bluuebunny/crossref_metadata_2025 huggingface dataset:
Nicotine Paradoxically Alters The Facilitatory Action Of Estrogen And Progesterone On Adenosine Receptor‐Mediated Renal Vasodilations
Authors: Gohar Eman M , El‐gowilly Sahar M , El‐Gowelli Hanan M , El‐Mas Mahmoud M
Published: June 2021

We tested the hypotheses: (i) nicotine modifies the interaction of ovarian hormones with adenosine receptor‐mediated renal vasodilations, and (ii) nitric oxide synthase (NOS) and heme oxygenase (HO) modulate this interaction. Vasodilations caused by NECA, adenosine analogue, in perfused kidneys were reduced in ovariectomized (OVX) rats and restored after estrogen (E2) or medroxyprogesterone acetate (MPA) supplementation. The E2 or MPA effect was abolished after blockade of respective receptors by ICI 182780 and mifepristone, and after NOS (L‐NAME) but not HO (ZnPP) inhibition. NECA vasodilations were increased and decreased by nicotine (1 mg/kg/day, 2 weeks) in OVX/E2 and OVX/MPA kidneys, respectively. By contrast, NECA responses were resistant to nicotine in rats with deficient (OVX) or balanced (sham or E2/MPA‐replaced OVX) hormonal milieu. Nicotine enhancement of NECA responses in OVX/E2 kidneys was abolished by L‐NAME but not ZnPP. Alternatively, nicotine attenuation of NECA responses in OVX/MPA kidneys disappeared in presence of hemin (HO inducer) but not L‐arginine (NOS substrate). The hemin effect was compromised by ZnPP. Thus, NOS facilitation and HO inhibition mediate the opposite effects of nicotine on NECA responses in E2‐ or MPA‐replaced OVX rats, respectively.

This project was supported financially by the Science and Technology Development Fund (STDF), Egypt, Grant No. 502.
Structural Determinants of RGS‐RhoGEF Signaling Critical to Entamoeba histolytica Pathogenesis
Authors: Bosch Dustin E , Kimple Adam J , Manning Alyssa J , Muller Robin E , Willard Francis S , Machius Mischa , Rogers Stephen L , Siderovski David P
Published: June 2021

G‐protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G‐protein a subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and GTPase‐accelerating protein (GAP), EhRGS‐RhoGEF, in a nucleotide state‐selective fashion. Co‐expression of EhRGS‐RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac‐dependent spreading in Drosophila S2 cells. EhRGS‐RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full‐length EhRGS‐RhoGEF reveals a putative inhibitory helix engaging the DH domain Rho‐binding surface and the PH domain. Mutational analysis of the EhGα1/EhRGS‐RhoGEF interface confirms a canonical RGS domain rather than a RhoGEF‐RGS (“rgRGS”) domain, suggesting a convergent evolution toward heterotrimeric and small G‐protein cross‐talk.
TAK1 is a Regulator of Sorafenib‐induced Keratinocyte Toxicity
Authors: Zimmerman Eric I , Gibson Alice A , Hu Shuiying , Berk Jeffrey , Orwick Shelley , Sparreboom Alex , Baker Sharyn D
Published: June 2021

Hand‐foot skin reaction (HFSR) is a debilitating toxicity resulting from treatment with the tyrosine kinase inhibitor (TKI) sorafenib. Molecular mechanisms underlying HFSR are unknown and there are no effective therapies other than sorafenib dose reduction. To identify signaling pathways associated with sorafenib‐induced keratinocyte toxicity, in situ kinome profiling (ActivX) was performed using human primary keratinocytes (HEKa). These data, in combination with microarray‐based HEKa gene expression analysis and literature‐based sorafenib target Kd analysis, resulted in 11 candidate kinases. Of these, we focused our efforts on TAK1 because it is implicated in the maintenance of skin homeostasis. Interestingly, compounds associated with HFSR were potent (0.15–1.3 μM Kd) inhibitors of TAK1 by in vitro kinase assay, whereas non‐HFSR‐associated TKIs had a Kd >;100 μM. Furthermore, sorafenib inhibited TAK1‐mediated signaling in HEKa cells in a dose‐ and time‐dependent manner, whereas non‐HFSR‐associated TKIs had no effect. Sorafenib treatment of HEKa cells induced ROS generation, caspase activation, and apoptosis; effects that were potentiated by TAK1 siRNA‐mediated knockdown. These data suggest that sorafenib inhibits TAK1 in HEKa cells, which at least in part mediates keratinocyte cell death. Ongoing preclinical studies will establish the role of TAK1 signaling in sorafenib‐induced keratinocyte toxicity in vivo.
Effects of Maternal Ethanol Intake during the Pregnancy and Lactation on the Expression of NMDA Receptors in Hippocampus of Pups at Different Stages of Brain Development
Authors: Cheregatto Nathálie Neves , Soler João Antonio , Mussi Sara Eleodoro , Fiorillo Henrique Mascioli , Borges Brunna Oliveira , de Oliveira Jamila Dib , Defendi Paola Galassi , da Silva Danilo Fernandes , Iyomasa Melina Mizusaki , Rosa Maria Luiza Nunes Mamede
Published: June 2021

