bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of dietary fatty acids on the recruitment of brown‐like adipocytes in inguinal white adipose tissue
Authors: Zhao Ming , Guo Hong , Chen Xiaoli
Published: June 2021

The physiological significance of brown adipose tissue (BAT) in energy metabolism in adult humans has been recently re‐evaluated. The evidence from recent studies indicates a potential of converting white to brown adipocytes in white adipose tissue (WAT). Herein, we explored the regulatory effect of dietary fatty acids on this conversion in WAT. We found that BAT‐specific UCP1 was expressed at a significantly higher level in inguinal adipose tissue (ING) compared to epididymal adipose tissue in mice. High fat diet (HFD) modulated UCP1 expression in BAT and ING in a different manner. After one week of HFD, UCP1 was increased in BAT, but not in ING of mice. However, UCP1 was dramatically decreased after chronic HFD feeding in both tissues. Additionally, we examined the effects of saturated and polyunsaturated fatty acids on the differentiation of brown‐like adipocytes in primary culture of stromal‐vascular (SV) cells from ING of normal mice. Fatty acids were added to SV cell culture either during 8 days of differentiation or after differentiation for 24 hours. In differentiated adipocytes, 24h treatment with palmitate significantly reduced UCP1, while n‐3 fatty acid (EPA) treatment did not change UCP1 expression. However, UCP1 and PGC1α expression was significantly increased in SV cell cultures after 8‐ day EPA treatment. Our data suggests that EPA is capable of inducing the differentiation of inguinal SV cells into brown‐like adipocytes, but failed to induce UCP1 expression in mature white adipocytes. We conclude that HFD or saturated FA reduces UCP1, while EPA has a potential to increase the recruitment of brown‐like adipocytes in inguinal WAT.

Grant Funding Source : General Mills Foundation, MN Obesity Consortium
S‐allylcysteine inhibits tumor growth and progression of oral cancer in both in vitro and in vivo models
Authors: Chang Chiao-Ying , Huang Wei-Yao , Chen Shih-Yan , Pai Man-Hui , Tang Feng-Yao
Published: June 2021

Oral cancer is a prevalent type of cancer in many countries. Although studies indicated that garlic extracts such as diallyl disulfide and diallyl trisulfide have anti‐cancer effects, the chemopreventive effects of aqueous garlic extracts, S‐allylcysteine (SAC), on oral cancer have not been studied yet. To this specific aim, we investigated the inhibitory effects of SAC on the proliferation and progression of oral cancer cells in both in vitro and in vivo models. In the present study, our results demonstrated that SAC dose‐dependently inhibited the proliferation of human oral squamous cancer CAL‐27 cells in vitro. The results indicated that SAC stabilized the adherent junction complex of E‐cadherin/β‐catenin and suppressed epithelial‐mesenchymal transition (EMT) in oral cancer cells through suppression of MAPK/ERK signaling pathways. By study end (4th week), consumption of SAC (at doses of 5 and 40 mg/kg of body weight per day) significantly prevented tumor growth of oral cancer in a mouse xenograft model. Histopathological and immunohistochemical (IHC) staining results indicated that SAC could effectively suppress tumor progression including EMT of oral cancer in vivo. The chemopreventive effects of SAC were associated with decreases in cyclin D1 expression and suppression of Akt, mTOR, IκBα, and ERK 1/2 signaling cascades in tumor tissues. SAC consumption could inhibit the expression of cyclooxygenase‐2 (COX‐2), Vimentin, and nuclear NF‐κB p65 (RelA) proteins. These results suggested that SAC could act as an effective agent to prevent tumor growth, progression, and EMT of oral cancer.

Grant Funding Source : NSC
Insufficient HO1 Response to Ischemic Stress Correlates with Increased Inflammation and Reduced Survival of EPCs in Diabetic Patients
Authors: Issan Yossi , Hochhauser Edith , Kornowski Ran , Leshem-Lev Dorit , Lev Eli , Sharoni Ram , Vanella Luca , Puri Nitin , Laniado-Schwartzman Michal , Abraham Nader G , Porat Eyal
Published: June 2021

Background

Diabetes is characterized by oxidative stress and systemic inflammation. Nuclear ‐E2‐related factor‐2 (Nrf2) is a transcription factor that activate of various antioxidant genes, including heme oxygenase‐1 (HO‐1). HO‐1 and adiponectin are cardioprotective ensuing ischemia reperfusion

Methods

adipose tissue from diabetic (n=15) and non‐diabetic (n=15) patients undergoing CABG was analyzed using RT‐PCR and immunoblotting for Nrf2 genes. Oxidative stress was evaluated by thiobarbituric acid‐reactive substance (TBARS). Peripheral blood, was evaluated for inflammatory cytokines and EPC markers.

Results

Compared to controls, TBARS were higher in diabetic patients. Although Nrf2‐dependent antioxidant proteins (TxR1, NQO1, GST) were upregulated, HO‐1 and adiponectin protein expression and serum levels of bilirubin were lower in diabetic patients. Inflammatory cytokines serum levels were higher in diabetic patients. EPC levels and function were significantly reduced with diminished viability in diabetic group.

Conclusion

These results demonstrate for the first time that there is an inadequate HO‐1‐adiponectin axis response in humans with chronic conditions of oxidative stress such as diabetic patients with severe coronary artery disease, leading to insufficient antioxidant and anti‐inflammatory outcome.
Cardiac‐specific overexpression of GTP cyclohydrolase 1 ameliorates cardiac dysfunction and remodeling after myocardial infarction
Authors: Ge Zhi-Dong , Vásquez-Vivar Jeannette , Baumgardt Shelley L. , Pratt Phillip F. , Warltier David C. , Kersten Judy R.
Published: June 2021

