bluuebunny/crossref_metadata_2025 huggingface dataset:
LPS Treatment Alters Oligodendrocyte Precursor Cell Migration
Authors: McKinley Raechel , Csoka Antonei B.
Published: April 2020

The etiology of multiple sclerosis (MS), is characterized by the immune system attacking specialized cells within the central nervous system known as oligodendrocytes. Cytosolic projections of oligodendrocytes wrap their processes around axons to insulate them so that fast salutatory conduction of action potentials can occur. However, in MS, myelin is damaged, compromising axonal integrity and salutatory conduction. This destruction is thought to be a combination of many factors such as activation of inflammatory pathways and epigenetic mechanisms. One of the inflammatory pathways that is thought to be involved in the disease pathology of MS is the Toll‐like receptor (TLR) pathway that leads to the release of pro‐inflammatory cytokines such as TNF‐alpha, NFkB, and series of ILs. Together, these pro‐inflammatory cytokines have been shown to cause cells to undergo apoptosis and cell death. Moreover, it has been found in previous studies that there is increased TLR expression in the lesions of MS patients as well as Experimental Autoimmune Encephalomyelitis, which is an animal model used to study MS and other demyelinating diseases. Along with increased TLR expression in MS lesions, DNA methylation changes, such as increased hypermethylation and demethylation have also been found. Although there have been studies looking into TLRs and DNA methylation in MS lesions, understanding the mechanisms of TLRs in the development of new myelin during remyelination needs to be studied. Previously, we found that using lipopolysaccharide (LPS) to activate TLR2 and TLR4 in induced pluripotent stem cells (iPS) derived oligodendrocyte precursor cells (OPCs) resulted in the demethylation of several genes associated with neural and stem cell migration. In this study, we analyzed if TLR2 and TLR4 activation alters OPC migration. To do this, we performed a chemotactic assay in LPS and 5‐Azacytidine treated OPCs and found that TLR2 and TLR4 activation, along with inhibited methylation, results in increased OPC migration.

Support or Funding Information

This project was supported by the National Institute of Health (NIH) R25 Resource Grant (1 R25 AG047843‐01)
Sensitivity of Affective Pain Circuits to Inhibitory G Protein‐Coupled Receptor Signaling
Authors: Jaeckel Elizabeth , Herrera Yoani , Arias-Hervert Erwin , Perez-Medina Alberto , Sanchez-Conde Fransisco , Birdsong William T.
Published: April 2020

Opioids modulate both sensory and affective components of pain, most significantly through agonist activity at the mu‐opioid receptor (MOR). While cellular mechanisms underlying opioid modulation of perception of stimulus intensity have been well characterized, mechanisms underlying opioid effects on pain‐related negative affect have not been well elucidated. The mediodorsal thalamus (MD) sends glutamatergic projections to the striatum as part of a thalamo‐cortical‐striatal loop that is involved in affective pain perception. Here, we determined the ability of various inhibitory coupled G‐protein coupled receptors (GPCRs), including MOR to inhibit glutamate release via presynaptic mechanisms. In mouse brain slices, MD terminals in the striatum were selectively activated using optogenetic techniques. Excitatory postsynaptic (EPSCs) were measured in striatal medium spiny neurons using whole cell patch‐clamp electrophysiology in the presence and absence of various inhibitory GPCR agonists. MOR full agonists met‐enkephalin (ME) and fentanyl, the delta opioid receptor agonist DPDPE, the adenosine A1 receptor agonist CPA, the GABAB receptor agonist baclofen, and the CB1 receptor agonist WIN 55,212 were tested. EPSC inhibition was seen with application of ME, fentanyl, CPA, and baclofen, indicating the presence of MORs, A1, and GABAB receptors. Inhibition was not seen with application of DPDPE or WIN 55,212, indicating delta opioid receptors and CB1 receptors are not present on these terminals. The effect of chronic opioid treatment on the function of these GPCRs will be discussed. Overall these data provide insight into how presynaptic GPCRs function to regulate neurotransmission in a circuit important for affective pain perception.

Support or Funding Information

NIH T32‐DA007281

R01DA042779
Co‐release of inhibitory neurotransmitters in the RVLM
Authors: Gao Hong , Desmoulins Lucie D. , Zsombok Andrea , Derbenev Andrei V.
Published: April 2020

Sympathetic nerve activity (SNA) and blood pressure (BP) are largely determined by the level of excitability of presympathetic neurons (C1 neurons) in the rostral ventrolateral medulla (RVLM). GABAergic inhibition in the RVLM is the primary mechanism for depressor and sympathoinhibitory responses; however, the role of glycinergic inhibition in the control of RVLM excitability is unknown. There is evidence that both, GABA and glycine efficiently inhibit RVLM neurons, but functional evidence for their co‐release or the mechanism of co‐inhibition is lacking. In our study we used both male and female glycine transporter 2 Cre (GlyT2‐Cre) mice. Glycinergic neurons sending projections to the RVLM were identified with viral labeling. Targeted expression of channelrhodopsin 2 (ChR2) in GlyT2‐expressing neurons and whole‐cell recordings from presympathetic RVLM neurons were used to study the mechanism of glycine release. We found that under steady‐state conditions spontaneous inhibitory postsynaptic current (sIPSC) are primarily mediated by GABA. Photostimulation of glycinergic fibers expressing GlyT2 in the RVLM resulted in evoked IPSCs with both glycinergic and GABAergic components. The time course of synaptic signaling were identical for the two transmitters. Bath application of GABAA receptors antagonist bicuculline (10 μM) or glycine receptor antagonist strychnine (1 μM) revealed that ~40% of the evoked inhibitory currents are GABAergic. High frequency photostimulation (20 Hz) of co‐releasing inhibitory fibers produced a short‐term synaptic depression. Blockade of GlyT2 prevented the glycinergic component of the light evoked IPSCs. In this study we showed that GABA and glycine are co‐released in the RVLM. Our findings suggest that the two neurotransmitters may be functionally interchangeable or complement each other, and thus provide diverse mechanisms for maintaining inhibitory homeostasis.
Small Molecule Inhibits Enterovirus 71 Replication by Targeting the IRES Domain
Authors: Davila-Calderon Jesse , Patwardhan Neeraj , Yuang-Chiu Liang , Sugarman Andrew , Cai Zhengguo , Penutmutchu Srinivasa , Li Mei-Ling , Brewer Gary , Hargrove Amanda , Tolbert Blanton S.
Published: April 2020

