bluuebunny/crossref_metadata_2025 huggingface dataset:
Mentoring Africa's PhD candidates in biomedical sciences: some tips from the FASEB Journal's publications
Authors: John Theresa Adebola
Published: June 2021

National growth depends on products of tertiary institutions. Contributions of graduates can be guided by good mentoring early in their career. In biomedical science research, factors that can guarantee career success of Africans need to be identified. Thirty three articles in the April 2012 issue of the FASEB Journal were examined for: a) number of printed pages of the journal; b) number of major studies described; c) number of figures and presentation of data within the figures; d) number of days the journal took to review the article before acceptance for publication; e) number of scientific references listed at the end of the article. The data showed that articles took up 10.5±1.52 printed pages, reported 8.03±0.36 major studies, utilized 49.76±7.4 references, and took 87.76 days to be accepted for publication. Data were presented as tables in 16/33 articles; diagrams in 18/33 articles; graphs in 21/33 articles; histograms (and other charts) in 31/33 articles; photos and pictures in 24/33 articles, blots (mainly western) in 20/33 articles; machine print‐outs in 10/33 articles; and sequences, arrays, or mappings in 7/33 articles. Biomedical/basic medical science PhD's need to be directed to take care of the following factors along their research career: a) quantity of work; b) quality of work; c) richness of evidence; d) planning and pacing of work; e) relationship of work with universal knowledge and development.
Evaluating the predictive value of the in vitro Platelet Toxicity Assay (iPTA) for the diagnosis of hypersensitivity reactions to sulfonamide drugs: A prospective case‐control study.
Authors: Elzzagallaai Abdelbaset A , Koren Gideon , Rieder Michael J
Published: June 2021

Drug hypersensitivity reactions (DHRs) are rare but potentially fatal types of adverse drug reactions that develop in susceptible patients following exposure to certain drugs including sulfonamides. Their diagnosis is challenging due to lack of safe and reliable test. The aim of this study was to evaluate the predictive value of the in vitro Platelet Toxicity Assay (iPTA) in diagnosis of DHRs to sulfonamides and to compare its performance to the conventional lymphocyte Toxicity Assay (LTA) test. Blood samples were obtained from 66 individuals (36 DHS‐sulfa patients and 30 controls) and LTA and iPTA were performed. Results were then analyzed to estimate the predictive value, sensitivity and agreement of the two tests. The concentration‐dependent toxicity was significantly greater in the cells from patients versus controls (p<0.05). The two tests had a high degree of agreement (correlation coefficient: R2 = 0.97). The iPTA was significantly more sensitive than the conventional LTA test in detecting the susceptibility of patient cells to in vitro toxicity (p<0.05). The iPTA has considerable potential as a diagnostic tool for DHS as it is cheaper to perform, requires no special reagents and has a greater sensitivity in detecting patients predisposed to DHRs to sulfonamides.
Pharmocogenetic predictors of inhaled budesonide response in asthma
Authors: Mackenzie Constance A , Kim Richard B
Published: June 2021

There is considerable interindividual variability in response to inhaled corticosteroids for treatment of asthma. Although there is evidence that some of this variability is mediated by genetic factors, as of yet, there are no clear predictors of clinical response to inhaled steroids, particularly in relation to drug metabolism and transport processes. The goal of our study was to better understand the contribution of pharmacogenetic differences for patients with asthma whose treatment regimen includes budesonide, a widely prescribed inhaled steroid, known to be metabolized by CYP3A4 and transported by MDR1(ABCB1). We have enrolled 50 patients with confirmed asthma who were on a stable dose of inhaled budesonide alone (Pulmicort) or in combination with a long acting beta‐agonist formoterol (Symbicort). We will genotype for known polymorphisms in CYP3A4 (*22) and CYP3A5*3, as well as commonly occurring polymorphisms in MDR1. Importantly, we will also measure beta‐hydroxycholesterol/cholesterol ratio as an in vivo probe of CYP3A activity and measure budesonide plasma levels using highly sensitive liquid chromatography‐tandem mass spectrometry. Pharmacogenetic markers will be correlated with drug levels, clinical efficacy, pulmonary function, and exhaled nitric oxide as a marker of airway inflammation. Our findings will lead to better pharmacogenetic predictors of inhaled steroid therapy efficacy. Supported by Program of Experimental Medicine, Department of Medicine, Western University.
Effect of surface charge and magnetic field on iron oxide nanoparticle permeability in a cell culture model of the blood brain barrier (BBB)
Authors: Sun Zhizhi , Yathindranath Vinith , Worden Matthew , Hegmann Torsten , Miller Donald
Published: June 2021