GTP cyclohydrolase (GTPCH) 1 is the major determinant of concentrations of myocardial tetrahydrobiopterin, an essential cofactor for nitric oxide synthase. We examined whether cardiac‐specific overexpression of GTPCH 1 beneficially affected cardiac remodeling and function after myocardial infarction. Myocardial infarction was produced by ligating the left anterior descending coronary artery in GTPCH 1 overexpressing and wild‐type mice. Sham control mice underwent all surgical procedures without coronary artery ligation. Echocardiography was used to evaluate left ventricular (LV) dimensions and function. There were no significant differences in the thickness of LV anterior wall and posterior wall, internal diameters, and fractional shortening between sham‐operated GTPCH 1 and wild‐type groups. Four weeks after surgery, LV wall became thinner and LV was markedly dilated in wild‐type mice subjected to coronary ligation compared with those of wild‐type mice subjected to sham operation, but this effect was attenuated in GTPCH 1 overexpressing mice. Masson trichrome stained cross sections at mid‐ventricular level showed an enlarged LV cavity and increased collagen scar tissue in infarcted wild‐type hearts compared with infarcted GTPCH 1 overexpressing hearts. Infarct size expressed as a percentage of infarct circumference/total LV circumference was significantly smaller in infarcted GTPCH 1 overexpressing group than in infarcted wild‐type group (22.3±2.7% vs. 38.1±3.8%, n=8–10 mice/group, P<0.05). Myocardial interstitial fibrosis was significantly increased in infarcted wild‐type group compared with infarcted GTPCH 1 overexpressing group. These results demonstrate that cardiac‐specific overexpression of GTPCH 1 improves cardiac function and remodeling after myocardial infarction.
Opioid receptor activation alters beta‐adrenergic receptor function in heart failure
Authors: Schultz Jo El Jean , Natter Amanda B , Dunn Caitlin , Bolte Craig S
Published: June 2021

Our lab previously demonstrated that opioid receptor (OR) stimulation significantly enhanced the decreased cardiac function in animal models predisposed to or with heart failure (HF). Opioid and beta receptors have opposing effects in the healthy heart, such that OR stimulation decreases and beta receptor signaling increases cardiac function. Yet, the interaction between the two receptors during HF is unknown. To investigate this, hearts from a hamster model predisposed to or with HF and their controls will be subjected to simultaneous agonism of opioid and β1‐adrenergic receptors. Dobutamine (DOB) increased contractility in 1 month hamster hearts which was blocked by propranolol; whereas, diminished beta‐adrenergic effect on contractility in 8 month failing heart was observed compared to the wildtype (p<0.05). A similar decrease in DOB‐induced contractility was seen in 1 month hearts upon kappa or delta OR activation. Failing hearts had a lesser decrease (20%) in DOB‐induced contractility upon kappa OR stimulation compared to wildtype (40%, p<0.05). Conversely, upon delta OR agonism, failing hearts had a significant decrease (81%) in DOB‐induced contractility compared to wildtype (28%, p<0.05). In conclusion, OR activation modulates beta‐adrenergic receptor function in HF and will provide a better understanding of these receptors as they pertain to HF treatment. Research funded by NIH/NIDA R21 DA0804.
Stimulation of Cyclooxygenase‐2 (COX2) Pathway in Human Coronary Artery Endothelial Cells by Histamine Involves ERK and p38 activation but not Early Growth Response Factor‐1 (EGR1)
Authors: Tan XIaoyu , Raveendran Vineesh V , Krishnan Harinivas H , Dileepan Kottarappat N
Published: June 2021

Histamine (HIS) is a major constituent of the mast cell and it regulates many vascular functions. We have shown that HIS acting through H1R stimulates prostaglandin I2 (PGI2) and PGE2 production by enhancing the expression of COX2, PGI2 synthase and PGE2 synthase. In this study, we examined the role of PKC isoforms, MAP kinases and EGR1 in HIS‐mediated COX2 expression in HCAEC. HCAEC were incubated with HIS (10 μM), and the expression of COX‐2 and EGR1 was analyzed by qRT‐PCR and Western blot, and the production of PGI2 and PGE2 was monitored by EIA. Stimulation of HCAEC with HIS led to marked increases in the expression of COX2 and EGR1, production of PGI2 and PGE2, and phosphorylation of p38, ERK, and JNK. The p38 (SB203580) and ERK (PD98059) inhibitors blocked HIS‐induced COX2 expression, and PGE2 and PGI2 production. However, JNK inhibitor (SP600125) increased COX2 expression, and PGI2 and PGE2 production. HIS did not increase phosphorylation of PKCα/β or PKC™. Furthermore, PKCα/β inhibitors (Ro‐31, Gö 6983) or PKC™ inhibitor (Rottlerin) did not inhibit HIS‐induced activation of p38, ERK, or JNK. Although HIS induces nuclear expression of Egr1, the silencing of Egr1 by siRNA did not abrogate HIS‐mediated COX2 expression. These results suggest that HIS‐induced COX2 expression in HCAEC is mediated through p38 and ERK MAP kinase activation, and EGR1 is not involved in this process. (Supported by NIH Grants R01‐ HL070101 and 3R01‐HL070101‐04S1, and by Carey Arthritis Fund from the KU Endowment Association)
The Genetic Basis for Normal Vision and Vision Disorders
Authors: Neitz Jay , Neitz Maueen
Published: June 2021

The genes encoding the long‐(L) and middle‐(M) wavelength sensitive cone photopigment opsins are located on the X‐chromosome, and the gene encoding the short‐(S) wavelength sensitive opsin is on chromosome 7. Inherited red‐green color vision deficiencies are quite common and are most often associated with deletions or rearrangements of the L and M cone opsin genes. The same genetic mechanism that produces the gene deletions and rearrangements of the L and M opsin genes also intermixes their sequences producing new opsins that are chimeras of the ancestral L and M opsins. Inherited colorblindness is usually presumed to be strictly congenital. Some of the chimeric opsins produce nonfunctional photopigments; however, others cause a progressive loss of cone function, and thus are associated with red‐green color vision deficiency that is inherited but not congenital. Inherited blue‐yellow color vision deficiencies are much rarer, and are associated with mutations of the S opsin gene. Mutations in the S opsin can cause an S cone dystrophy and color vision defects that are also inherited but not congenital.
The role of colour in knowledge retention: a body‐painting study
Authors: Finn Gabrielle Maria , White Pamela M
Published: June 2021