Aim

To investigate whether ethanol consumption during pregnancy and lactation might induce changes on the expression of NR1 and NR2A in the hippocampus of pups at different stages of brain development. Female rats were exposed to water (controls, n=8) or ethanol 10% (alcoholic, n=8) for 60 days, from mating to weaning. The pups were studied at PND30–90 (n=8–13). Immunohistochemistry. Positive cells were quantified. ANOVA. Maternal ethanol intake induced changes in NR1 expression only in pups at PND21 (p<0,001) increasing in CA1 (30%) and decreasing in both CA3 (25%) and HDG (28%). Controls or alcoholic pups compared within the same treatment showed discrepant results in CA1, CA3 or HDG along the development. NR2A expression was reduced in CA1 of alcoholic pups at PND21 and 60 (25 and 44%, respectively), reduced in CA3 at PND30 (23%), increased in CA3 at PND90 (37%) (p<0,001) and no changed in HDG. Comparisons within the same treatment showed predominant reduction in NR2A expression along the development of both controls and alcoholic pups (18–38%, p<0,001). Maternal ethanol intake during pregnancy and lactation induces different changes on the expression of NMDA receptors in hippocampus of pups in different periods of development, which may reflect alterations in both glutamatergic neurotransmission during the brain maturation and hippocampal function at adulthood. Support: FPA.
Role of GRK6 in the addictive effect of psychostimulant drugs
Authors: Li Lingyong , Gurevich Eugenia V
Published: June 2021

The dopamine innervation of the nucleus accumbens (NAc) is the key neural substrate mediating the primary reinforcing and psychomotor stimulant effects of drugs of abuse. Psychostimulant drugs (PDS) enhance the dopamine concentration by blocking dopamine transporter and/or inducing non‐vesicular dopamine release in the brain, which increases the activity of dopamine receptors. G protein‐coupled receptor kinases (GRKs) are the key proteins regulating the signaling of G protein‐coupled receptors (GPCR) via the mechanism of homologous desensitization. The aim of the present study was to investigate the role of GRK6‐dependent GPCR desensitization in PSD addiction. Overexpression of GRK6 in NAc using lentivirus‐mediated gene transfer reduced cocaine and amphetamine‐induced conditioned place preference (CPP). The effect of down‐regulation of GRK6 in NAc on PSD‐induced CPP was also tested. To evaluate the role of GRK6 in drug craving and relapse, the drug‐induced reinstatement of CPP was performed. Our results shift the traditional focus away from receptors themselves and put the GRK/arrestin‐based receptor desensitization machinery as the main regulatory mechanisms involved in PSD reward. A pharmacological strategy based on increasing activity and/or availability of GRK6 may be a promising therapeutic approach for PSD addiction. Work supported by NIH grants NS065868 and R21DA030103.
In Vivo Binding of N‐Substituted Benztropine Analogs and Antagonism of Cocaine Self‐Administration
Authors: Wilkinson Derek , Hiranita Takato , Mereu Maddalena , Tanda Gianluigi , Newman Amy H. , Katz Jonathan L.
Published: June 2021

Previous studies indicate that benztropine (BZT) analogs bind to the dopamine transporter (DAT), inhibit DA uptake, are less effective than cocaine in a variety of procedures, and block cocaine self administration. Previously studied BZT analogs have a low apparent rate of association, which has been hypothesized to contribute to their lower cocaine‐like effectiveness. Recent studies have revealed BZT analogs with a relatively fast onset of action, but it is unclear if these actions were related to DAT binding. Therefore, the present study assessed the in vivo displacement of [125I]RTI‐121 binding to the DAT by the N‐substituted BZT analog JHW 013, its self administration, effects on cocaine self administration and effectiveness in stimulating DA levels in the nucleus accumbens shell. Maximal in vivo binding of JHW 013 was achieved at 30 min, compared to 45 min with cocaine, indicating a relatively fast rate of association. In addition, JHW 013 dose‐dependently increased DA in the nucleus accumbens shell. JHW 013 failed to maintain self‐administration at levels greater than saline and its pretreatment blocked cocaine self administration. The relatively fast onset of action of JHW 013 at the DAT indicates that a low rate of association is not necessary for the reduced cocaine‐like effects of BZT analogs and their potential as therapeutics for stimulant abuse. Supported by NIDA IRP
Grape powder supplementation improves blood pressure, anxiety‐like behavior and learning and memory function in ovariectomized rats.
Authors: Salim Samina , Patki Gaurav , Chugh Gaurav , Bohat Ritu , Allam Farida , Jafri Faizan , Saleem Mohammad , Asghar Mohammad , Alkadhi Karim A
Published: June 2021

Estrogen is of prime importance to normal brain function; and its depletion at menopause may account, at least in part, for cognitive decline as well as hypertension. Here, we examined the effect of grape powder treatment on anxiety‐like behavior, learning and memory impairment and blood pressure in ovariectomized (OVR) rats. Four groups of Wistar female rats were used. Group 1 and 2 included controls for sham and sham‐grape powder treated, group 3 was OVR, and group 4 was OVR and grape powder treated for 2 weeks. At the end of the 2 weeks, body weights, food and water intake, blood pressure were recorded, anxiety‐like behavior (open‐field and light‐dark) and learning‐memory (radial water maze) tests were conducted. We observed a significant increase in systolic and diastolic blood pressure in OVR rats as compared to sham‐controls. Furthermore, ovariectomy increased anxiety‐like behavior and caused learning and memory impairment in rats as compared to the sham‐control group. Interestingly, supplementation with grape powder to OVR rats for 2 weeks was marked with restoration of both systolic and diastolic blood pressure, improvement in the anxiety‐like behavior and learning and memory function. This supports our hypothesis that grape powder may be an attractive target for estrogen replacement therapy. Oxidative stress data points towards interesting mechanistic clues to these observations.

