 mM) for 6 days. Western blots of the whole cell lysates were conducted to evaluate Orai1 expression level. We found that both MG132 and ammonium chloride increased abundance of Orai1 protein in MCs incubated with NG. The MG132 response was further increased by HG treatment. However, HG failed to cause additional increase in Orai1 protein expression in ammonium chloride‐treated cells. Furthermore, fura‐2 fluorescence ratiometry study showed that both MG132 and ammonium chloride increased the cyclopiazonic acid (25 μM)‐stimulated SOCE. Simultaneous treatment with HG only enhanced the MG132 response, but not the ammonium chloride response. Taken together, our results indicate that HG increased expression of Orai1protein by inhibiting its degradation through the lysosomal pathway. (Supported by NIH)
One Degree, Many Job Opportunities
Authors: Bond Judith
Published: June 2021

The objective of the PhD is to empower the recipient to generate new knowledge, NOT to train for a specific job. PhD training increases one's ability to: think critically about science and scientific knowledge, understand state‐of the art technologies, advance science, solve problems, design experiments, communicate, recognize what is feasible, make conclusions on the basis of what we know from data, envision what is possible. These are powerful, important skills and abilities needed by society, and yet only 1% of American adults have a PhD. A few decades ago a large percent of PhDs in biomedical sciences went into tenure‐track positions in research‐intensive universities; the percentage has been decreasing, and some scientists/administrators are calling for producing fewer PhDs in the biomedical sciences. Alternatively, institutions, faculty and trainees are exploring and implementing programs that lead to a wide range of appropriate careers and jobs for PhDs including teaching at all levels, working in industry/biotech, research administration and management, regulatory agencies, public health, science testing, science policy, intellectual property, and entrepreneurship. Graduate programs and individuals are increasingly implementing curricular changes to prepare PhD candidates for a variety of jobs and helping graduates find satisfying career paths that use their scientific training.
The Development of Angiotensin II ‐ induced Hypertension During Various Salt Intakes in Cre/lox Conditional Knockout Mice for Bradykinin Receptors in The Collecting Duct
Authors: Kopkan Libor , Huskova Zuzana , Jichova Sarka , Cervenka Ludek , Saifudeen Zubaida , El‐Dahr Samir
Published: June 2021

Bradykinin is an important antihypertensive peptide acting mostly via the kinin B2 receptor (gene: Bdkrb2; protein: B2R). We elucidated the role of collecting duct B2R in the development of salt‐sensitivity and angiotensin II (ANG II)‐induced hypertension. We used a Cre‐Lox recombination strategy to generate mice lacking Bdkrb2 gene for B2R in the collecting duct (Hoxb7‐Cretg/+:Bdkrb2flox/flox). In 3 groups of control (Bdkrb2flox/flox) and 3 groups of UBBdkrb2‐/‐ mice, systolic blood pressure (SBP) responses to high salt intake (4 or 8% NaCl; HS) were monitored by radiotelemetry in comparison with standard salt diet (0.4% NaCl) prior to and during subcutaneous ANG II infusion (1000 ng/min/kg) via osmotic pumps. High salt intakes alone for 2 weeks did not alter SBP in either strain. ANG II significantly increased SBP equally in control (121 ± 2 to 156 ± 3 mmHg) and UBBdkrb2‐/‐ mice (120 ± 2 to 153 ± 2 mmHg). The development of ANG II‐induced hypertension was exacerbated by 4%HS in both control (125 ± 3 to 164 ± 5 mmHg) and UBBdkrb2‐/‐ mice (124 ± 2 to 162 ± 3 mmHg) during 2 weeks. Interestingly 8%HS caused a more profound and earlier ANG II‐induced hypertension in UBBdkrb2‐/‐ (129 ± 2 to 166 ± 3 mmHg) as compared to control (128 ± 2 to 158 ± 2 mmHg) and it was accompanied by body weight loss and increased mortality. In conclusion, targeted inactivation of B2R in the collecting duct does not cause salt‐sensitivity; however, collecting duct B2R attenuates the hypertensive actions of ANG II action under conditions of very high salt intake.
Prevention of High Fat‐induced Podocyte Injury and Glomerular Sclerosis in Mice Lacking Nod‐like Receptor Protein 3: Role of Inflammasome Extinction
Authors: Boini Krishna , Xia Min , Conley Sabena , Li Guangbi , Gehr Todd , Li Pin‐Lan
Published: June 2021