Surface charge and applied magnetic fields affect cellular uptake of iron oxide nanoparticles (IONPs). However, their effects on permeability of IONPs across the BBB remain unexplored. The aim of this study was to determine the impact of IONP surface charge on permeability in a cell culture model of the BBB in the presence and absence of a magnetic field. Permeability of positively (AmS‐) and negatively charged (TMSPEDT)‐IONPs was evaluated in confluent bEnd.3 monolayers grown on polycarbonate membrane inserts (3 μm pore). Our results show neither IONPs were not transported across the monolayer even in presence of magnetic field. When tight junctions were disrupted using D‐mannitol, 44% and 28% of TMSPEDT‐IONPs were found across the monolayer in presence and absence of magnetic field, respectively. Under the same conditions, there was only 10% permeability of the AmS‐IONPs detected. In conclusion, negatively charged IONPs have a more favorable permeability profile in brain endothelial cells via paracellular route following osmotic disruption. Use of TMSPEDT‐IONPs during transient disruption of BBB, may improve nanoparticle based drug delivery to the brain. Support provided by Manitoba Medical Services Foundation, Thorlakson Foundation and NSERC.
Expression and Function of Hepatic Drug Metabolizing Enzymes and Transport Proteins in Livers of Patients with End‐Stage Renal Disease (ESRD)
Authors: Feere David Alexander , Velenosi Thomas , Gaspar Melisa Lynn , Urquhart Brad
Published: June 2021

Objectives

This study aims to determine the changes in expression and function of hepatic drug metabolizing enzymes (DMEs) and drug transport proteins in liver samples from patients with end‐stage renal disease (ESRD).

Methods

Liver samples were collected from nine recently deceased patients with ESRD and six recently deceased controls. Relative gene expression of DMEs and transport proteins were determined by qPCR.

Results

P‐gp and CYP2E1 expression were significantly decreased (P<0.05) in ESRD livers compared to control livers (21% and 17% of control, respectively). There was a trend of decreased expression of CYP2D6, CYP2C9, OATP1B1 MRP2 and MRP3, although this failed to reach significance.

Conclusions

Our results demonstrate that DMEs and transport proteins are down‐regulated in ESRD. Current studies are evaluating the enzymatic activity of DMEs in hepatic microsomes from ESRD patients using the specific probe substrates midazolam (CYP3A4), tolbutamide (CYP2C9), chlorzoxazone (CYP2E1) and dextromethorphan (CYP2D6). Our results help explain the large decrease in non‐renal drug clearance observed in patients with ESRD.

This research is supported by the Lawson Health Research Institute.
FUNCTIONAL ACTIVITY IN HUMAN HEART EXPRESSING CYP1A1
Authors: Huguet Jade , Gaudette Fleur , Michaud Véronique , Turgeon Jacques
Published: June 2021

Background

Drug metabolism by CYP450s expressed in cardiac myocytes can modulate cardiac drug levels and actions. CYP1A1 and 2C8 are importantly expressed in the human heart. A cocktail approach with 7‐Ethoxyresorufin (7‐ET) and repaglinide (REP) were chosen to study selective metabolism of CYP1A1 and 2C8, respectively.

Methods

Incubations were first performed in Supersome™ of each CYP450s founded in the heart. Then, cytosolic (S1) and human heart microsomes (HHM) were isolated by differential centrifugations from one human heart. Resorufin and REP metabolite were quantified by LC‐MS/MS. Western blot analysis was performed using a monoclonal antibody raised against CYP1A1.