Grant Support: NIH R15 G103327
Antiproliferative mechanism of novel imidazoline compound in PC12 cells
Authors: McLean Lancelot Stuart , Crane Louis , Baziard‐Mouysset Genevieve , Edwards Lincoln
Published: June 2021

Common anti‐diabetic agents that cause release of insulin such as sulfonylurea drugs are linked to an increased risk of cancer. Clinically useful novel I1‐imidazoline receptor agonists such as moxonidine have pleiotropic effects, including antihypertensive and insulin sensitization effects. Recently, some of these compounds have been associated with cell death in vitro, however the mechanism of action is not fully elucidated. Our objective was to investigate the antiproliferative mechanism of the novel imidazoline compound, S43126 in PC12 pheochromocytoma cells. One group of cells were treated time dependently with vehicle or S43126 (1mM). A second group of cells were pretreated for 2 hours with z‐VAD‐fmk, NAC, SP600125, SB202190, followed by S43126 (1mM). Cells were then analyzed for apoptosis, ROS formation, mitochondrial membrane permeabilization, caspase activation and MAPK phosphorylation. Our results showed that in PC12 cells, stress activated MAPKs and caspases are involved in the antiproliferative activity of S43126. Our data also showed that in PC12 cells, S43126 increased oxidative stress and mitochondrial membrane permeabilization. These results indicate that S43126‐ induced PC12 cell death is partly mediated by oxidative stress, stress activated MAPKs, and caspases.

These studies were supported in part by the Center for Dental Research at Loma Linda University
Antiapoptotic Effects of 2,3‐Dichloro‐5,8‐dimethoxy‐1,4‐ naphthoquinone on Salsolinol‐induced Toxicity in Human SHSY‐5Y Cells: A Mechanistic Study
Authors: White Shannan Tonette , Copeland Robert L.
Published: June 2021

2,3‐dichloro‐1,4 naphthoquinone (2,3DCNQ) derivatives possess a wide variety of biological responses including antibacterial and anti‐inflammatory. However, these compounds have biphasic effects and at low nM concentrations have neuroprotective activity. This study was designed to evaluate the neuroprotective effect of 2,3DCNQ derivatives against SAL‐induced cytotoxicity in the human neuroblastoma cell line, SH‐SY5Y, by inhibition of apoptosis. Exposure of SH‐SY5Y cells to 0.2 and 0.5 mM SAL for 24 hours resulted in 40 to 70 % cell death respectively as determined by MTT assay. Cells pretreated for 60 minutes with 15 nM 2,3DCNQ resulted in attenuation of salsolinol‐induced cytotoxicity. Flow cytometry analysis showed the highest number of early plus late apoptotic cells in SAL treated group exposed for 24 h. The 2,3DCNQ pretreatment reduced the apoptotic cell death. Western Blot analysis showed increased Akt activation. In nM concentrations, 2,3DCNQ appears to be specific for the antiapoptotic pathway, particularly PI3/Akt. This study suggests that 2,3DCNQ protect SH‐SY5Y cells from SAL‐mediated cytotoxicity by suppressing apoptosis.
Monitoring AMP production in diverse biochemical reactions using HTS‐formatted bioluminescent assays
Authors: Goueli Said , Hsiao Kevin , Zegzouti Hicham
Published: June 2021

Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. Many biochemical reactions that generate AMP as a reaction product are pivotal players in a variety of signaling pathways and thus are considered validated drug targets. These include enzymes involved in protein ubiquitination, aminoacyl tRNA synthetases in protein synthesis, cAMP‐Phosphodiesterases in second messenger regulation, DNA ligases in DNA synthesis and DNA repair, etc. Thus an assay that can monitor the concentration of AMP generated by these enzymes is desirable in the search for modulators of these enzymes and the development of novel therapeutics. In addition, enzyme reactions that generate products which can be converted to AMP can be also monitored using this assay. We have developed a homogenous bioluminescent assay that is antibody free, sensitive, free from fluorescent compound interference, and easy to use. We will show its utility in various applications such as cAMP‐phosphodiesterases, ubiquitin ligases, DNA ligases, methyltransferases, etc
Development of cell‐based high‐throughput screening assay for DNase I inhibitors or activators
Authors: Kharade Sujay V. , Basnakian Alexei G.
Published: June 2021

Deoxyribonuclease I (DNase I) is an integral part of the universal pathway responsible for irreversible cell death in response to injury in normal and cancer cells. However, there are no specific small molecule inhibitors or activators of DNase I that could be used therapeutically to protect vital organs from toxicity or increase the effectiveness of the cancer therapy. Available DNase assays are lengthy and not precise enough to be used for high throughput screening (HTS) of chemical libraries. Thus, we have developed a new cell‐based assay to measure the DNase I activity using a proprietary fluorescent DNA probe. Here, we report the initial optimization of the key assay parameters. NRK‐52E cells were grown in 96‐well plate overnight to form a monolayer. The probe was added to the medium and the fluorescence intensity was measured at 665ex and 703em using a plate reader. The EC50 of the probe is 125 nM. The medium with 5% FBS had no apparent effect on the signal compared to serum‐free medium. Similarly, DMSO at concentrations up to 1% did not affect the signal. DNase I inhibitor, zinc chelate of 3,5‐diisopropylsalicylic acid, decreased the fluorescence in dose‐dependent manner with IC50 value of 60 μM. Further, the assay was validated with excellent Z′ factor (>;0.5). Thus, this new assay can be used to screen chemical libraries to identify possible DNase I inhibitors or activators. NIH R01 DK078908 & VA Merit Review grants to AGB
Comparison of mass‐action law algorithm‐based pharmacodynamics with the conventional pharmacokinetic studies
Authors: Chou Ting‐Chao
Published: June 2021