This study tested student claims that bright color plays a significant role in retention of knowledge from body painting sessions. Medical students (n=117) were divided into two conditions: block color (CLR) and black outlines (BLK). Students completed a pre‐test before a body painting teaching session on abdominal referred pain. CLR students used bright block colors, and BLK students mapped outlines in black. Immediately afterward, students sat a mid‐test to compare the conditions. Students completeda post‐test to compare long‐term retention of knowledge four weeks later. There was no difference in knowledge between the two conditions immediately after the teaching(P =0.653). There was no significant difference in long‐term retention of knowledge between those using black outlines and those using color (P=0.278). The use of color had no impact on short‐term or long‐term retention of knowledge, despite previously collected qualitative data that color helped memorization. Despite there being no immediate difference in the amount of information retained, the students’ enthusiasm for body painting and the use of bold colors warrant body painting's inclusion within the anatomy curriculum. Acceptability plays a significant role in the success of any teaching modality.

Grant Funding Source : Internal funding
Validation of the digestive tolerance of Soluble Gluco Fibre, a new fibre
Authors: Housez Beatrice , Cazaubiel Murielle
Published: June 2021

To assess consumers’ acceptance of Promitor™ Soluble Gluco Fibre (SGF), we determined its digestive tolerance in healthy subjects. A composite score of gastrointestinal (GI) tolerance was expressed in arbitrary units (AU) and integrated GI symptoms, stool frequency and consistency. In order to statistically validate this composite score, the GI tolerance of inulin (10 g vs. 20 g) was assessed in 18 healthy volunteers in a randomized double‐blind placebo‐controlled cross‐over study. In a second double‐blind placebo‐controlled cross‐over study with 20 healthy volunteers, the GI tolerance of increasing doses of SGF in both a single and multiple administrations of consumption was assessed and the AU compared to the scores set by the inulin testing. In contrast to 10‐g inulin, 20‐g of inulin demonstrated a significant difference in composite score to placebo (p<0.001, diff=7.6 AU [3.8;11.3]). Using single dose administration, 40 g SGF was the highest dose tested that the composite score difference from placebo. The tolerance score was lower than 7.6 AU and the upper limit of the 95% CI was lower than 11.3AU, the clinically comparable tolerance determined from inulin scores. In multiple doses totalling 65 g SGF in a day was the threshold tolerance dose (p<0.001, diff=6.5 AU [3.4;9.5]). The study demonstrated that 40g SGF bolus and 65g SGF in multiple doses consumption are well‐tolerated by healthy volunteers. Funding source: Tate & Lyle Health & Nutrition Sciences
Role of Rac1 in A2B Adenosine Receptor and Epac1 signaling to Extracellular Signal‐Regulated Kinase in human endothelial cells
Authors: Olah Mark , Miller Lauren
Published: June 2021

Endothelial cell proliferation is required for angiogenesis. Our laboratory has previously shown that activation of the A2B adenosine receptor (A2BAR) through the cAMP effector Epac1 activates extracellular signal‐regulated kinase (ERK1/2), a key protein for endothelial cell proliferation. Presently, we hypothesize that the small GTPase Rac1 is involved in signaling from the A2BAR and Epac1 to ERK1/2 activation. In human microvascular endothelial cells (HMVEC), the Rac inhibitor NSC23766 diminished ERK1/2 activation by the A2BAR agonist 5‐N‐ethylcarboxamidoadenosine (NECA) and forskolin by 61.7 ± 10.4% and 60.3 ± 7.9%, respectively. Targeting of Rac1 with siRNA reduced NECA‐ and forskolin‐induced ERK1/2 stimulation. NECA, forskolin and a direct activator of Epac1 each promoted an ~2.2‐fold stimulation of Rac1 as assessed in a Rac1‐specific pull‐down assay. Knockdown of Epac1 with siRNA reduced NECA‐ and forskolin‐stimulated Rac1 activation by 65.4 ± 9.0% and 78.6 ± 10.1% respectively. A putative downstream target of Rac1 in HMVEC is NADPH oxidase with production of reactive oxygen species a possible mediator of ERK1/2 activation. The NADPH oxidase inhibitor diphenyleneiodonium reduced ERK1/2 activation by NECA and forskolin by 55.6 ± 7.1% and 78.5 ± 6.6%, respectively. These results indicate that the A2BAR activates ERK1/2 in endothelial cells via an Epac1‐Rac1‐NADPH oxidase signaling cassette.
Kinase activity of phosphoinositide 3‐kinase gamma (PI3Kγ) is important for neutrophil infiltration in inflammation
Authors: Sablad Marciano , Castro Glenda , Rozenkrants Natasha , Ngo Karen , Rao Tadimeti , Zhang Yan , Fung-Leung Wai-Ping
Published: June 2021

Phosphoinositide 3‐kinase (PI3K) is a family of lipid kinases that generates phosphatidylinositol 3, 4, 5‐trisphosphate in the plasma membrane to recruit and activate downstream signaling molecules. PI3Kγ is the only member of the class IB subfamily and it is composed of a catalytic subunit p110γ and one of the two regulatory subunits, p101 or p84. PI3Kγ activation is driven by G‐protein coupled receptors responding to chemotactic ligands. In autoimmune diseases, neutrophils are one of the major cell types causing the pathological outcome and tissue damage. Infiltration of neutrophils to site of inflammation is mediated by a wide variety of chemotactic factors. To demonstrate the role of PI3Kγ in neutrophil migration in vivo, knock‐in mice with inactive PI3Kγ were tested in two acute local inflammation models, arachindonic acid‐induced dermal inflammation and chemokine (RANTES)‐induced peritoneal cellular influx. Neutrophil infiltration was significantly reduced in PI3Kγ inactive mice when compared to wild‐type control mice in both models. The results suggest that PI3Kγ kinase activity plays an important role in neutrophil migration. Thus, inhibition of PI3Kγ kinase activity with small molecules could represent a promising approach for the treatment of inflammation and autoimmune diseases.
Role of endothelial Ca2+ in LPS‐induced ICAM‐1 expression in lung microvessels
Authors: Kandasamy Kathirvel , Parthasarathi Kaushik
Published: June 2021