Results

REP metabolism was selective towards 2C8 and 7‐ET for 1A1 in Supersome™. For 7‐ET, Km was 0.36 vs. 0.4μM and Vmax was 1640 vs. 1944 pmoles of resorufin/mg of protein/min when incubated alone vs. with REP, respectively. For REP, Km was 3.7 vs. 3.35μM and Vmax was 1283 vs. 1120 pmoles REP metabolite/mg of protein/min when incubated alone vs. with 7‐ET, respectively. No activity was observed when 7‐ET and REP were incubated in HHM but there was a low activity in the S1 fraction (0.3 pmoles of resorufin/mg prot/min). This was confirmed by Western blot analysis where CYP1A1 protein was found in S1 but not in HHM.

Conclusion

This cocktail approach allows us to study the CYP450 activities in the heart. Research funded by the CIHR, HSF and FRSQ.
Genetic and clinical determinants of CYP3A4 activity in patients using 4β‐hydroxycholesterol as an in vivo probe
Authors: Gong Inna Ying , DeGorter Marianne K , Schwarz Ute I , Yin Peter , Choi Yun Hee , Tirona Rommel G , Kim Richard B
Published: June 2021

There is marked interindividual variability in cytochrome P450 3A (CYP3A) activity. Plasma concentration of 4β‐hydroxycholesterol (4βOH‐C), an endogenous metabolite formed by CYP3A‐mediated hydroxylation of cholesterol, has been proposed as in vivo probe of CYP3A activity. Thus, we characterized the clinical and genetic variables associated with 4βOH‐C concentration in patients (n = 532). Subjects were consented to provide a brief medical history, plasma and DNA for genotyping. 4βOH‐C was measured by liquid‐chromatography tandem mass spectrometry. Predictors of molar ratio of 4βOH‐C to cholesterol (4βOH‐C/C) were assessed by multiple linear regression analysis. The mean 4βOH‐C concentration was 29.3 ng/mL (SD 20.6). 4βOH‐C/C did not appear to be significantly affected by concomitant administration of drugs with CYP3A inducing or inhibitory properties. Of the demographic variables assessed, 4βOH‐C/C was significantly related to body mass index (p < 0.001). Importantly, 4βOH‐C/C was associated with CYP3A4*22 (p < 0.01), CYP3A5*3 (p < 0.005), and POR*28 (cytochrome P450 oxidoreductase, p < 0.05) genotypes. Finally, 4βOH‐C/C concentrations were associated with concentrations of the CYP3A substrates atorvastatin and tamoxifen (p < 0.01). Our data demonstrate 4βOH‐C levels can be effectively used to quantify the in vivo relevance of CYP3A genetic variations and clinical variables.
Subarachnoid Hemorrhage Suppresses KV1 and KV2 Currents via Different Mechanisms in Rat Parenchymal Arteriolar Myocytes
Authors: Koide Masayo , O'Connor Kevin P. , Pappas Anthony C. , Syed Arsalan U. , Wellman George C.
Published: June 2021

Subarachnoid hemorrhage (SAH) leads to membrane potential depolarization in arteriolar myocytes and enhanced arteriolar tone in brain parenchymal arterioles (Nystoriak et al, 2011). However, the mechanism underlying this augmented constriction is currently unknown. Here, we studied the impact of SAH on voltage‐gated potassium (KV) currents. In rat parenchymal arterioles from control animals, KV1.2, 1.5, 2.1 and 2.2 mRNA was expressed, and KV1 and KV2 currents were detected by conventional whole cell patch clamp electrophysiology. Both KV1 and KV2 currents as well as total KV (4‐aminopyridine‐sensitive) currents were significantly suppressed in arteriolar myocytes after SAH, however KV channel subtype expression was not changed. Our previous work demonstrated that a blood component oxyhemoglobin causes KV current suppression through heparin binding EGF‐like growth factor (HB‐EGF). HB‐EGF caused significant K+ current suppression in myocytes from control animals, but not after SAH. Further, HB‐EGF suppressed K+ currents in the absence and presence of KV2 channel inhibition, but failed to alter currents in the presence of 4‐aminopyridine or during KV1 channel blockade. These data suggest SAH causes KV1 current suppression through HB‐EGF shedding, while KV2 current suppression is independent of the HB‐EGF pathway. Supported by NIH P01 HL095488, Totman Medical Research Trust and the Peter Martin Fund.
Circulating factors induced by caloric restriction in the non‐human primate Macaca mulatta activate angiogenic processes in endothelial cells
Authors: Csiszar Anna , Sosnowska Danuta , Tucsek Zsuzsanna , Gautam Tripti , Toth Peter , Colman Ricki J , Weindruch Richard , Anderson Rozalyn M , Sonntag William E , Ungvari Zoltan
Published: June 2021