Human Enterovirus 71 (EV71), one of the major etiological agents of the hand, foot, and mouth disease (HFMD), represents a serious global health threat against which there is no FDA‐approved treatment. Enteroviruses utilize a highly structured RNA domain, known as a type I Internal Ribosome Entry Site (IRES), to facilitate cap‐independent translation and genome replication by recruiting multiple host RNA‐binding proteins (ITAFs) that fine‐tune translation. Of particular interest, the stem‐loop II (SLII) IRES domain has been shown to specifically interact with several ITAFs such as hnRNP A1 and AUF1, having the SLII‐bulge as the binding surface to effectively modulate viral translation. Further studies showed that deletions and mutations to the bulge motif inhibit EV71 replication by offsetting IRES‐driven translation. Altogether, the results provide strong basis to classify the EV71 SLII domain as a promising target to develop novel antiviral agents. Here, we screened an RNA focused small molecule (rfsm) library against the EV71 SLII domain which yielded in the identification of six rfms that specifically bind to the bulge motif when detected by NMR spectroscopy and titration calorimetry. One of the aforementioned rfms impairs IRES‐dependent translation and displays dose‐dependent antiviral properties in cell culture. Furthermore, the NMR‐derived 3D structure of the SLII‐rfsm complex provides evidence that the small molecule binds to the bulge to induce a conformational change that exposes key nucleotides on the binding site for AUF1. This was further validated both in an in vitro biochemical assay and in vivo. Collectively, these results suggest an allosteric mechanism of inhibition in which the rfsm stabilizes the SLII‐AUF1 complex to repress translation. Therefore, this study provides structural and functional insight to develop novel RNA‐focused small molecule inhibitors of the Enterovirus genus.

Support or Funding Information

NIH R01GM126833
Intestinal Gene Expression of Na+/HCO3− cotransporter 1 (nbce1) Isoforms in Atlantic Salmon During Smoltification and Seawater Acclimation
Authors: McKay Ian , Koltenyuk Victor , Lema Sean , McCormick Stephen , Breves Jason
Published: April 2020

In preparation for migration from freshwater to marine habitats, Atlantic salmon undergo smoltification, a springtime transformation that includes the development of seawater tolerance. The ability of smolts to inhabit seawater is attained through the coordinated development of ion and water‐transporting processes in key organs such as the gill, kidney, and intestine. To promote solute‐linked water absorption, the intestinal epithelium of fishes acclimated to SW secretes HCO3− to form luminal Ca2+ and Mg2+ precipitates. As a source of HCO3− for apical anion exchange, Na+/HCO3− cotransporter 1 (Nbce1) facilitates the transport of plasma HCO3− into the interior of epithelial cells across the basolateral membrane. While Atlantic salmon (Salmo salar) express three nbce1 isoforms (nbce1.1, nbce1.2a, and nbce1.2b), there is no information on whether their expression corresponds with the development of intestinal fluid absorption that occurs during smoltification. We first determined that nbce1.1 and nbce1.2b gene transcripts were highly expressed in pyloric cacae and anterior intestine of smolts, whereas nbce1.2a showed low expression in the intestine but robust expression in gill. Next, we sampled salmon undergoing parr‐smolt transformation and observed elevated branchial Na+/K+‐ATPase activity in May characteristic of smoltification. Moreover, smolts exhibited an improved ability to maintain ionoregulatory balance when transferred to seawater in May versus March. We will report corresponding patterns of nbce1.1 and nbce1.2b expression in pyloric cacae and anterior intestine during smoltification and following seawater transfer to resolve whether their transcriptional patterns correspond with the acquisition of intestinal fluid absorptive processes.