In sepsis, ICAM‐1 expression is augmented in endothelial cells of lung microvessels. However, the role of endothelial cytosolic Ca2+ in the induction of ICAM‐1 expression is not known. Toward this, we blood‐perfused isolated rat lungs. We introduced a microcatheter through the left atrial cannula and then infused LPS (100μg/ml) into microvessels for 30 min. After 60 min, we determined ICAM‐1 expression by indirect in situ immunofluorescence. We recorded confocal images of surface microvessels using a LSM‐710 imaging system and quantified fluorescence intensity along the wall of microvessels. Intensity of ICAM‐1 expression was higher in LPS‐treated microvessels (venule 132.2±9.2; capillary 56.9±4.3, p< 0.001, n=3) compared to saline‐treated controls (12.6±1.8; 7.0±0.6 mean±SE, n=3). To establish the role of Ca2+, we infused Xestospongin‐C (25 μM), an inhibitor of inositol‐1,4,5‐trisphosphate (IP3) receptor on the endoplasmic reticulum (ER), into microvessels 10 min prior to LPS infusion. Xestospongin markedly reduced LPS‐induced lCAM‐1 expression in both microvessels (28.3±1.7; 8.2±0.4, n=2), indicating a role for IP3 in the process. We interpret from these data that LPS induction of ICAM‐1 expression in microvessels depends on IP3‐mediated ER Ca2+ release. Thus, we show for the first time that endothelial Ca2+ increase may be important in LPS‐induced responses in lung microvessels. (NIH HL75503)
Biomechanical Determinants of Endothelial Sprouting and Morphogenesis
Authors: Song Jonathan W , Munn Lance L.
Published: June 2021

Angiogenesis requires the coordinated growth and migration of endothelial cells (ECs), and each EC residing in a vessel wall integrates local signals to determine whether it remains quiescent or undergoes morphogenesis. Among these signals include vascular endothelial growth factor (VEGF), a well characterized morphogen which induces the formation of specialized “tip cells” that guide sprouts, and mechanical forces due to blood flow such as shear stress tangential to the endothelial surface and interstitial plasma flow across the vessel wall. Using a microfluidic tissue analogue of angiogenic sprouting, we found that physiological fluid shear stress (3 dyn/cm2) inhibits EC morphogenesis (sprouting and invasion into the 3‐D matrix) in a nitric oxide‐dependent manner and interstitial flow (2.5–35 μm/s) increases the rate of morphogenesis. Surprisingly, invading ECs not only detected the direction of VEGF gradients but also showed dramatic differences in morphogenesis depending on the direction of interstitial flow. Endothelial tip cells producing filopodia preferentially protruded against the direction of interstitial flow and in the direction of an increasing VEGF gradient. However, dominant‐negative RhoA ECs maintain the ability to respond to VEGF gradients by producing filopodia but no longer respond to interstitial flow. These results suggest that ECs integrate signals from fluid forces and local VEGF gradients to mediate endothelial sprouting and that RhoA is involved in flow‐induced tip cell morphogenesis. Research support: NIH T32CA073479 and NIH R01CA149285.
Effects of vitamin A supplementation during lactation on infant's antibody response to hepatitis B vaccine in China
Authors: Wang Zhixu , Sun Zhongqing , Fan Ping , Li Yonghua , Wu Jieshu , Zhong Chunmei
Published: June 2021

This study was designed to assess effects of maternal VA supplementation during lactation on their offspringˈs vitamin A status and immune response to Hepatitis B vaccine. Total 180 lactating women were recruited into a randomized, double‐blind, controlled intervention trial and were given one of the following three VA supplements: 1) large doses VA supplements (60mg) each at 2 weeks and 3 months postpartum; 2) daily VA supplements (1.2 mg) for 6 months; or 3) no any VA supplements. At the end of trial, 125 blood samples were collected at 3 month after the 3rd Hepatitis B vaccine from infants of mothers of three groups (42, 40 and 43, respectively) and analyzed for serum anti‐Hepatitis B antibody, retinol and acute phase proteins. The results showed there were significant differences in average logarithms of serum anti‐Hepatitis B antibody concentration among infants from mothers of three groups (2.79±0.38, 2.59±0.41, 2.53±0.42, F=4.488, P=0.013), although the difference in serum retinol was not significant (1.31±0.25, 1.28±0.30, 1.22±0.25, F=1.334, P=0.267) among the infants of the mothers with different VA intervention. The experiment gave a conclusion that VA supplements on lactating mother could benefit Hepatitis B vaccination of breast‐fed infant and large doses VA supplements works more effectively.
Resveratrol and grape seed extract combination elevates apoptosis in the colon cancer stem cells, even in the presence of IGF‐1, via P53 dependent pathway
Authors: Vanamala Jairam , Charepalli Venkata , Radhakrishnan Sridhar , Reddivari Lavanya
Published: June 2021

Cancer stem cells are contributors of chemotherapeutic resistance. We have previously shown that resveratrol (RSV) potentiates grape seed extract (GSE) induced apoptosis and suppression of proliferation in human colon cancer cell lines, but not in normal colonocytes, via p53 dependent mitochondrial apoptotic pathway. However, no information is available on whether grape compounds elevate apoptosis in the cancer stem cells. We hypothesized that RSV‐GSE combination will elevate apoptosis in human colon cancer stem cells (CD34+, CD44+, CD133+, ALDH1b1+), even the presence of IGF‐1 (a mitogen growth factor elevated during obesity), via elevation of p53 dependent pathway. RSV‐GSE combination elevated (p<0.05) apoptosis (Caspase 3/7 Glo and TUNEL assay) in the colon cancer stem cell line similar to that of 5‐fluorouracil (5‐FU), a standard colon cancer chemotherapeutic drug. P53 inhibition by pithifrin‐α (150 μM), a transcriptional inhibitor, and p53 shRNA (2 × 105 lentiviral particles) suppressed (p<0.05) RSV‐GSE and 5‐fluorouracil induced apoptosis. RSV‐GSE elevated (p<0.05) apoptosis, even in the presence of IGF‐1, indicating its potential efficacy in obese condition. This study demonstrates RSV‐GSE induced apoptosis in colon cancer stem cells via p53 dependent pathway. Results suggest that consumption of RSV‐GSE could contribute towards colon cancer prevention.
Electroacupuncture increases cerebral blood flow in a clinically relevant model of ischemic stroke
Authors: Lei Hong , Davis-Gorman Grace , McDonagh Paul , Ritter Leslie
Published: June 2021