Moderate caloric restriction (CR) increases healthspan in virtually every species studied, including nonhuman primates. In mice, CR exerts significant microvascular protective effects resulting in increased microvascular density in the heart and the brain, which likely contribute to enhanced tolerance to ischemia and improved cardiac performance and cognitive function. Yet, the underlying mechanisms by which CR confer microvascular protection remain elusive. To test the hypothesis that circulating factors triggered by CR regulate endothelial angiogenic capacity, we treated cultured human endothelial cells with sera derived from Macaca mulatta on long‐term (>;10 years) CR. Cells treated with sera derived from ad libitum fed control monkeys served as controls. We found that factors present in CR sera up‐regulate VEGF signaling and stimulate angiogenic processes, including endothelial cell proliferation and formation of capillary‐like structures. Treatment with CR sera also increased cellular migration and adhesion. Collectively, we find that circulating factors induced by CR promote endothelial angiogenic processes, suggesting that increased angiogenesis may be a potential mechanism by which CR improves cardiac function and prevents vascular cognitive impairment
Development of the elastin network in the walls of resistance arteries from neonatal and adult rats
Authors: Collins Melissa J , Nourian Zahra , Stupica Aaron , Min Li , Clifford Philip S , Krenz Maike , Baines Christopher , Meininger Gerald A , Hill Michael A
Published: June 2021

Elastin distribution within the walls of resistance vessels is complex, with discrete fibers varying in orientation across the adventitia and media as well as the more sheet‐like structure of the internal elastic lamina (IEL). These structural patterns likely subserve specific functional properties including mechanosensing, control of forces, cellular positioning, and communication between vascular cells. To understand the impact of elastin on small artery function we initiated analyses of mRNA level expression patterns measured by qPCR, and protein distribution determined by fluorescence 3D confocal imaging. Studies were performed on 2nd order mesenteric arteries from rats aged 3, 7, 11, 14 and 19 days as well as 2 mths. Elastin mRNA peaked at day 11 before declining at later times. During the postnatal period, the elastin component of the IEL of mesenteric vessels accumulated/matured progressing from a punctate/fibrous appearance into a continuous sheet. Analysis of adventitial and medial elastin during this period showed accumulation of fibers as well as re‐organization of fiber orientation. Collectively these data show marked remodeling and development of elastin networks in the walls of small arteries as they mature. The impact of elastin remodeling on vessel function, and its relationship to concurrent changes in hemodynamics, require further study. (*Co‐first authors; NIH HL095486 to GAM)
Histamine is novel endothelium‐derived relaxing factor in rat mesenteric lymphatic vessels
Authors: Gashev Anatoliy , Nizamutdinova Irina Tsoy , Nagai Takashi , Maejima Daisuke , Bridenbaugh Eric , Thangaswamy Sangeetha , Chatterjee Victor
Published: June 2021