Support or Funding Information

Supported by the National Science Foundation (IOS‐1755131).
Integration of GPCR‐induced endothelial cytoprotection signaling by β‐arrestin‐2
Authors: Molinar-Inglis Olivia , Zhang Lili , Nicholas Dequina , Chen Buxin , Cisneros Metztli , Grimsey Neil J. , Coronel Luisa J. , Lin Ying , Rada Cara , Chi Neil , Lawson Mark A. , Trejo JoAnn
Published: April 2020

Vascular dysfunction is a hallmark of inflammation and results in endothelial barrier disruption, vascular leakage and apoptosis. G protein‐coupled receptors (GPCRs) are highly druggable and mediate inflammatory responses associated with endothelial dysfunction. Specifically, two GPCRs, protease‐activated receptor‐1 (PAR1) and sphingosine‐1‐phosphate receptor‐1 (S1PR1) promote endothelial cytoprotection including barrier protection, Akt‐mediated cell survival, and anti‐inflammatory responses. Activated protein C (APC) activates PAR1 to enhance endothelial barrier stability, whereas S1PR1 contributes to basal barrier stability, whether the two receptors coordinate signaling to promote endothelial cytoprotection is not known. Our previous work showed that APC/PAR1 signaling via the β‐arrestin‐2 and Dishevelled‐2 (Dvl2) scaffolds promote Rac1 signaling and endothelial barrier protection. However, whether β‐arrestin‐2 and Dvl2 are required for integration of S1PR1 into APC/PAR1‐mediated cytoprotection is not known. Here, we report that APC/PAR1 protection against TNF‐α‐induced apoptosis requires S1PR1, whereas S1PR1 is not necessary for APC/PAR1‐mediated barrier protection. We further show that APC/PAR1 transactivation of S1PR1 is mediated by β‐arrestin‐2 dependent activation of sphingosine kinase 1 (Sphk1) and is pivotal for anti‐apoptotic Akt signaling. S1PR1, is activated by sphingosine‐1‐phosphate generated by Sphk1 and we hypothesized that β‐arrestin‐2 and Dvl2 were required for Sphk1 activity. We found that APC promotes Sphk1 phosphorylation and translocation to the membrane. In addition, loss of β‐arrestin‐2 but not Dvl2 abolished APC‐mediated Sphk1 activation. Pharmacological antagonism of S1PR1, inhibition of Sphk1, and β‐arrestin‐2 depletion abolished APC/PAR1‐mediated Akt signaling. Moreover, inhibition of Sphk1, and β‐arrestin‐2 depletion also resulted in loss of APC/PAR1‐mediated cell survival suggesting that Sphk1 and β‐arrestin‐2 are involved in APC/PAR1‐mediated cytoprotection. Our mechanistic studies were corroborated in vivo using a Sphk1 gene knockout zebrafish model. Loss of Sphk1 ablated APC/PAR1‐mediated cell survival and we are currently in the process of validating a β‐arrestin‐2 knockout zebrafish line to test its role in vivo in cytoprotection. In summary, our work demonstrates that APC/PAR1 signaling via the β‐arrestin‐2 multi‐functional scaffold segregates into two distinct pathways: 1) β‐arrestin‐2‐Sphk1 activation induces transactivation of S1PR1 to promote Akt‐mediated cell survival and 2) β‐arrestin‐2/Dvl2‐Rac1 activation promotes endothelial barrier protection.

Support or Funding Information

Supported by NIH/ NIGMS R35 GM127121 and NIGMS IRACDA K12 GM068524.
Lactobacillus reuteri alleviates Glucocorticoid induced Avascular Necrosis of Femoral Head.
Authors: Kang Ho Jun , Schepper Jonathan , Vidal-Franco Monica , Parameswaran Narayanan , McCabe Laura R.
Published: April 2020

Glucocorticoids (GC) are well‐known anti‐inflammatory drugs that are widely used to manage many diseases including autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. Chronic GC use can have significant side effects including 1) dysbiosis (an imbalance of gut microbiota composition), 2) increased intestinal permeability which is linked to increased serum endotoxin levels, 3) trabecular bone loss and 4) osteonecrosis (i.e., avascular necrosis of the femur head). Especially, patients with GC‐induced osteonecrosis of femur head suffer from pain and stiffness, resulting in long‐term disability. Our lab and others have shown that the gut (microbiome and barrier function) regulates bone density. Recently, we have also shown that the probiotic Lactobacillus reuteri 6475 (LR) prevented GC‐induced trabecular bone loss in male and female mice. Based on these findings, we examined if LR treatment can prevent GC‐induced osteonecrosis (GCON) of the femur head. Male C57BL/6J (16‐weeks‐old) mice were treated for 8‐weeks with GC (2.5 mg/kg/day, subcutaneously slow release pellet) and supplemented with or without 3.3×108 cfu/ml LR in their drinking water. Femur immunohistochemistry examining von Willebrand factor positive blood vessels revealed that GC‐induced avascularization of the femur head was prevented by LR supplementation. Specifically, compared to controls, GC treatment caused a 50% reduction in the number of blood vessels in the femur head – this was completely prevented by LR treatment. Similarly, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses determined that LR treatment prevented GC‐induced apoptosis of trabecular osteoblast and osteocytes. In addition, LR treatment prevented GC‐induced marrow adiposity. Consistent with these findings, microcomputed tomography indicated that femoral head bone loss was also prevented by LR treatment. Gene expression analysis of the femur head identified that GC‐induction of tumor necrosis factor alpha and Bcl‐2‐associated x protein (pro‐apoptotic) was prevented by LR. Together, these data indicate that probiotic LR supplementation prevents the pathological bone effects of GCs (avascularization, cell apoptosis, adiposity, bone loss) and may be a straightforward cost‐effective way to mitigate avascular necrosis of femoral head.