Effects of EA at clinically relevant times in evolving ischemic stroke remain essentially unexplored. The purpose of this study was to determine if electroacupuncture (EA) initiated after the onset of ischemic stroke and during reperfusion can increase CBF. Middle cerebral artery occlusion (MCAO) was performed in anesthetized, ventilated male rats via the filament method. EA treatment consisted of 5–10 min. of stimulation at 4Hz using standard needles placed in the scalp (GV20) and the paw (LI4) ipsilateral to the MCAO. EA was applied before and during ischemia and during reperfusion. CBF (mean % increase +/SD over baseline values) was measured via doppler flowmetry. Mean arterial pressure (MAP) was monitored. In an exploratory experiment, the affects of EA on the microcirculation was observed using standard cranial window and fluorescence microscopy methods. Needle placement without EA had no effect on CBF or MAP nor did EA affect MAP. EA resulted in an increase in CBF before MCAO (93% +/− 64%) (N=4), during MCAO (82 +/− 42%) (N=3) and during reperfusion (26.1 +/− 2%) (N=3). EA increased blood flow, leukocyte flux and adhesion in the cerebral microcirculation, which reversed after withdrawal of stimulation. EA treatment applied during ischemic stroke, reperfusion, or both may improve perfusion in the microcirculation and is worthy of further investigation.
Smooth muscle cell TMEM16A channels contribute to myogenic constriction in cerebral arteries
Authors: Bulley Simon , Neeb Zachary P , Thomas-Greenwood Candice , Burris Sarah K , Jaggar Jonathan H
Published: June 2021

Transmembrane 16A (TMEM16A) channels are Ca2+‐activated Cl− (ClCa) channels that are expressed in smooth muscle cells of several vascular beds. Physiological functions of vascular TMEM16A channels are unclear. Here, we demonstrate that cell swelling (hypoosmolar bath solution) activates Cl− currents in cerebral artery myocytes. Swelling activated Cl− currents were inhibited by a pore‐targeting TMEM16A antibody, TMEM16A knockdown using siRNA, extracellular Ca2+ removal, and substitution of EGTA for BAPTA in the pipette solution. In contrast, swelling‐activated Cl− currents were not altered by SR Ca2+ load depletion (thapsigargin). TMEM16A knockdown attenuated pressure‐induced vasoconstriction in cerebral arteries when compared to arteries treated with control siRNA. In contrast, TMEM16A knockdown did not alter depolarization (60 mM K+)‐ induced vasoconstriction. These data indicate that membrane stretch activates a local sub‐plasma membrane Ca2+ signal in myocytes that stimulates TMEM16A channels. Such TMEM16A channel activation contributes to the myogenic response in cerebral arteries.
Elucidating the Proteolytic Regulation of Bioactive Peptides
Authors: Saghatelian Alan
Published: June 2021

Peptide hormones and neuropeptides are an important class of physiological regulators that mediate a number of important processes, such as glucose metabolism, blood pressure and pain. Understanding the enzymatic regulation of peptides can reveal the biochemical function of enzymes and inform us how these peptides are regulated. Despite transformative advances in many areas of biological research we still lack a general approach assigning the enzymes that regulate specific bioactive peptides in vivo. We solve this problem by developing an integrated strategy that combines peptidomics, biochemistry and genetics to elucidate the peptidases that regulate physiological levels of bioactive peptides.
Menopause status explains large individual variation in cardiovascular disease risk marker response to different dietary choline intake levels
Authors: Spencer Melanie Davis , Zheng Xiaojiao , King Sarah M. , Xie Guoxiang , Da Costa Kerry-Ann , Fischer Leslie M. , Jia Wei , Zeisel Steven H.
Published: June 2021

Excess dietary choline has been implicated in increased cardiovascular disease (CVD) risk, a process mediated by bacterial catabolism of choline to trimethylamine N‐oxide (TMAO). To determine whether variation in dietary choline leads to differences in plasma TMAO and CVD risk in human subjects, we enrolled twenty female participants in a two‐month, controlled dietary intervention study where they were exposed to three different intake levels of choline‐equivalents (choline, phosphatidylcholine and other choline‐containing compounds). Plasma samples collected at the end of each exposure were analyzed using liquid chromatography time‐of‐flight mass spectrometry to determine TMAO levels. Plasma oxidized low density lipoprotein (oxLDL), a marker for CVD risk, was measured using sandwich ELISA to establish whether changes in dietary choline levels were associated changes in CVD risk. Our analysis identified significant dose‐response relationships between choline equivalent intake and both plasma TMAO response (P=2.15e‐05 – MANOVA) and plasma oxLDL levels (P=0.0285). TMAO and oxLDL response variation were largely explained by menopause status (P=0.0003 and P=0.0014 respectively). These results suggest the possibility that bacteria‐mediated TMAO production may be important in the increased CVD risk observed in post‐menopausal women. NIH DK055895, DK056350 and RR00046 supported this work.
Homotropic Cooperativity During The Mechanism‐Based Inactivation Of Cytochrome P450 2B4 By 9‐Ethynylphenanthrene
Authors: Zhang Haoming , Gay Sean C , Shah Manish , Foroozesh Maryam , Halpert James R , Hollenberg Paul F
Published: June 2021