Our recent studies (Nagai et al., 2011) suggested the existence of yet undiscovered, shear‐dependent, but NO‐independent regulatory mechanism in rat mesenteric lymphatic vessels (MLV). In this study we performed for the first time the detailed evaluation of potential involvement of histamine in such regulation of MLV contractility in F‐344 rats of various age groups (3‐mo, 9‐mo and 24‐mo). Using confocal and epifluorescent IHC labeling, we confirmed the presence of histidine decarboxylase (HDC), histamine‐producing enzyme, within the lymphatic endothelial cells in MLV. These results were re‐confirmed by Western blot analysis. Using Vivo‐Morpholino technology we were able to knock‐down effectively HDC protein within MLV. In experiments with isolated MLV, we found that only combined blockade of eNOS (NO production) by L‐NAME and HDC (histamine production) by α‐methyl‐DL‐histidine dihydrochloride was able to completely eliminate all shear‐dependency of lymphatic contractile self‐adjustment during periods of steady increases of imposed flow. Such regulatory role of histamine appears to be not severely altered by aging. Up‐to‐date results shows that histamine is not involved in fast phasic contraction‐generated shear‐dependent contractile regulation. Our data provide ground for new understanding on the role of histamine in regulation of lymphatic function. NIH R01 AG030578, HL094269.
Effects of gender and race on interleukin‐6 production in response to CRP in HUVECs
Authors: Ling Chenyi , Kretzschmar Jan , Grimm Heather , Brown Michael
Published: June 2021

Background

Interleukin‐6 (IL‐6) is a pro‐inflammatory cytokine that can be excreted by endothelial cells. Elevated C‐reactive protein (CRP) level is a risk factor for the development of endothelial dysfunction that can lead to hypertension. The study was to determine whether race or gender affected the IL‐6 response to CRP in HUVECs.

Methods

Eight HUVEC lines from African American (AA) and Caucasian (CA) donors with gender split evenly were cultured and incubated with CRP for 24‐hrs. Doses of CRP were 0, 25, 50 and 100 μg/mL. IL‐6 was measured in the media that was collected after the incubation and was normalized to protein concentration (pg/mg).

Results

The female group had higher IL‐6 production than the male group for each CRP condition and there was a significant difference between genders at the 50 μg/mL CRP condition (P=0.03). Though the IL‐6 production tended to be higher in the AA group under 25, 50, and 100 μg/mL CRP conditions, the differences were not significant.

CRP level (μg/mL)
IL‐6 production (pg/mg)

Female
Male

0
66.96 ± 30.88
82.73 ± 28.75

25
144.86 ± 50.15
92.03 ± 40.77

50
177.97 ± 20.73
110.98 ± 25.56

100
179.15 ± 59.41
151.94 ± 103.41

Conclusion

It suggests that females with high CRP levels may have a greater risk of developing endothelial dysfunction due to a greater IL‐6 response. Whether the gender difference is related to differences in CRP receptor function or downstream signaling mechanisms is unknown.
Increased Circulating Microparticles in Diabetic Rats are not only a Result of Cell Activation but Actively Serve as Mediators of Inflammatory Signaling
Authors: Stork Christian , He Pingnian
Published: June 2021

Diabetes is commonly associated with microvascular dysfunction. Our previous study found that microvessels of diabetic rats had increased basal permeability and enhanced permeability responses to inflammatory mediators. We also found that diabetic rats had increased circulating microparticles (MPs). MPs are small vesicles released by activated or apoptotic cells and carry multiple antigens from activated cells. We hypothesized that MPs serve as vectors to actively disseminate inflammation to the whole vascular system. We used flow cytometry to quantify the numbers of MPs and their cell origins. We tested differences between MPs generated by diabetic rats and MPs from normal rats by directly perfusing them into intact microvessels. The effects of MPs on microvessel walls were examined by perfusing matched numbers of isolated Annexin V positive MPs into normal venules for 30 min. After 10 min resumed blood flow in MP perfused vessel, we found significantly higher number of adherent leukocytes in diabetic MP perfused vessels than in normal MP perfused vessels. Diabetic and normal MP‐ mediated leukocyte adhesion were 17 ± 2.9 and 5.7 ± 0.5 leukocytes per 100 μm of vessel length, respectively. Flow cytometry analysis showed that diabetic MPs had an average 7‐fold increase in phosphatidylserine exposure per MP and a 12‐fold increase in platelet‐leukocyte MP aggregates from those in normal plasma MPs. Our results suggest that the population differences and the larger surface area of PS exposed diabetic MPs may contribute to the greater leukocyte adhesion induced by diabetic MPs. These results support our hypothesis that increased MPs in diabetic rats are more than simply biomarkers of the disease but that they are able to directly mediate leukocyte adhesion and actively disseminate inflammation to remote regions.