Pharmacodynamics (PD) of dose‐effect is governed by the physico‐chemical principle of the mass‐action law. Its systematically derived equations can be deduced to the general median‐effect equation for single entity, and the combination index equation for multiple entity interactions (Chou TC. Pharmacol. Rev. 58: 621–681, 2006, free web access). However, pharmacokinetic (PK) formula are obtained for conventional utilities, such as t1/2, Cmax, Vdis, and clearance rate that are associated with “time”, but not efficacy nor toxicity. PK offers the proper use of drug, e.g., interval/frequency of doses, and precaution for renal or hepatic deficiencies. Contemporary R&D has emphasized PK and ADME, however, rigorous investigation on PD has been grossly neglected which is attributable to the high rate of new drug R&D failure. PD should be the primer. Unfortunately, PD is an underdeveloped area, e.g., bio‐researchers are still drawing the empirical curves to fit numerous data points rather than using a small number of data points to fit the mass‐action law. Furthermore, drug combinations are widely used in treating cancer and AIDS, however, most bio‐researchers are still confused by nearly 20 different definitions for synergy claims without knowing that the mass‐action law algorithm is the key (Chou, Pharmacol. Rev. 58: 621–681, 2006; Scientist 21: 15, 2007; Cancer Res. 70: 440–446, 2010). This confusion leads to inefficiency and waste in research. This author concludes that the mass‐action law PD algorithms and software provide efficient, quantitative econo‐green bio‐research as illustrated in Chou, Integr. Biol. 3: 548–559, 2011 and in Am. J. Cancer Res. 1(7): 925–954, 2011.
How do students acquire rational prescribing competence? A Bernsteinian analysis
Authors: Moch Shirra
Published: June 2021

There is global concern about the quality of prescribing training in medical schools and the rational prescribing skills of junior doctors. In the Faculty of Health Sciences, University of the Witwatersrand, curricular change sought to reduce the factual burden of learning and enhance evaluative capacities through the introduction of an integrated syllabus delivered via problem‐based learning. Despite the new curriculum, graduating students reported a lack of confidence to prescribe. To explore this finding, this paper analyzes prescribing competence in relation to Bernstein's theoretical framework of the structure of knowledge. Prescribing is described as a regionalized skill in relation to several singular disciplines. For successful students, the disciplinary knowledge base necessary for rational prescribing decisions is a Bernsteinian vertical discourse with conceptual coherence. Other students, however, cannot distinguish beyond the context of each clinical problem presented and their resultant learning is segmented and not transferrable. In addition, this contextual learning leads to a subjective increase in volume, since disciplinary knowledge is not incorporated into inclusive principles Thus, curricular components including problem‐based learning and horizontal integration constrain epistemic access to the structure of rational prescribing knowledge for some students.
Pharmacogenomics of vincristine‐induced neurotoxicity in pediatric cancer patients
Authors: Amstutz Ursula , Loo Tenneille T , Gylling Helene M , Rassekh Rod , Hukin Juliette , Zada Yassamin Faroz , Dubé Marie‐Pierre , Brown Andrew M , Phillips Michael S , Hayden Michael R , Ross Colin J , Carleton Bruce C
Published: June 2021

The use of the anticancer agent vincristine is limited by debilitating neurotoxicity (VIN), for which a genetic basis has been suggested. Here, we investigated variation in 315 genes involved in drug absorption, metabolism, distribution, excretion (ADME) and toxicity in the context of VIN in children with cancer.

We genotyped 4536 ADME variants in a pilot cohort of 140 children with Wilms tumor and rhabdomyosarcoma. To replicate findings, known variants in CYP3A5 and ABCB1 as well as variants with evidence for association (p<0.01) in the pilot cohort were analyzed in 422 patients with acute lymphoblastic leukemia (ALL).

No association of variants in CYP3A5 or ABCB1 with VIN was observed. Of the candidates identified in the pilot cohort, the association of a variant in ABCA4 with VIN was replicated in patients with ALL (combined cohort OR: 1.7; P = 2.9×10−4).

This study was the first to broadly screen genetic variation in the context of VIN in pediatric cancer patients and serves as a starting point for further study of the genetic basis of VIN, providing novel hypotheses for mechanisms underlying the susceptibility to VIN and the possibility for predictive genetic testing to identify patients at risk.

Funded by CIHR/CFI
Decreased Nuclear Receptor Activity Mediates Down‐ Regulation of Drug Metabolizing Enzymes in Chronic Kidney Disease Through Epigenetic Modulation.
Authors: Velenosi Thomas , Feere David , Sohi Gurjeev , Hardy Daniel , Fu A , Urquhart Brad
Published: June 2021

Objective

To determine the mechanism of hepatic drug metabolizing enzyme down‐regulation in chronic kidney disease (CKD).

Methods

Hepatic CYP3A1, CYP3A2 and CYP2C11 mRNA expression were determined in rats with surgically induced CKD. Chromatin Immunoprecipitation (ChIP) was performed to determine nuclear receptor and epigenetic mediated differences in the promoter region of these enzymes.

Results

Hepatic CYP3A and CYP2C11 mRNA expression was significantly decreased in CKD rats compared to controls (P<0.05). RNA polymerase II binding to the CYP3A and CYP2C11 promoter regions was decreased in CKD rats (P<0.05). ChIP also revealed a decreased PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). HNF4α binding to the CYP3A and CYP2C11 promoter regions was also decreased compared to controls (P<0.05). The decrease in PXR and HNF4α binding was concurrent with diminished histone 4 acetylation in the CYP3A2 promoter locus for nuclear receptor activation.

Conclusions

We demonstrate a novel mechanism of drug metabolizing enzyme regulation in CKD. Our results show that decreased CYP3A and CYP2C11 mRNA expression is secondary to decreased PXR and HNF4α binding as a result of histone modulation in CKD. This research is supported by the Natural Sciences and Engineering Research Council.
Regulation of Cardiolipin Remodeling in Human Lymphoblasts
Authors: Mejia Edgard , Taylor William , Vandel Marilyne , Hatch Grant
Published: June 2021

Background

Barth Syndrome (BTHS) is a rare X‐linked genetic disorder caused by mutations in the TAZ gene. The TAZ gene product, tafazzin, is responsible for remodeling Cardiolipin (CL) with the necessary acyl species. TAZ mutations can result in cardiomyopathy. Monolysocardiolipin acyltransferase‐1 (MLCL AT‐1) is another enzyme capable of CL remodeling.

Objective

To examine if MLCL AT‐1 complements tafazzin in the remodeling of CL.