Support or Funding Information

These studies were supported by funding from the National Institute of Health, grants RO1 DK101050, AT007695 and by Michigan State University Foundation.
Impact of Redox Modification on MAPK Global Substrate Selection
Authors: Hampton Laquaundra , Postiglione Anthony , Keyes Jeremiah , Poole Leslie B. , Newman Robert H.
Published: April 2020

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which “arm” of a branched pathway is activated in response to a given signal. Specifically, we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D‐recognition site (DRS) and the F‐recognition site (FRS). Interestingly, MAPKs contain redox‐sensitive cysteine residues within or in close proximity to their substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following pre‐treatment of the kinases with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pre‐treatment, both ERK2 and p38α exhibited an increase in their relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 leads to substantial changes in Km and, to a lesser extent, kcat for both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). These findings suggest that redox modification of the cysteines located within the MAPK DRS may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.

Support or Funding Information

This research was supported by the NIH National Institute of General Medical Sciences through grants SC2GM113784 and SC1GM130545 (to RHN) and R01GM119227 (to LBP). Likewise, LM is supported through a Title III HBGI grant from the U.S. Department of Education.
Late‐In‐Life Treadmill‐Training Improves Autophagy, Protein Clearance, and Contractile Function in Mouse Hearts
Authors: Ly Kellsey , Cho Jae Min , Hansen Michele , Ramous Caroline , Thompson Lauren , Pires Karla Maria , Ferhat Maroua , Whitehead Kevin , Carter Kandis , Buffolo Marcio , Ghosh Rajeshwary , Park Seul-Ki , Boudina Sihem , Symons J. David
Published: April 2020

Evidence exists that a progressive decline of the cellular quality control mechanism called macroautophagy occurs during the process of primary aging. This leads to the accumulation of protein aggregates (i.e., proteotoxicity) that would otherwise be transported to and degraded in the lysosome. Post‐mitotic cells like cardiomyocytes rely heavily on macroautophagy because of their inability to clear defective proteins via cell division. While voluntary wheel running improves cardiac autophagy, lessens cardiac proteotoxicity, and attenuates myocardial dysfunction in mice with desmin‐related cardiomyopathy (Bhuiyan et al., JCI, 2013), the ability for a physiological intervention to improve these indexes in aged mice is unknown. We hypothesized that late‐in‐life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs adult mice. As expected, 24‐month old male C57Bl6 mice (old) exhibited repressed autophagosome formation in the heart, myocardial protein aggregate accumulation, systolic and diastolic dysfunction, and reduced exercise capacity vs. 6‐month old (adult) mice (all p< 0.05; n=10 per group) Separate cohorts of 21 month old mice completed a 10‐week progressive resistance / duration treadmill‐running program (old‐ETR) that improved (all p<0.05): (i) body composition; (ii) maximal workload capacity; and (iii) soleus muscle citrate synthase activity vs. age‐matched sedentary mice (old‐SED). Of note, (iv) mRNA and protein expression of autophagy markers indicated trafficking of the autophagosome to the lysosome increased, (v) protein aggregate clearance improved, and (vi) contractile function was enhanced (all p<0.05), in hearts from old‐ETR vs. old‐SED mice. Dietary maneuvers (e.g., caloric restriction, nutraceutical supplementation) and pharmacological interventions (e.g., rapamycin) are reported to elevate myocardial autophagy and mitigate / reverse age‐associated cardiac dysfunction. Here we show the first evidence that a physiological intervention i.e., late‐in‐life exercise training, improves autophagic flux, protein aggregate clearance, and contractile function in hearts from aged mice.

Support or Funding Information

KL [APS STRIDE, UU Undergraduate Research Program (UROP)], JMC (UU Research Fellowship), SKP (AHA 17POST33670663), CR (APS UGRF, UROP), LT (UROP), SB (NIDDK R01‐DK‐098646‐01A1, R01‐DK‐099110, AHA 16GRNT30990018), JDS (AHA16GRNT31050004, NIH RO3AGO52848, NIHRO1HL141540).
Caloric restriction increases hepatic glucose in obese, insulin‐resistant OLETF rats
Authors: Emery Janna Marie , Cornejo Manuel Alejandro , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy Martin
Published: April 2020

Approximately 40% of adults in the United States are classified as obese. Inappropriately elevated body fat in humans contributes to the impairment of insulin signaling, ultimately resulting in alterations to glucose metabolism associated with insulin resistance (IR) and diabetes, both of which are linked to metabolic syndrome (MetS). MetS typically presents with obesity, as a result of overnutrition and a sedentary lifestyle, and causes adverse effects on metabolism, namely on glucose tolerance and insulin response. Caloric restriction (CR) may improve glucose uptake and cellular metabolism, and consistently increases the responsiveness of insulin, resulting in improved insulin sensitivity. The effects of CR on hepatic glycolytic enzymes are not well defined although it is suggested that CR inhibits hepatic glycolysis. More specifically, the effects of CR on hexokinase are not defined. Hexokinase IV (HK IV) catalyzes the first committed step of glycolysis, which is the phosphorylation of glucose to generate glucose‐6‐phosphate (G6P). To evaluate the effect of CR on glucose metabolism and hepatic HK activity during early onset of type 2 diabetes (T2D), sixteen 12‐week old rats were separated into 4 groups (n=4 per group): (1) control strain, LETO, (2) calorically restricted (30%) LETO, (3) obese insulin‐resistant, OLETF, and (4) calorically restricted OLETF. Prior to dissection, an oral glucose tolerance test (oGTT) was performed, and at dissection livers were harvested for measurements of glucose, G6P, and HK activity. Fasting glucose increased with CR in both LETO (63 ± 3 vs 78 ± 2 mg/dL) and OLETF (86 ± 4 vs 92 ± 4 mg/dL). The area under the curve (AUC) for glucose and insulin concentrations were used to estimate the insulin resistance index (IRI). Mean IRI was higher with CR in OLETF (11.7 ± 1.0 vs 13.7 ± 0.89). Hepatic glucose content increased with CR in OLETF (13.2 ± 3.1 vs 17.1 ± 1.0 nmol/μL). Neither hepatic G6P content nor glucose:G6P ratio were significantly changed with CR. While CR improves systemic glucose tolerance and insulin resistance, it increased hepatic glucose content in insulin resistant OLETF rats suggesting that CR inhibits glycolysis via reduced HK activity. Thus, the robust systemic improvements in glucose tolerance with CR during insulin resistant conditions are likely achieved by enhancing peripheral insulin signaling and glucose metabolism.
In Vitro Rat intestinal α‐glucosidase inhibitory and Antioxidant Activities of Phenolic Compounds isolated from Colored Rice Keun‐nun‐Jami (Oryza sativa L.) Germ Extracts
Authors: Kim Tae-Yang , Kang Hanna , Yu Jin-a , Mitiku Haimanot , Hong Sung-Gil , Ryu Su-Noh , Kwon Young-In
Published: April 2020