Supported by: 1F32HL114376, P20GM103434, HL56237, HL084338
Vascular islands increase during microvascular network regression and proliferate during angiogenesis
Authors: Kelly‐Goss Molly Rose , Sweat Richard Shelton , Lee Murfee Walter
Published: June 2021

Angiogenesis is commonly associated with capillary sprouting and intussusception. Recent work implicates vascular island incorporation as another endothelial cell dynamic. The objective of this study was to characterize the presence of vascular islands temporally correlated with vascular regression. Mesenteric tissues were harvested from adult male Wistar rats according to the following groups: unstimulated, 10 days post compound 48/80 mast cell degranulation stimulation, 70 days post stimulation, 70 days post stimulation + 3 days re‐stimulation, and 70 days post stimulation +10 days re‐stimulation (n= 16 tissues per group). Tissues were labeled for PECAM, NG2, and Collagen IV. Vascular length density increased at day 10 post stimulation and decreased by day 70. Compared to day 10 networks, the number of vascular islands, defined as disconnected endothelial cell segments, was dramatically increased at day 70. Vascular islands associated with regressing networks at day 70 contained NG2 positive pericytes and were typically bridged to nearby networks by both pericytes and collagen IV labeling. By day 3 post re‐stimulation proliferating endothelial cells were observed along islands, and by day 10 post re‐stimulation the number of vascular islands again decreased. The results from this study suggest that vascular islands associated with regression contribute to microvascular network re‐growth.
Impact of redox signaling inhibition on collateral growth in young, healthy rats
Authors: Miller Steven J , Wenning Mary Jo , Bills Randall G , Sliva Petr , Labarrere Carlos A , Kelley Mark R , Fishel Melissa L , Unthank Joseph L
Published: June 2021

Previous results demonstrated inhibition of collateral growth (CG) by antioxidant (apocynin) treatment in young healthy rats. The aim of this study was to determine if normal CG was regulated by redox‐mediated signaling and transcription factor activation. CG (% luminal expansion) was assessed with a well‐established mesenteric artery ligation model in Wistar Kyoto (WKY; 2–3 mo‐of‐age) rats. NFkB nuclear translocation indicated by percent p65‐positive intimal nuclei was significantly greater in collateral than control arteries (51.2±1.2 vs. 4.5±2.2, respectively, p<0.01), and apocynin decreased translocation to untreated control values (5.2±2.6). Various inhibitors were used to further assess the potential role of redox‐dependent regulation of transcription factors and signaling pathways, including: NFkB (p65 siRNA), APE1/Ref‐1 redox signaling function (E3330), PI3‐kinase (LY294002), and mTOR (rapamycin). CG was inhibited by p65 siRNA vs. vehicle control (3.3±0.44 vs. 25.1±2.7, respectively, p<0.001) as well as by E3330 vs. vehicle control (19.7±2.1 vs. 31.3±4.2, respectively, p<0.05). CG in LY294002‐ and rapamycin‐treated WKY also was significantly less compared to vehicle (5.24±0.93; p<0.001, and 22.0±2.0; p<0.05 vs. 33.5±4.5, respectively). Taken together, the results suggest an important role for redox‐mediated signaling and NFkB activation in physiological CG. Support: HL092012
High glucose induces pro‐inflammatory phenotype in human astrocytes and enhances neurotoxicity
Authors: Bahniwal Manpreet , Little Jonathan P. , Klegeris Andis
Published: June 2021