Methods

Lymphoblasts from normal or BTHS patients were transfected with TAZ and/or MLCL AT‐1 RNAi or an MLCL AT‐1 carrying plasmid. TAZ and MLCL AT‐1 gene expression were analyzed. Other analyses included measuring CL mass, MLCL AT‐1 enzyme activity and radiolabelling studies. Our studies also include using BN PAGE to analyse mitochondrial complexes in normal and BTHS lymphoblasts.

Results

MLCL AT‐1 gene expression increased when TAZ was knocked down. Expression of MLCL AT‐1 elevated CL levels, increased [1‐14C] Linoleic acid incorporation into CL and raised MLCL AT‐1 enzyme activity in normal and BTHS cells in which TAZ was knocked down. The double knockdown results are not lower than TAZ knockdown alone. Results show that MLCL AT‐1 over‐expression is able to elevate mitochondrial complex I formation in BTHS lymphoblasts.

Conclusion

MLCL AT‐1 over‐expression may serve as a potential therapeutic approach to treat BTHS.

Funding:

CIHR, Barth Syndrome Foundations of USA and Canada
Polydrug use among pregnant methadone users
Authors: Delano Kaitlyn , Gareri Joey , Koren Gideon
Published: June 2021

Methadone maintenance treatment (MMT) is used to manage opioid dependence. However, the common practice of polydrug use poses a serious concern with women co‐abusing substances during pregnancy. The objective of this study was to assess polydrug use in pregnant methadone users by meconium result analysis. Meconium samples were collected from the Motherisk Laboratory for testing as per requests by children's aid societies and hospitals. Over 22 months, samples were tested for substances of abuse through immunoassay (ELISA) or GC‐MS analysis. All methadone positive samples were used to assess frequency of polydrug use in a high‐risk pregnant population with methadone negative samples serving as controls. Of the tested samples, 117 were positive and 88 were negative for methadone. Opioids were the most prevalent drug class detected for both groups (44.4 and 50% respectively). No statistical difference was found for the prevalence of any individual or class of drug, with the exception of codeine (p=0.042), nor for the average minimum number of drugs detected (1.33 (SD=1.05) and 1.55 (SD=1.30) respectively). With opioids being the most prevalent drug class, this could suggest that opioid dependence is not effectively managed in a substantial fraction of individuals on MMT. With a high rate of polydrug use, it is pertinent to study the effects this can have on the fetus in future research.
The mechanism of protective effects of branched‐chain amino acids on hepatic ischemia/reperfusion‐induced liver injury in rats
Authors: Kitagawa Tomomi , Yokoyama Yukihiro , Kokuryo Toshio , Nagino Masato
Published: June 2021

We determined whether there is a protective effect of branched‐chain amino acid (BCAA) on hepatic ischemia/reperfusion (IR)‐induced acute liver injury. Wister rats were divided into four groups: simple laparotomy with vehicle; simple laparotomy with BCAA; IR (30 min clamp) with vehicle; and IR with BCAA. Serum liver function tests and the gene expression of adhesion molecules and endothelin‐1 in the liver were examined. In the in vivo study, portal venous pressure, leukocyte adhesion, and hepatic microcirculation were evaluated. Furthermore, Kupffer cells were isolated and cultured with BCAA in lipopolysaccharide (LPS). Increased levels of liver function tests, the expression of adhesion molecules and endothelin‐1, portal venous pressure, enhanced leukocyte adhesion and deteriorated hepatic microcirculation following IR were significantly attenuated by BCAA treatment. In the experiment using isolated Kupffer cells, the expression of inflammatory cytokine, and endothelin‐1 in response to LPS stimulation was attenuated by BCAA. Oral administration of BCAA has excellent therapeutic potential for IR‐induced liver injury. These effects may result from the direct attenuation of Kupffer cell activation.
Hypoxia Preconditioning Lowers Myocardial Matrix Metalloproteinase Activity
Authors: Gonzalez Leticia , Estrada Juan , Mallet RT , Downey HF , Caffrey JL
Published: June 2021

Intermittent hypoxia conditioning (IHC) decreases infarct size in dogs from 40% to below 5% of the area at risk. The protective effect appears to involve delta‐opioid (DOR) receptors. Reperfusion injury is attributed in part to the early incursion of neutrophils whose entry and transit in the myocardium is facilitated by the matrix proteinase, MMP‐9. This led to the hypothesis that IHC might reduce reperfusion injury by reducing MMP‐9 activity. The IHC protocol consisted of progressively increasing cycles from five 5 min cycles of 10% oxygen and 4 min reoxygenations on day one to the maximum of seven cycles of 10 min hypoxia and 4 min reoxygenations on days 11–20. Adult dogs were sham conditioned, IHC conditioned or IHC conditioned with DOR blockade for 20 days. On Day 21, dogs were evaluated physiologically and then cardiac tissues were collected and frozen in liquid nitrogen. Myocardial proteins were extracted, separated by electrophoresis in collagen impregnated gels, incubated, stained and quantified by densitometry. Setting sham MMP‐9 activity in the left ventricle to 1.0, the comparable activity in IHC ventricle was 0.5 and that in the DOR blocked animals was 1.25. These findings are internally consistent with the hypothesis that the cardioprotective effect of IHC is mediated in part by restricting the incursion of neutrophils and/or their intra‐myocardial transit following ischemia‐reperfusion.
Blood Platelet Insulin sensitivity in Atherogenic Dieted Rabbits Fed Fish Oils with Comparable Eicosapentanoic Acid Content
Authors: Beard Elizabeth L. , Skelly Stephanie E. , Bergens Brian M.
Published: June 2021