Recent studies have demonstrated that NLRP3 inflammasome formation and activation are an important early mechanism responsible for podocyte injury and glomerular inflammation in obese mice. However, it remains unknown whether Nlrp3 gene is critical for the activation of inflammasomes in glomeruli of obese mice. To answer this question, the Nlrp3 knockout (Nlrp3‐/‐) and wild type (Nlrp3+/+) mice were fed a high fat diet (HFD) or normal chow (ND) for 10 weeks to produce obesity. Confocal microscopic analysis showed that HFD increased the colocalization of NLRP3 with Asc or caspase‐1 in glomeruli of Nlrp3+/+, but not in Nlrp3‐/‐ mice, suggesting that obesity‐enhanced formation of Nlrp3 inflammasomes. Furthermore, colocalization of Nlrp3 with podocin indicates the enrichment of Nlrp3 in podocytes. In addition, biochemical analysis demonstrated that HFD significantly increased the caspase‐1 activity and IL‐1β production in Nlrp3+/+ mice, but not in Nlrp3‐/‐ mice. Correspondingly, the urinary protein and albumin excretion were significantly higher in Nalp3+/+ mice compared to ND fed mice. However, the HFD‐induced proteinuria were significantly lowered in Nlrp3‐/‐ compared to Nlrp3+/+ mice. In addition, Western blot analysis showed that HFD significantly increased the desmin, HMGB‐1 and RAGE expression in glomeruli of Nlrp3+/+ mice, but not in Nlrp3‐/‐ mice.Based on these results, it is concluded that Nlrp3 is an essential component of Nlrp3 inflammasomes and that extinction of Nlrp3 inflammasomes in podocytes protects podocytes and glomeruli from obesity‐induced injury (supported by NIH grant DK54927).
Angiotensin AT2 Receptor Agonist Prevents Salt‐Sensitive Hypertension in Obese Zucker Rats
Authors: Ali Quaisar , Patel Sanket , Hussain Tahir
Published: June 2021

High sodium intake is a risk factor for the pathogenesis of hypertension, especially in obesity. The present study was designed to investigate whether angiotensin type‐2 receptor (AT2R) activation with selective agonist C21 prevents high‐sodium diet (HSD)‐induced hypertension in obese animals. Male obese rats were treated with AT2R agonist C21 (1mg/kg/day, oral) while maintained on either normal‐sodium diet (NSD, 0.4%) or HSD (4%) for 2‐weeks. Radio‐telemetric recording showed a time dependent increase in systolic blood pressure in HSD‐fed obese rats, being maximal increase (~27 mmHg) at day 12 of the HSD regimen. C21 treatment completely prevented the increase in blood pressure in HSD‐fed rats. Compared to NSD controls, HSD‐fed obese rats had greater natriuresis/diuresis, which were further increased by C21 treatment. HSD‐fed rats expressed higher level of cortical Ang II, which was reduced to 50% by C21 treatment. HSD feeding and/or C21 treatment had no effects on cortical renin activity and the expression of ACE and chymase, both AngII producing enzymes. However, Ang(1‐7) concentration and ACE2 activity in the cortex were reduced by HSD feeding, but C21 treatment rescued both the parameters. Also, C21 treatment reduced the cortical expression of AT1R in HSD‐fed rats, but had no effect of AT2R expression. We conclude that chronic treatment with the AT2R agonist C21 prevents salt‐sensitive hypertension in obese animals and a reduction in the renal AngII/AT1R levels may play a potential role in this phenomenon.

Acknowledgement: This study was supported by NIH R01grant DK61578 to TH.