Excessive carbohydrate‐based meals may result in postprandial hyperglycemia of type 2 diabetes patients, a modern human metabolic disorder. Therefore Inhibition of α‐glucosidase involved in the digestion and absorption of carbohydrates can significantly decrease the postprandial increase of blood glucose level after a carbohydrate diet. In the screening of anti‐hyperglycemic and antioxidant compounds from the extract of Keun‐nun‐jami Germ (KGE), several bioactive ingredients were isolated and identified from ethyl acetate extraction of KGE using HPLC and MPLC systems. Their structures were elucidated by extensive spectroscopic analysis, including 1D and 2D NMR, and FTICR‐MS. To prove the anti‐hyperglycemic effect of purified compound A and B, inhibitory activities of these compounds against rat intestinal α‐glucosidases (sucrase) were investigated. Furthermore, antioxidant activities of these compounds were also evaluated by oxygen radical absorbance capacity (ORAC) method. A major bioactive compound A in KGE had high rat intestinal sucrase inhibitory activity as well as antioxidant activity. Although we need to identify full structure analysis of these compounds and in‐vivo assay, a major bioactive compound A which has high antioxidant and anti‐hyperglycemic activities has the potential to contribute as a useful food supplements for controlling postprandial hyperglycemia.
The novel high‐frequency variant of TRPV3 p.A628T in East Asians showing faster sensitization in response to chemical agonists
Authors: Choi Si Won , Choi Seong Woo , Chae Jeesoo , Yoo Hae Young , Kim Jong-il , Kim Sung Joon
Published: April 2020

TRPV3, a member of the thermosensitive Ca2+‐permeable TRPV channel subfamily expressed in skin and sensory nerves, is also activated by chemical agonists such as 2‐aminoethyl diphenylborinate (2‐APB). Repetitive stimuli induce sensitization of TRPV3 activation, characterized by the cumulative increase in current amplitude and linearization of current‐voltage relation (I/V curve). Through genomic analysis of various populations, we found non‐rare TRPV3 mutation (p.A628T) in East Asian people with an allele frequency of 0.249 while 0.007 in Caucasian. Slope‐conductance of unitary channel was not different between WT and p.A628T. Whole‐cell patch clamp study of wildtype TRPV3 (WT) and p.A628T overexpressed in HEK293T cells showed similar sensitization by the repetitive increase in temperature from 23 to 37 °C, while slightly higher sensitization to 43 °C in p.A628T. In contrast, the repetitive application of 2‐APB (10 μM) or carvacrol (100 μM) induced faster sensitization in p.A628T than WT. However, 1 μM farnesyl pyrophosphate, an intrinsic lipid metabolite agonist induced similar level of slow activations in WT and p.A628T. In Fura‐2 microspectrofluorimetry, the 2‐APB pulses induced a faster increase of [Ca2+]c in p.A628T than WT. In terms of ionic selectivity of channels, WT and p.A628T showed similar Ca2+ permeability (PCa/PNa) calculated from the reversal potential of I/V curves. Taken together, p.A628T shows faster sensitization to chemical agonists that are reflected as higher [Ca2+]c signaling. Based on the intriguing pharmacological sensitivity, the physiological implications of p.A628T in the East Asian population require further investigation. (Published in Pflugers Archiv (2019) 471:1273–1289)

Support or Funding Information

This work was supported in part by the National Research Foundation of Korea (NRF) Grants funded by the Korean government (NRF‐2018R1A5A2025964 and NRF‐2018R1D1A1B07048998).
A Single Amino Acid in ABCG2 is Essential for Clamping Substrates and Inhibitors into the Binding Pocket
Authors: Gose Tomoka , Shafi Talha , Fukuda Yu , Das Sourav , Wang Yao , Lynch John , McHarg Ailsa Gavan , Shelat Anang , Ford Robert C. , Schuetz John D.
Published: April 2020

ABCG2 is an ATP‐binding cassette transporter that, by exporting diverse structurally unrelated anticancer drugs, was identified as a strong contributor to cancer cell multidrug resistance. Subsequently, it was revealed to have important physiological roles in restricting the penetration of multiple xeno‐ and endobiotics into cells and playing a protective role in vivo against cytotoxins for susceptible host tissues. Although ligand binding is the obligate initial step in the transport cycle, it is unknown if any one residue has an indispensable role in binding either substrates and/or inhibitors. The ABCG2 structural studies indicate that substrates interact with fewer residues in the binding pocket than do inhibitors. This finding suggests that a single residue that strongly impacts the binding of both substrates and inhibitors (we will refer to as ligands) would not be found.