The effects of two fish oils added to an atherogenic diet fed to New Zealand strain, male rabbits were studied for 9 weeks. Cod liver oil (CLO) containing 8–10% eicosapentaneoic acid (EPA) or MaxEPA, a concentrated mix of sardine and anchovie oil, with 18–20%EPA, was added to the atherogenic diet (AD) of 2% Cholesterol, 6% Corn Oil and 92% Purina Rabbit Chow and fed to the two experimental animal groups. There were 3 groups of 5 rabbits in the study: an ADC control group fed AD alone, an AD/CLO group fed AD with 8% CLO added and an AD/MaxEPA group fed AD with 4% MaxEPA. Blood Plasma Cholesterol assay mean values were 862mg/dl for ADC, 549mg/dl for AD/CLO and 781mg/dl in AD/MaxEPA animals and for blood platelets were 461mg/dl for ADC, 277mg/dl in AD/ CLO and 440mg/dl in AD/MaxEPA platelets. Plasma plasminogen activator activity (PAA) assays yielded mean %fibrinolysis values of 17.4% in ADC, 20.2% in AD/CLO and 18.2 %. in AD/ MaxEPA plasma . Platelet insulin sensitivity assays found glucose levels of 285.8mg% in ADC, 345.3 mg% in AD/CLO and 342.6 mg% in AD/MaxEPA platelets. Insulin sensitivity increased comparably in platelets of rabbits fed AD/CLO or AD/MaxEPA with the same EPA content. Both fish oils yielded moderately reduced blood plasma and platelet cholesterol and increased blood PAA compared to ADC tissues. At autopsy, aortae from rabbits fed AD/CLO or AD/ MaxEPA showed reduced atherosclerotic plaque development vs. ADC aortae. Funded by Louisiana Heart Association
Ischemic treatment hyperpolarizes cochlear arteriolar smooth muscle and endothelial cells via different channel mechanisms
Authors: Jiang Zhi‐Gen , Yang Yuqin
Published: June 2021

Regional acute ischemia generally induces a compensatory vasodilation in the most vascular beds including the cochlear arterioles but the underlying cellular mechanism remains controversial. Using conventional intracellular and whole‐cell recording from smooth muscle (SMC) and endothelial cells (EC) in situ of and dispersed from segments of guinea pigs cochlear spiral modiolar artery, we analyzed the membrane actions of acute ischemic treatment (AIT, hypercapinic hypoxia, pO2 =0 mmHg, pH=6.2). The AIT for 2 to 20 min caused a reversible and partially repeatable vasodilation and a hyperpolarization in the majority of cells. ACh‐ and 10 mM K+‐induced dilation was significantly reduced, lost or reversed to a constriction during and after the AIT. Holding at −20 mV, AIT induced a reversible 10–65 pA outward current within 1–2 min in the majority of SMCs and ECs. During the AIT, the whole‐cell I/V curve became stronger outwardly rectifying at voltages >; −40 mV. The AIT‐induced net current in isolated SMCs exhibited a delayed outward rectification upon depolarization steps and partial blockade by 1–10 mM TEA or 4‐AP, suggesting an activation of both voltage‐gated (KV) and big conductance Ca2+‐activated (BK) K+‐channels. In contrast, the AIT‐induced current in isolated ECs was blocked by 30 μM niflumic acid but not by TEA, 4‐AP and linopirdine; the net current showed a voltage and time dependence typical of Ca2+‐activated Cl−‐channels (CaCC): slowly activating outwardly rectifying with a reversal potential near −30 mV. AIT (>;10 min) also reduced gap junction coupling current in in situ SMCs and ECs. We conclude that the acute ischemia in the cochlear arteriole induces hyperpolarization, dilation and loss of EDHF function via activation of KV and BK in smooth muscle cells and activation of Ca2+‐activated Cl−‐channels in endothelial cells, as well as an inhibition of gap junction coupling. Supported by NIH NIDCD DC 004716, P30 05983.
Is Human Double Minute‐2 a new marker of angiogenesis in exercised human skeletal muscle?
Authors: Aiken Julian , Slopack Dara , Gouzi Fares , Mercier Jacques , Hayot Maurice , Haas Tara L , Gustafsson Thomas , Birot Olivier , Roudier Emilie
Published: June 2021

Protein expression of the E3 ubiquitin ligase Murine Double Minute‐2 (Mdm2) is increased in response to exercise training in mouse skeletal muscle. By regulating endothelial cell proliferation and migration in the muscle tissue, Mdm2 is indispensable for exercise‐induced angiogenesis to occur (Roudier et al., FASEB J 2012).

Objective

Here, we investigated in human skeletal muscle if exercise training exerts a similar effect on Human Double Minute‐2 (Hdm2), the human homologue of Mdm2 protein, and if a link is present between Hdm2 and the endothelial marker Platelet Endothelial Cell Adhesion Molecule‐1 (PECAM‐1).

Method

Protein levels for Hdm2 and PECAM‐1 were measured by western blot in vastus lateralis muscle biopsies from 16 young and 14 senior subjects enrolled in a 6‐week training program.

Results

Exercise training significantly increased Hdm2 and PECAM‐1 protein expression. In the young population, a significant correlation was observed between exercise‐induced increases in Hdm2 and PECAM‐1 proteins.

Conclusion

Results from our previous study in rodents translate here to human subjects and suggest that Hdm2 might represent a key regulator of exercise‐induced angiogenesis in human skeletal muscle.