Chronic neuroinflammation caused by activation of microglia and astrocytes in the brain contributes to neuronal loss and disease progression in Alzheimer's disease (AD). High glucose has been shown to increase release of pro‐inflammatory mediators from various immune cells, including microglia. We investigated the effects of glucose (5.5–30.5 mM) on astrocytic cells. High glucose increased expression and secretion of pro‐inflammatory cytokines interleukin (IL)‐6 and IL‐8 (P<0.05) in human primary astrocytes and U‐118 MG astrocytoma cells. This astrocytic proinflammatory response to elevated glucose may involve increased phosphorylation of the signal transducer and activator of transcription (STAT)‐3. High glucose also increased the susceptibility of human SH‐SY5Y neuroblastoma cells to toxicity induced by hydrogen peroxide and Alzheimer amyloid‐β1–42 (P<0.05). We hypothesize that brain hyperglycemia in type 2 diabetes contributes to the observed increased risk of AD by exacerbating astrocyte‐mediated neuroinflammation and neuronal injury caused by disease‐specific agents.
Locomotor training improve metabolic function, body composition and trunk motor control in spinal cord injured individuals
Authors: de Paleville Daniela Terson , Lorenz Douglas , Aslan Sevda , Harkema Susan
Published: June 2021

Obesity and cardiovascular problems are some of the most common complications in chronic spinal cord injury (SCI). Lower resting metabolic rate and oxygen consumption in individuals with SCI individuals may be related to higher risk of obesity, hyperlipidemia, diabetes and coronary artery disease. Improved postural and motor of the trunk muscles are related to independence in activity of daily living. We propose that locomotor training (LT) is a therapeutic intervention that can improve these outcomes. LT is a task‐specific rehabilitation, in which participants are placed on a treadmill with partial body weight bearing supported by a harness, and therapists manually facilitate the participant to accomplish gait. Our hypotheses are that: individuals with SCI will have lower percentage of body fat as measured by dual energy x‐ray absorptiometry (DEXA) and skinfold subcutaneous fat assessment; will have higher resting metabolic rate as measured by basal oxygen consumption (open circuit spirometry) and will improve in trunk postural control and neural activation of the trunk muscles after LT as measured by the trunk functional neurophysiologic assessment (EMG). This is preliminary data from an ongoing project from individuals with chronic SCI (n=5). These participants show lower percentage of body fat, maintained or increased lean body mass; improved metabolic function at rest and during exercise and enhanced motor control of the trunk muscles during different voluntary maneuvers after 60 sessions of locomotor training (60 min. sessions, 3–5 times per week).
Chronic DOCA‐salt treatment alters hypothalamic, but not cortical or brainstem, Na+/K+/Ca2+ content
Authors: Osborn John W , Abrams Joanna , Dubinsky Janet
Published: June 2021

The present study was conducted to establish whether chronic salt loading alone, or in combination with the mineralocorticoid deoxycorticosterone acetate (DOCA), alters the content of sodium (Na+), potassium (K+) and calcium (Ca2+) in the brain. Male Sprague Dawley rats were implanted with a silicone pellet containing 0 (n=6) or 100 (n=6) mg DOCA and given 0.9% saline to drink ad libitum. The control group received 0mg DOCA and water ad libitum (n=6). After 21 days of treatment rats were anesthetized and brains were excised. The brainstem was removed and the remainder of the brain was bisected midsagitally. The right half of the brain was weighed intact and the left half was dissected into hypothalamus and cortex. Samples were analyzed for Na+/K+/Ca2+ content (ppm/gram) by inductively coupled argon plasma optical emission spectrometry. Compared to the control group, saline loading alone had no effect on Na+/K+/Ca2+ in any brain region. However, DOCA‐salt treatment increased Na+ (p < 0.05), and tended to decrease K+ content (p >; 0.05), in the hypothalamus but had no effect in the cortex or brainstem. DOCA‐salt treatment also increased hypothalamic (p < 0.05), but not cortical or brainstem, Ca2+ content. We conclude that regulation of hypothalamic Na+/K+/Ca2+ content is unique compared to the cortex and brainstem. HL64176–05
Selective knockout of novel intracellular renin isoform results in reduced adiposity
Authors: Folchert Matthew D. , Littlejohn Nicole K. , Liu Xuebo , Grobe Justin L. , Sigmund Curt D.
Published: June 2021