We investigated the mechanism by which 9‐ethynylphenanthrene (9EP) inactivates cytochrome P450 2B4 (CYP2B4). Our results demonstrated that 9EP is a potent mechanism‐based inactivator (MBI) of CYP2B4 with a partition ratio and kinact of 0.2 and 0.25 min−1, respectively. More importantly the kinetics of the MBI exhibit homotropic cooperativity with a Hill coefficient of 2.5 and S50 of 4.5 μM. To the best of our knowledge, this is the first report of homotropic cooperativity in the mechanism‐based inactivation of P450s. A fully modified CYP2B4 was purified to homogeneity, and its structure was solved by X‐ray crystallography. Based on this crystal structure, 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, resulting in inward rotation of Phe 297 and Phe 206. It is unlikely that the active site of CYP2B4 can accommodate two 9EP molecules. To explore other binding site(s) responsible for cooperativity, fluorescence quenching resulting from 9EP binding to CYP2B4 was investigated. These studies revealed two distinct binding sites for 9EP in CYP2B4. A high affinity site with a Kd of ~46 nM was observed in the modified CYP2B4, which likely arises from the binding of 9EP to a peripheral site. In contrast, a high affinity site as well as a low affinity site were associated with the binding of 9EP to the unmodified CYP2B4. These results suggest that 9EP binds to two distinct sites in CYP2B4 that function cooperatively.
IL‐1β Induces Brain Microvascular Endothelial Cell Hyperpemeability Through Caspase‐3
Authors: Wilson Rickesha L , Sawant Devendra A , Stagg Hayden W , Davis Matthew L , Smythe W. Roy , Tharakan Binu
Published: June 2021

Traumatic brain injury (TBI) is one of the most prevalent causes of morbidity and mortality worldwide. TBI is associated with brain edema, neuroinflammation and upregulation of proinflammatory cytokines. Interleukin‐1β (IL‐1β) is a major cytokine implicated in the adverse consequences of TBI. We hypothesized that IL‐1β induces brain microvascular hyperpermeability and edema via caspase‐3 mediated barrier dysfunction. Our prior studies have demonstrated caspase‐3 to be a key mediator in cell junction disruption. Rat brain microvascular endothelial cells were exposed to IL‐1β following caspase‐3 inhibitor Z‐DEVD (10μM) pretreatment. The permeability was determined using FITC‐dextran flux across the Transwell monolayer. Caspase‐3 activity was measured fluorometrically. The endothelial cell tight junction integrity was studied by immunofluorescence of zonula occludens‐1, claudin‐5 and occludin. The f‐actin stress fiber formation was studied using rhodamine phalloidin staining. Z‐DEVD attenuated IL‐1β‐induced hyperpermeability significantly (p<0.05). IL‐1β induced caspase‐3 activity significantly (p<0.05). Z‐DEVD decreased IL‐1β induced disruption of the tight junctions and f‐actin stress fiber formation. IL‐1β induces disruption of the brain endothelial cell barrier leading to hyperpermeability through caspase‐3‐mediated cleavage of the tight junction proteins.
No cardiac hypertrophy induced by T3 in a mouse model of the syndrome of resistance to thyroid hormones
Authors: Império Güínever Eustáquio , Pereira Guilherme Faria , de Góes Freitas Isalira Peroba Ramos , Santiago Letícia Aragão , dos Santos Almeida Norma Aparecida , Fuziwara Cesar Seigi , Kimura Edna Teruko , Olivares Emerson Lopes , Ortiga‐Carvalho Tania Maria
Published: June 2021

Triiodotironine (T3) is critical for cardiac function. Heart expresses TRα1 e TRβ1, the two main thyroid hormone (TH) receptors (TR). We aimed to investigate the role of TRβ1 on T3‐induced cardiac hypertrophy.

Adult male mice, wild‐type (WT) or homozygous (HO) for Δ337T mutation on TRβ (unable to bind T3), were used. Animals were studied at baseline, after hypo‐ (PTU) and hyperthyroidism (PTU+T3) induction. Echo‐ and electrocardiogram were performed. Hearts were collected, weighed and total RNA was extracted. Expression of mRNA and miRNAs were quantified by real time RT‐PCR.

T3 caused cardiac hypertrophy in WT, but not in HO animals when evaluated by cardiac index (P<0.001) or by echocardiography (25%, P<0.05). Genes responsive to T3 as ARb1, SERCa2 and HCN2 were responsive to T3 in WT but not in HO. Absence of a functional TRβ1 altered several parameters in HO cardiac function. Stroke volume and ejection fraction dropped in HO‐PTU+T3 vs. HO‐PTU. P wave reduced (50%, P<0.001) and heart rate increased (P<0.05) on WT‐PTU+T3 vs. WT‐PTU; but not in HO. Analysis of MicroRNAs miR‐1 and miR‐208, involved in cardiac hypertrophy, showed that T3 treatment increased both (2 to 3 times) in a different fashion, being miR‐1 TRβ independent and miR‐208 TRβ dependent.

TH‐induced cardiac hypertrophy is TRβ‐dependent and involves miR‐208, while the absence of T3 ligation on TRβ promotes a significant deterioration in cardiac function.
Comparison of TaqMan and SYBR Green qPCR Methods for Quantitative Gene Expression in Animals
Authors: Cao Heping , Roussel Anne‐Marie , Anderson Richard A.
Published: June 2021