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).
A fluorescent staining technique for studying vascularity and angiogenesis in interdigitated maternal and fetal villi of sheep placenta
Authors: Redmer Dale A , Dorsam Sheri T , Grazul‐Bilska Anna T , Borowicz Pawel P
Published: June 2021

The objective of this study was to improve detection of the blood vessel network in placenta while simultaneously distinguishing maternal from fetal villi for image analysis. Therefore, we developed a technique for dual fluorescent labeling of endothelial cells and fetal trophoblast. Placentomes (d90 and d130 of pregnancy) were fixed (10% formalin or Carnoy's soln) and paraffin embedded. Following antigen retrieval (50mM glycine, 1mM EDTA, 0.05% Tween 20, pH 9.0; 120 C, 10 min), maternal and fetal blood vessels were identified by immunofluorescent labeling with an antibody (c/n:ab28364, ABCAM) against CD31 antigen, and CF 633 conjugated IgG (c/n:20122, Biotium). The trophoblast layer was stained with FITC labeled BS1 lectin (c/n:FL‐1101; Vector; 20 μg/ml). CD31 labeling resulted in bright fluorescent localization of all blood vessels. Positive lectin staining of fetal trophoblast allowed differentiation of fetal and maternal compartments. Thus, CD31 staining along with fluorescently labeled lectin combined with microscopy and imaging techniques provide an excellent tool to study angiogenesis and vascular architecture in sheep placenta, and other tissues. Grants: Hatch ND1748; USDA‐NIFA 2011–67016‐30174.
Lag and Causality in Baroreceptor Response during Passive Head‐up Tilt
Authors: Shykoff Barbara E , Florian John P
Published: June 2021

Multiple methods measure baroreflex sensitivity as effect of blood pressure (BP) on heart rate or interbeat interval (IBI). However, most consider only magnitude, not phase or lag. Also, in the closed‐loop IBI‐BP system, many methods do not differentiate between cause and effect. We postulated that long lag or loss of causality between changes in BP and IBI would predispose to unstable BP. Beat‐to‐beat systolic BP (Finometer) and IBI signals were considered as input and output respectively. Cross covariance (XCV) functions calculated using 40 to 80 heartbeats from cardiovascular steady state were examined for lag for one quarter of their length. Lag, the beat number at maximum XCV with changes in BP leading, was converted to time using average IBI. Significant (p<0.05) correlation at that lag indicated causality. Of 21 subjects with 15 minutes of 70° head‐up tilt, correlation in early tilt (ET) was significant in 19, with lag (mean ± SEM) 0.9±0.2 s. In late tilt, correlation was significant in 16, with lag 2.2±0.4 s. After 5 days of 6‐hour water immersion in 7 subjects, 2 became presyncopal during tilt and lost correlation. ET causal lag was 2.2±0.4 vs. 0.4±0.2 s pre‐immersion (p<0.01).

Support: ONR and NAVSEA DSBDP
Acute Alcohol Intoxication is Associated With Sustained Neuroinflammation Without Exacerbation of Early Neurobehavioral Outcomes Post Traumatic Brain Injury
Authors: Teng Sophie Xu , Molina Patricia
Published: June 2021

Approximately 1.7 million people sustain a traumatic brain injury (TBI) annually, and acute alcohol intoxication (AAI) contributes to 36% to 51% of TBI incidents in the U.S. Studies indicate that the risk of injury is positively correlated with blood alcohol concentrations (BAC). However, conflicting reports from animal and clinical studies have failed to establish whether AAI significantly impacts outcomes from TBI. The aim of the study was to determine whether AAI aggravates neurobehavioral and neuroinflammatory sequelae from TBI. Male Sprague‐Dawley rats were surgically instrumented with gastric and vascular catheters prior to fitting with a female Luer‐lock over a 5 mm left lateral craniotomy. After a 3 day recovery, animals received a primed (2.5 g/kg) 15 h constant (300 mg/kg/h) intragastric alcohol infusion achieving BAC of 266±10 mg/dl. TBI was induced by lateral fluid percussion (~1.4J, ~30 ms) 30 minutes following discontinuation of the infusion. AAI at the time of TBI did not alter apnea duration (15.3±5.3 sec vs. 26.5±8 sec), righting reflex (716±118 vs. 522±83), and neurobehavioral outcomes (neurological Δ2.2+0.6 vs. Δ3.9+0.6; behavioral Δ1.7+0.8 vs. Δ1.3+0.1 scores). TBI resulted in a localized inflammatory response reflected in increased myeloperoxidase (MPO) activity in the ipsilateral (10‐fold; p<0.05) cortex 24 h post‐TBI in dextrose treated animals. AAI did not alter MPO activity but markedly exacerbated neuroinflammation as reflected by significantly (P<0.05) higher IL‐1, IL‐6, TNFα, and MCP‐1 mRNA expression as compared to the dextrose‐treated animals at 24 h post‐TBI. These results show dissociation between neuroinflammation and clinical neurobehavioral measures following TBI in AAI. The clinical implications of enhanced neuroinflammation to long term recovery in the AAI TBI victim warrant further investigation.
Neuroprotective Mechanisms of Methylene Blue
Authors: Ryou Myoung‐Gwi , Choudhury Gourav Roy , Mallet Robert T , Yang Shaohua
Published: June 2021

Background

The phenothiazine dye methylene blue (MB) has antioxidant properties and augments ATP production, and is selectively internalized by neurons after systemic injection into rats.

Hypothesis

MB protects neurons against hypoxia‐reoxygenation (H/R) injury by enhancing ATP production and activating the Akt/mTOR signaling pathway.

Methods

HT22 cells were subjected to 3h oxygen‐glucose deprivation (OGD; 0.5% O2) followed by 24 h reoxygenation (H/R protocol). Cell viability (Calcein AM), and contents of hypoxia inducible factor‐1α, Akt/mTOR pathway components, and actin (immunoblot), erythropoietin (EPO; immunocytochemistry) and ATP content (microplate assay) were analyzed. P values <0.05 were considered statistically different.

Results

OGD reduced cell viability by 40% vs. normoxia, but 50 and 100 nM MB prevented H/R induced cell death. MB (10–1000 nM) concentration‐dependently increased cellular ATP content, in parallel with increased HIF‐1α content and EPO expression. MB (500 μM) increased Akt phosphorylation fourfold and mTOR phosphorylation by 45% vs. H/R control.