To evaluate ABCG2 ligand binding, we performed a thermal shift assay using isolated plasma membranes. Among the tyrosine kinase inhibitors (ABCG2 ligands) tested, lapatinib showed the strongest stabilization of ABCG2 against thermal denaturation. Structure‐based docking identified potential lapatinib binding residues. With this knowledge as a guide, mutagenesis and biochemical studies (thermal shift, viability, and transport assays), revealed that a single highly conserved phenylalanine residue was critical for binding, not just lapatinib, but all tested substrates and inhibitors. The alanine substitution for phenylalanine resulted in the loss of ABCG2 interaction with all ligands tested. Conversion of the phenylalanine to amino acids with similar planar, aromatic side‐chain rings, i.e., tryptophan or tyrosine, restored binding and transport activity comparable to wildtype ABCG2. Importantly, amino acid substitution at other individual binding pocket residues did not result in a complete loss of ABCG2 binding and function. Structural modeling using ABCG2 (PDB: 6ETI) showed that the π‐π interactions from the phenylalanine of each monomer were capable of mediating the binding of a surprisingly diverse array of structurally unrelated therapeutic substrates and inhibitors. Apparently, this symmetrical π‐π interaction “clamps” the ligand into the binding pocket. From the molecular features of ABCG2 ligands that use the π‐π clamp, along with structural studies, we created a “global” ABCG2 pharmacophore model that will be invaluable in predicting ABCG2 interacting drugs and xenobiotics.

These findings have important therapeutic implications by revealing a key structural feature that ABCG2 uses to engage and initiate transport of multiple drugs and xenobiotics.

Support or Funding Information

This work was supported by NIH and by ALSAC.
Persistent Changes in Salt Intake by Estrogen‐Treated Rats after Repeated Episodes of Dietary Sodium Deficiency
Authors: Ehresman Emily , Curtis Kath
Published: April 2020

Aims

Previous research from our lab and others demonstrated sex differences in sodium intake, though estrogen’s role in these differences is unclear. Further, previous studies analyzing salt intake with repeated episodes of sodium depletion using furosemide showed that stimulated salt intake was similar between males and females; however, there were clear sex differences in sodium intake after rats were no longer sodium depleted. We aimed to further elucidate the long‐term effects of estrogen on sodium intake using repeated episodes on a low salt diet, a more physiological challenge.

Methods

Ovariectomized rats were placed on two‐week periods of access to regular chow (NaR) or sodium deficient chow (NaD), which we alternated for a total of two exposures to each diet. Estrogen benzoate (EB) or oil injections were given twice weekly throughout the experiment. At the end of each diet period, rats were given a 2‐hour two bottle test (water and 0.5 M NaCl). At the end of the experiment, rats were sacrificed and trunk blood was collected and centrifuged to collect plasma. Plasma protein levels were measured. Uteri were collected and 1 cm segments of the uterine horns were weighed.

Results

We confirmed the efficacy of EB treatment on body weight, uterine weight, and plasma proteins. As expected, EB‐treated rats maintained body weight throughout the experiment, with transient decreases, while oil‐treated rats gained weight. Both plasma proteins and uterine weights were greatly increased in EB‐treated compared to oil rats. During two‐bottle tests, both EB‐ and oil‐treated rats had similar baseline intakes of 0.5 M NaCl and water. When salt intake was stimulated by dietary sodium deficiency, both EB‐ and oil‐treated rats drank similarly increased amounts of 0.5 M NaCl. Interestingly, EB‐treated rats drank more 0.5 M NaCl and water than oil‐treated rats after returning to regular chow. The period of dietary sodium deficiency again elicited similar intakes of 0.5 M NaCl and water by EB‐ and oil‐treated rats.

Conclusions

Our study demonstrated that estrogen did not alter induced salt intake induced by the physiological challenge, dietary sodium deficiency. However, the low salt diet led to persistent changes in behavior, as demonstrated by the continued consumption of salt and water by EB‐treated rats even after lost dietary sodium was replenished.
Lipidomic Profiling of Host‐Pathogen Interactions Using Untargeted and Targeted Lipidomics Approaches in a Sepsis Cell Culture Model
Authors: Amunugama Kaushalya , Ford David A.
Published: April 2020