Quantitative real‐time‐PCR (qPCR) such as TaqMan and SYBR Green qPCR assays are widely used for gene expression analysis. The drawbacks of SYBR Green assays include that the dye is nonspecific which can generate false positive signals if non‐specific products or primer‐dimers are present in the assay and that the length of the amplicon affects the intensity of the amplification. However, little was reported in the literature on direct comparison of both methods using the same pairs of primers and biological samples. We therefore compared both assays using 24 RNAs from mouse 3T3‐L1 adipocytes, RAW264.7 macrophages, and rat adipocytes, brain, kidney, liver, and muscle tissues. High quality RNA with high rRNA ratio and RNA integrity number was isolated from these animal cells and tissues and used for cDNA synthesis and qPCR amplification. The surprising finding was that SYBR Green qPCR over‐estimated the expression levels by several folds in most of the genes and tissues tested. This was unlikely due to non‐specific PCR products or the length of PCR products. The results demonstrate that both assays are reliable for determining gene expression levels in cells and tissues, but that TaqMan assay generally generates lower calculated expression levels than SYBR Green assay. This study suggests that any discussion of gene expression levels requires specification of qPCR methods used in the analysis.
Dietary Supplementation with Branched‐Chain Amino Acids (BCAA) to Lactating Sows Enhances the Availability of Glutamate and Glutamine in Milk
Authors: Rezaei Reza , Knabe Darrell , Gabriel Ana San , Nakamura Eiji , Uneyama Hisayuki , Wu Guoyao
Published: June 2021

Thirty multiparous sows (Landrace × Large White) were assigned randomly into one of three groups: 1) control (basal diet), 2) basal diet + 1.53% BCAA; and 3) basal diet + 3.07% BCAA. Diets were made isonitrogenous by addition of appropriate amounts of alanine. Lactating sows had free access to the drinking water and their respective diets. The number of live‐born piglets was normalized to 9 per sow at Day 0 of lactation. On Days 3, 15 and 29 of lactation, body weight and milk consumption of piglets were measured and blood samples obtained from sows and piglets at 2h after feeding. Data were analyzed by 1‐way ANOVA. Feed intake did not differ among the three groups of sows (P >; 0.05). Concentrations of aspartate, glutamine, citrulline, arginine, and BCAA were higher (P < 0.05) in the plasma of BCAA‐supplemented sows and their piglets than those in the control. Compared with the control, dietary supplementation with 1.53% and 3.07% BCAA increased (P < 0.05): 1) concentrations of free glutamate plus glutamine in milk by 18% and 35%; 2) milk intake of piglets by 8.2% and 13.7%; 3) daily weight gains of piglets by 6.5% and 10.3%; and 4) prenatal survival by 6.5% and 7.2%, respectively. These results indicate that dietary supplementation with up to 3.07% BCAA enhances milk production to support piglet growth in lactating sows. (Supported by Ajinomoto Inc. and Texas AgriLife Research)
Bendroflumethiazide a potent opener of human BK channel : a new perspective for an old drug in the treatment of periodic paralysis
Authors: Cannone Gianluigi , Calzolaro Sara , Mele Antonietta , Camerino Giulia M , Dinardo Maria M , Latorre Ramon , Conte Diana C , Tricarico Domenico
Published: June 2021

Carbonic‐anhydrase inhibitors and related drugs are used in the neuromuscular diseases but their effects on human Ca2+activated‐ K+‐(BK) channels are not known. The effects of acetazolamide(ACTZ), dichlorphenamide(DCP), methazolamide(MTZ), bendroflumethiazide(BFT), ethoxzolamide(ETX) and hydrochlorthiazide(HCT) on the human BK channel alpha‐subunit(hslo) expressed in HEK293 cells were investigated using patch‐clamp technique. The data were compared with that of NS1619, quercetin(QUERC) and resveratrol(RESV). We showed that the hslo whole‐cell current was potentiated by all drugs in the range of concentration tested(10−11–10−3M) with different potency and efficacy, with the exception of HCT. The ranking of the openers at −60 mV(Vm) was BFT(EC50=1.1±3×10− 9M)>; ACTZ(EC50=1.21±2×10−7M) >;DCP(EC50=4.3±1×10−7M) ≥RESV(EC50=5.5±1×10−7M)≥ ETX(EC50=7.1±0.1×10−7M)>; NS1619(EC50=5.5±1×10−5M)>; MTZ(EC50=1.17±1×10−4M)≥ QUERC(EC50=1.5±0.1×10−4M). We found that BFT and ACTZ were the most potent drugs at −60 mV(Vm). Patch depolarization potentiated the drugs action. The observed ACTZ and DCP actions on hslo channel at −60 mV(Vm) at sub‐micromolar concentrations explain their use in hypokalemic‐PP. BFT may be used in the hyperkalemic‐PP. This work is supported by Telethon Grants.
Amphetamine potentiates NMDA receptor currents in midbrain dopamine neurons
Authors: Ingram Susan L. , Li Minghua , Underhill Suzanne M. , Amara Susan G.
Published: June 2021

Amphetamine (AMPH) is known to increase extracellular concentrations of dopamine and glutamate. AMPH enhances extracellular dopamine primarily by competing with dopamine for the dopamine transporter (DAT); however, it is not known how AMPH alters concentrations of glutamate. Recently, we determined that acute AMPH exposure leads to internalization of the glutamate transporter, EAAT3, in dopamine neurons in a DAT‐dependent manner. Electrophysiological studies determined that AMPA:NMDA ratios are decreased with AMPH superfusion primarily due to potentiation of NMDA responses. NR2B‐selective inhibitors, ifenprodil and Ro25–6981, abolish the AMPH‐induced increase in NMDA currents. Blockade of synaptic NMDA receptors with the use‐dependent NMDA antagonist, MK‐801, blocked basal evoked NMDA currents but did not inhibit AMPH‐and NR2B‐dependent evoked NMDA currents. These results indicate that that new NMDA receptors are activated by AMPH. Further, Group I metabotropic glutamate receptor (mGluR) antagonists also inhibit AMPH potentiation of NMDA currents. Additional studies are focused on determining if NR2B‐containing receptors are activated by synaptic glutamate spillover, recruited to the synapse, or modified by second messenger signaling in midbrain dopamine neurons.