Conclusion

MB protects HT22 cells by both enhancing ATP production and activating EPO's anti‐apoptotic signaling.
Hydrogen sulfide (H2S) in nucleus tractus solitarii (nTS) modulates the cardiorespiratory system and its response to hypoxia
Authors: Kline David D. , Hasser Eileen M.
Published: June 2021

H2S modulates the chemosensory and respiratory response to hypoxia. We previously demonstrated H2S produced by cystathionine beta synthase (CBS) tonically augments synaptic transmission in nTS, the central site for chemosensory integration. Here, we sought to determine the role of H2S in nTS on respiratory, cardiovascular and autonomic parameters and their response to hypoxia. Phrenic (Phr) and splanchnic sympathetic nerve (sSNA) activity, as well as mean arterial pressure (MAP) and heart rate (HR) were evaluated in anesthetized rats exposed to brief hypoxia (10% O2, 2 min). Compared to control, bilateral nTS microinjection of the CBS activator, SAM (100 μM, 90 nL) had no effect on basal Phr frequency but increased Phr amplitude, MAP and sSNA. Hypoxia increased Phr and sSNA, consistent with chemoreflex activation. SAM also augmented sSNA, HR and Phr responses to hypoxia. By contrast, bilateral nTS microinjection of the CBS blocker AOA (1 mM, 90 nL) decreased Phr amplitude but not sSNA during hypoxia, suggesting an effect of endogenous H2S on hypoxic ventilatory responses in vivo. Altogether, these data suggest H2S modulates the cardiorespiratory system, in part, via its actions in the nTS. HL55306, HL085108.
Effects of Transcranial Direct Current Stimulation on Hemodynamic Responses to Pain
Authors: Hamner Jason W. , Villamar Mauricio , Fregni Felipe , Taylor J. Andrew
Published: June 2021

Painful (noxious) stimuli cause increases in heart rate blood pressure, and peripheral vascular resistance. This response is due to a both direct nociceptive reflex responses and conscious pain perception. Transcranial direct current stimulation (tDCS) is effective in modulating pain perception threshold in healthy volunteers and thus might be used to dissociate afferent (skin pain receptors) and efferent (cortical pain perception) components in the response to pain. We examined systemic (heart rate and blood pressure) and regional (calf blood flow/resistance) responses to cold pressor tests at 14, 7, and 0 °C before and after tDCS (active and sham) in 15 volunteers. All hemodynamic responses were graded with temperature (14<7<0 °C, P<0.05). tDCS reduced the perceived pain score, but only at 14 °C (P<0.05). This coincided with a slight reduction in peak heart rate response of ~1–2 beats. However, aside from this, tDCS had no effect on hemodynamic responses. Hence, tDCS may alter the pain perception threshold but this has minimal impact on nociceptive activation of cardiovascular centers.
Pro‐inflammatory cytokines interact with brain reninangiotensin system in the subfornical organ to induce sympathetic and hemodynamic responses in rat
Authors: Wei Shun‐Guang , Yu Yang , Zhang Zhi‐Hua , Felder Robert B.
Published: June 2021

Our previous work indicated that subfornical organ (SFO) is an important brain sensor mediating the effects of blood‐borne pro‐inflammatory cytokines (PIC) on autonomic and cardiovascular function. However, the mechanisms by which SFO mediates these central effects of circulating PIC remain unclear. In this study, we examine whether the brain renin‐angiotensin system (RAS) in the SFO contributes to the excitatory effects of PIC on blood pressure (BP, mmHg), heart rate (HR, bpm) and renal sympathetic nerve activity (RSNA, %). In urethane anesthetized SD rats (n=6), microinjection of interleukin‐1β (IL‐1β, 20 ng) into the SFO significantly (*p<0.01) elevated BP (19.3 ± 2.0*), HR (70 ± 7*) and RSNA (84.6 ± 4.2*). Pretreatment of SFO with the angiotensin II type 1 receptor (AT1‐R) blocker losartan (5 μg, n=6) or the angiotensin converting enzyme inhibitor captopril (5 μg, n=6) significantly reduced IL‐1β induced BP, HR and RSNA responses. SFO microinjection of tumor necrosis factor‐α (20 ng, n=4) elicited similar increases in BP, HR and RSNA that were also significantly attenuated by SFO pretreatment with losartan or captopril. AT1‐R mRNA expression in the SFO was markedly increased (from 1.03 ± 0.11 to 2.05 ± 0.24*) 4 hr after intravenous injection of IL‐1β (1 μg/kg). These data suggest that circulating PIC elicit hemodynamic and sympathetic responses at least in part by upregulating RAS activity in the SFO.
Enhanced levels of proteases in tubular fluid activate ENaC in chronic heart failure
Authors: Zheng Hong , Liu Xuefei , Li Yulong , Sharma Neeru M , Patel Kaushik P
Published: June 2021

One of the key mechanisms involved in renal sodium retention in chronic heart failure (CHF) is activation of epithelial sodium channels (ENaC) in collecting tubules. Proteolytic cleavage and ubiquitylation/deubiquitylation have important roles in activating ENaC. We hypothesized that enhanced levels of proteases in tubular fluid activate ENaC resulting in renal sodium retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF compared to sham rats (increased fold: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham, P<0.05). Whole‐cell patch‐clamp was conducted in cultured renal collecting duct M‐1 cells to record Na+ currents. Protease‐rich urine (from rats with CHF vs. sham rats) significantly increased the Na+ inward current in M‐1 cells (7.2 folds). Ubiquitin assay demonstrated reduced ubiquitylated γ‐ENaC in the kidneys of CHF rats. We also found that ubiquitin‐protein Nedd4 was decreased (79%) and deubiquitylation enzyme Usp2–45 was increased (75%) in the isolated pure populations of tubular fragments from CHF rats. These findings demonstrate for the first time that increased proteases in the tubular fluid contribute to the enhanced ENaC activity, providing a novel mechanistic insight for sodium retention commonly seen in CHF.