Sepsis is a life‐threatening organ dysfunction caused by a dysregulated host response to an infection. When pathogens disseminate into the systemic blood circulation from a localized infection, the body initiates a complex series of immune and biochemical reactions. Neutrophils are the main leukocytes that are recruited to an infection site to eliminate bacteria. In sepsis, systemic neutrophil activation is also evident. One of the main pathologies of sepsis is the damage of the endothelial cell integrity by immune stimuli leading to leaky blood vessels. Although numerous research has been conducted on sepsis, the characteristic heterogeneity of sepsis has challenged the identification of specific biomarkers and treatments. We have designed an in vitro co‐culture model of endothelial cells, human neutrophils and, E.coli to identify lipid biomarkers of sepsis. The three cell types in the model allow us to dissect lipidome of individual cell systems under a cell culture sepsis‐like state. EA.hy926 cells were initially incubated with urosepsis E.Coli strain CFT073 and further incubated after adding neutrophils. We compared the lipidome of co‐culture samples with non‐co‐culture samples to identify altered lipid profiles. In addition, we have performed similar experiments with non‐pathogenic JM109 E.Coli strain. We first used an untargeted lipidomics platform with high‐resolution mass spectrometry in both positive and negative ion modes. Potential lipid biomarkers identified in untargeted lipidomics were furthered confirmed by a targeted lipidomics approach using shotgun lipidomics. In CFT073 co‐cultures, lipids were changed mainly in 4 lipid classes; phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine and triglycerides. Minor changes were observed in sphingomyelin, phosphatidylserine and phosphatidylglycerol classes. Several of the changes in molecular species in each lipid class included significant increases in PE 32:1, PE 34:1, PE 32:2, PC 36:0, PC 40:8 and PC 32:0. Lipid profiles were also altered in non‐pathogenic JM109 co‐cultures. Interestingly, there are marked species variations depending on E.coli strain used in co‐cultures such as PC 32:1, PC 36:1 are only elevated in CFT073 co‐cultures. Collectively, our studies demonstrate that lipid profiles are altered during inflammation initiated by bacterial exposure and subsequent neutrophil activation. We have also shown that pathogenicity of bacteria may lead to distinct alterations in lipidomes. These studies represent initial insights into the complex lipidomic alterations during host‐pathogen interactions and may provide new targets to examine as biomarkers and mediators during in vivo sepsis.

Support or Funding Information

R01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697, U01 ES028182
Zoledronic Acid Activates TRPV1 Channels: Possible Role in Cell Proliferation and Pain
Authors: Scala Rosa , Maqoud Fatima , Latorre Ramon , Scilimati Antonio , Tricarico Domenico
Published: April 2020

Bisphosphonates (BPs) are the most used bone specific anti‐resorptive drugs. Lately there is an increasing interest in BPs capability to prolong osteoblasts lifespan and mineralize; however, the mechanism of action which underpin these effects are nowadays poorly understood. In addition, an increasing concern about BPs pain‐relieving effects is now emerging. Empirical evidence showed BPs may exert anti‐nociceptive, anti‐hyperalgesia and anti‐allodynic properties against multiple stimuli; targets involved in these analgesic effects remains elusive. FDA alert showed that the chronic use of BPs is linked with an increased risk of musculoskeletal pain; mechanisms underlying this sensation are not known. TRPV1 channel is actually recognized to play a key role in bone formation process; as well, it appeared to be involved in bone pain sensation. Capsaicin, a selective agonist of TRPV1 channel, is used for treating different bone pain conditions. We have recently demonstrated that the BP Zoledronic acid (ZOL) can activate TRPV1 currents on osteoblasts. In the mineralization assay, the co‐application of ZOL with capsazepine, an antagonist of TRPV1 ion channel, reduced ZOL‐induced mineralization (Scala et al., Cancer 2019). We tested ZOL effects on TRPV1 ion channel in differentiated neuronal SH‐SY5Y cells and in HEK293 cells transfected with cDNA codifying for WT and PI(4,5)P2‐binding site mutant TRPV1 ion channels. In whole‐cell patch clamp experiments on neuronal SH‐SY5Y cells, ZOL (100μM) activated strong outward currents both at negative and positive voltages. At positive membrane voltages ZOL (100μM)‐ evoked currents were closed after the application of capsazepine (1μM). On WT TRPV1 channel expressed on HEK293 cells, ZOL (100 μM) activates currents by 37% at −60 mV, by +81% at +60 mV and +80% at +180 mV (Vm). As well, on K694A mutant, ZOL was able to activate currents by +10%, +63% and +76% at −60 mV, +60 mV and +180 mV (Vm), respectively. On the contrary, no activation of currents was observed on the R579A mutant.

The ZOL‐induced activation of TRPV1 channel contributes to the mineralization of osteoblasts and to the pain‐relieving effect of ZOL in neurones. Experiments on mutant TRPV1 ion channel suggest that the arginine in the S4–S5 linker of the channel is essential for the drug interaction.

Support or Funding Information

This research was funded by Reg. Puglia (Italy) project “Cluster in Bioimaging” code QZYCUM0 D.T and A.S., and Fondecyt grants 1150273, 1190293 R.L.. R.S. was supported by GLOBAL THESIS Study (2016–2017) by Univ. degli Studi di Bari “Aldo Moro” at CINV “Centro Interdisciplinario De Neurociencia De Valparaíso”.
IgM Antibodies with atheroprotective properties in heart transplants are IgM anti‐phosphorylcholine
Authors: Labarrere Carlos Alberto , Hardin James W. , Jaeger Beate R. , Bhatt Deepak L. , Kassab Ghassan S.
Published: April 2020

Introduction and Objective

Although the etiology of transplant coronary atherosclerosis (TCA), the major cause of long‐term morbidity and mortality in heart transplantation (tx), is unknown, several immunological and non‐immunological causes (innate immunity, inflammation, cell activation, coagulation) have been proposed. Our group and others found that high circulating levels of C‐reactive protein (CRP), a major soluble pattern recognition receptors (sPRR) in humans, associate with development and progression of native atherosclerosis and TCA; although the pathogenic role of different isotypes of CRP (native, non‐native and proinflammatory monomeric) in atheroprotection or atherogenesis remains unclear. Immunoglobulin (Ig) M natural antibodies (NAbs), another innate sPRR, conveys an atheroprotective function, since high titers of IgM anti‐phosphorylcholine (IgM anti‐PC) NAbs associate with reduced atherosclerosis. Our objective was to determine the protective role of sPRR, more specifically IgM anti‐PC, in TCA.