Supported by ARRA‐funded NIH grant DA07595 (SGA) and DA024041 (SLI).
Isolation of an osmoreceptor (TRPV4) from the avian lower gastrointestinal tract
Authors: Warner Paige , Braun Eldon
Published: June 2021

Avian osmoregulation differs from that of mammals in that not only do the kidneys regulate water and electrolyte levels but the lower gastrointestinal tract does as well. From the kidney, urine is conveyed to the cloaca where a reverse peristalsis of the GI tract brings the urine in contact with epithelial tissues of the colon where its composition can be modified. Brummermann and Braun (AJP, 268, 1995) demonstrated that this reverse peristalsis is not centrally regulated but rather locally regulated in the lower gastrointestinal tract. We suggest that the sensor of luminal fluid osmolarity is a member of the vanilloid‐receptor subfamily of transient receptor potential channels (TRPV4) a moderately selective Ca2+ channel. In the present study, tissues from the kidney, cloaca, and colon, were harvested from mourning dove (Zenaida macroura) and homogenized for analysis. Using Western Blotting and Immunohistochemistry, TRPV4 was visualized as the reaction product of DAB as well as a secondary antibody labeled with a fluorescent tag. To show the specific binding of the primary antibody, a blocking peptide was used to disrupt the above reactions. The results of immunohistochemistry show that TRPV4 is localized in the muscularis mucosa of the urodeum and similarly in the muscularis mucosa of the colon. In the avian kidney, TRPV4 appears to be localized in the ascending limbs of Henle similar to that of the mammalian kidney.
The effect of vitamin D supplementation during pregnancy and lactation on maternal & infant 25‐hydroxyvitamin D (25OHD) concentration
Authors: Chen Nancy N , March Kaitlin , Innis Sheila M , Shand Antonia , von Dadelszen Peter , Lyon Michael , Weiler Hope , Green Tim J
Published: June 2021

Attaining adequate vitamin D status during pregnancy and lactation is important for maternal and infant health. Our objective was to determine the effect of three dose regimens of vitamin D on maternal and infant 25‐hydroxyvitamin D concentrations. 163 healthy pregnant women between 13–22 wks gestation were recruited from Vancouver, Canada and randomized to one of three doses of supplemental vitamin D3 (400 IU/d, 1000 IU/d or 2000 IU/d) to take until 8 wks postpartum. Maternal blood was collected at baseline, 36 wks gestation, and maternal and infant blood were collected 8 wks postpartum. Mean 25OHD was 68 nmol/L at baseline and 17% of participants had a 25OHD < 50 nmol/L. At 36 wks gestation, maternal serum 25OHD [mean (95%CI)] concentrations, after adjusting for baseline 25OHD, were significantly higher in the women who received 2000 IU/d [79 (75,84) nmol/L] and 1000 IU/d [78 (74, 82)] than the 400 IU/d [69 (65, 74)]. At 8 wks postpartum, maternal serum 25OHD concentrations were highest in the 2000 IU/d [88 (83, 92)] followed by the 1000 IU/d [80 (75, 84)] and the 400 IU/d [71 (67, 76)] group. Likewise, serum 25OHD concentrations were highest in infants at 8 wks whose mothers received 2000 IU/d 75 (66, 84)] followed by the 1000 IU/d [54 (45, 62)] and the 400 IU/d (44 (35, 52)]. Generally, vitamin D supplementation increased maternal and infant 25OHD concentrations in a dose response manner. (Canadian Institutes of Health Research)
The impact of geranylgeraniol on the differentiation of murine 3T3‐F442A preadipocytes
Authors: Elfakhani Manal , Yeganehjoo Hoda , Shah Anureet , Hussein Deema , Mo Huanbiao
Published: June 2021

The statins, competitive inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, suppress the differentiation of adipocytes via mevalonate deprivation. We hypothesize that geranylgeraniol, a diterpene shown to accelerate the degradation of HMG CoA reductase, mimics the impact of lovastatin in adipocytes by suppressing adipocyte differentiation and adipogenic gene expression. Adipo‐Red assay and oil Red O staining showed that a 7‐d incubation with 2.5 – 20 μmol/L geranylgeraniol dose‐dependently reduced the intracellular triglyceride content of murine 3T3‐F442A adipocytes. Concomitantly, geranylgeraniol down‐regulated the expression of peroxisome proliferator‐activated receptor γ (PPARγ), a key regulator of adipocyte differentiation as analyzed by real‐time qPCR. The expression of adipocyte marker genes including adiponectin, leptin, fatty acid binding protein 4 and lipoprotein lipase was also suppressed by geranylgeraniol. No changes in cell viability were observed in 3T3‐F442A cells incubated with geranylgeraniol (0–400 μmol/L) for 24 and 48 h. Mevalonate‐derived metabolites have essential roles in promoting adipocyte differentiation and adipogenic gene expression. Dietary mevalonate suppressors may have potential as anti‐adipogenesis compounds.
Regulation of Cardiopharyngeal Fate Specification in a Simple Chordate
Authors: Christiaen Lionel , Razy‐Krajka Florian , Wang Wei , Kaplan Nicole , Lam Karen
Published: June 2021

In vertebrates, the pharyngeal subset of head muscles shares a common clonal origin with cardiomyocytes in early embryos. The ascidian Ciona intestinalis offers an opportunity to study cardiopharyngeal fate specification with cellular resolution in a simple chordate model. In embryos, bipotent cardiopharyngeal progenitors undergo asymmetric cell divisions to produce first and second heart precursors and pharyngeal muscle precursors following a stereotyped clonal pattern. Targeted perturbations, Fluorescence Activated Cell Sorting (FACS), transcriptome profiling and fluorescent in situ hybridization showed that multipotent cardiopharyngeal progenitors express a mixture of early cardiac and pharyngeal muscle regulators. This multilineage transcriptional priming is resolved through regulatory cross‐antagonisms, whereby Tbx1/10 inhibits Gata4/5/6 and heart specification in the pharyngeal muscle precursors, thus promoting Ebf expression and muscle fate specification; while, in the heart precursors, Nk4/Nkx2‐5 inhibits Tbx1/10 and Ebf, permitting Gata4/5/6 expression and cardiac specification. In the pharyngeal muscles, Ebf activates an Mrf‐associated muscle differentiation program and maintains muscle precursors' stemness through Notch‐mediated lateral inhibition. Finally, polarized signaling events regulate the initial asymmetries and trigger the above transcriptional cascades underlying fate choices.