Methods

Adult heart transplant patients (n=172; 5.2±1.0 serial biopsies/patient in first 3‐months post‐tx) followed 8.9±5.0 years were studied. CAV and CAV severity were evaluated with angiograms (5.3±1.0/patient). Percentage of microvascular IgM was calculated from double staining (IgM/CD31 or Ulex europaeus lectin). IgM and IgM anti‐ PC from biopsy eluates were evaluated with immunoblotting and ELISA. Analyses used logistic regression; and Kaplan‐Meier curves for different tertile groups.

Results

Patients had low (<30% positive microvessels in all biopsies; n=67), high (>50% positive microvessels in all biopsies; n=64) or oscillatory IgM (IgMosc; low and high in all biopsies; n=41). Steady high IgM had an atheroprotective effect at 1‐, 5‐ and 10‐years (p<0.001) post‐tx compared with steady low IgM; while IgMosc was only atheroprotective at 5‐years (p<0.04). To determine if IgM was bound to PC, we incubated biopsies with high IgM (n=5) with PC, CRP or albumin, and immunoblots from eluates showed that myocardial IgM was removed by PC and CRP but not albumin. ELISA studies of biopsy eluates confirmed IgM anti‐PC following PC (56.7±20.0 U/ml) and CRP (60.5±18.3 U/ml), but not albumin (0.0±0.0 U/ml) incubation. Immunohistochemistry of biopsies post‐elution showed a complete absence of IgM.

Conclusions

Atheroprotective IgM antibodies are NAbs to PC and future research needs to demonstrate if enhancing IgM anti‐PC NAbs ameliorates TCA, and if native CRP enhances atheroprotection.
High Expression of F11R/JAM‐A is Predominant in Triple‐negative Breast Cancer Subtype and Correlates with Poor Prognosis in Breast Cancer Patients
Authors: Fang Chih-Yeu , Chen Chi-Long , Hsiao Michael
Published: April 2020

Breast cancer (BrC) is the leading type of cancer in women, accounting for 25% of all female tumor cases. It affects about 13% of women worldwide and is the most common cause of cancer‐related death in women. Breast cancer is a heterogeneous disease that contains several subtypes with substantial differences in pathology and clinical outcome. F11 receptor (F11R), also known as the junctional adhesion molecules‐A (JAM‐A), is one of the members of an immunoglobulin superfamily of cell adhesion receptors. F11R is a component of tight junction molecules and involved in a variety of physiological processes including cell polarity, cell morphology and leukocyte migration. Overexpression of F11R has been reported in BrC, but its correlation with clinical prognosis is controversial. In this study, we investigated and clarified the role of F11R in breast carcinogenesis. Two BrC cohorts with a total of 542 cases were used to confirm the correlation of F11R expression and the clinical outcome. High expression of F11R was found to be associated with tumor size (p=0.035), grading (p=0.001), and recurrence (p=0.001) in BrC. Cox model analysis revealed that high expression of F11R is significantly associated with poor overall and disease‐free survival in BrC patients. Interestingly, high expression of F11R is predominant in triple‐negative breast cancer (TNBC, >65%) subtype and is inversely correlated with the expression of estrogen and progesterone receptors. Our data validated the expression of F11R in BrC, suggesting that it is a valuable biomarker for prognostic prediction of recurrence and survival, especially for TNBC.

Support or Funding Information

This research was supported by Academia Sinica [AS‐SUMMIT‐108].
Contribution of the ventral tegmental area to spontaneous coactivation of the motor and cardiovascular systems in decerebrate rats
Authors: Ishii Kei , Asahara Ryota , Komine Hidehiko , Liang Nan , Matsukawa Kanji
Published: April 2020

Central command is a feedforward signal from higher brain centers, regulating the cardiovascular system in association with exercise. Although the concept is widely accepted, neural circuits responsible for central command were still unclear. We hypothesized that the ventral tegmental area (VTA) contributes to generating spontaneous motor activity and concomitant cardiovascular responses. To test the hypothesis, a GABAergic receptor agonist (muscimol) and saline were injected into the bilateral VTA in 9 decerebrate paralyzed rats. In the rats, spontaneous motor activity occurred at frequency of 80 ± 15 times/h (mean ± SE) and accompanied increases in heart rate (by 11 ± 2 beats/min) and mean arterial blood pressure (MAP, by 36 ± 4 mmHg). Muscimol into the VTA reduced the frequency of spontaneous motor activity (19 ± 8 times/h) and the baseline MAP, but not HR. The concomitant tachycardiac and pressor responses (by 12 ± 3 beats/min and 29 ± 3 mmHg) were similar (P > 0.05) before and after the muscimol injection. In contrast, saline into the VTA had no effects (P > 0.05) on the frequency of spontaneous motor activity and the baseline and peak values of HR and MAP. These findings suggest that the VTA plays a significant role in the synchronized activation of the motor and cardiovascular systems in decerebrate rats.

Support or Funding Information

Support of this work was provided by Grant‐in‐Aid for Research Activity Start‐up (grant number 17H07393) from the Japan Society for the Promotion of Science.