Renin gene expression is regulated by two distinct promoter / first exon combinations that target renin for either secretion (exon 1a) or for cytoplasmic retention (exon 1b). Renin‐1b is expressed predominantly in the brain and is differentially regulated from renin‐1a in response to stimuli such as deoxycorticosterone (DOCA)‐salt. Mice were generated that lack exon 1b (and thereby intracellular renin, icREN‐KO) but retain exon 1a. Male icREN‐KO mice were similar in size (n=4, 14.7±0.9 wk, 27.94±1.08 g) compared to littermate controls (n=7, 14.2±0.8 wk, 27.96±0.82 g, P=0.988), but by NMR exhibited reduced total and relative total fat mass (1.83±0.15 vs 3.00±0.12 g, or 6.6±0.7 vs 10.9±0.5 %, both P<0.001). Interestingly, interscapular brown adipose (289±77, n=5 vs 68±61 mg, n=9 P=0.04) and heart (162±12 vs 129±9 mg, P=0.04) masses were both increased in icREN‐KO mice, possibly suggesting increased resting metabolic rate (RMR) and blood pressure. Liver and kidney masses were normal. Food intake was normal (3.25±0.28, n=4 vs 3.14±0.14, n=8 P=0.7), but preliminary tests uncovered trends toward increased RMR (7.67±0.75, n=4 vs 6.93±0.59 g/day, n=4 kcal/kg lean/hr, P=0.47) and glucose tolerance (2g/kg, 25768±3994, n=3 vs 33734±2854 mg/dL*min, n=8, P=0.157). Together these data suggest that the novel icREN isoform contributes to metabolic control.
Activation of nucleus tractus solitarii (nTS) neurons that project to the rostral ventrolateral medulla (RVLM) or hypothalamic paraventricular nucleus (PVN): Role of acute hypoxia (AH)
Authors: Ruyle Brian Christopher , Kline David D , King T Luise , Heesch Cheryl M , Hasser Eileen M
Published: June 2021

The nTS receives and integrates chemoafferent input signaling arterial O2, and sends this information to the RVLM and PVN to produce coordinated cardiorespiratory responses to AH. The importance of the nTS in this reflex is understood, but the relative contribution of these two pathways is unknown. Retrograde tracers were microinjected to label RVLM‐ and PVN‐projecting nTS neurons. After recovery, conscious rats underwent 2 hr normoxia (21% O2) or AH (14, 12, 10 or 8% O2). We used Fos‐immunoreactivity (IR) as an index of neuronal activation and tyrosine hydroxylase (TH)‐IR to identify catecholaminergic neurons important in cardiorespiratory function in the nTS. AH progressively increased Fos‐IR in nTS cells. The number of Fos‐IR nTS‐RVLM cells was greater compared to nTS‐PVN cells after 14% ‐ 10% O2 (e.g. 12% O2: RVLM, 25±5; PVN, 14±4 cells) but was similar at 8% O2. However, the percentage of cells that were Fos‐IR was greater in nTS‐PVN neurons than nTS‐RVLM neurons after 12–8% (e.g. 10% O2: RVLM, 15±3; PVN, 25±4%). Very few nTS‐RVLM cells were catecholaminergic. However, 42±3% of nTS‐PVN cells was catecholaminergic; these cells were progressively activated by AH. We conclude that the nTS‐RVLM pathway is important as an early response to mild AH. The nTS‐PVN pathway has an important neuromodulatory role during severe AH, and these cells are likely catecholaminergic. HL98602
Role of renal innervation and endothelin‐1 for the regulation of upper urinary tract contractions
Authors: Grisk Olaf , Schaper Katrin , Steinbach Antje , Schlüter Torsten , Zimmermann Uwe , Rettig Rainer
Published: June 2021

Contractions of isolated rodent upper urinary tract (UUT) can be stimulated by norepinephrine (NE) and endothelin‐1 (ET‐1) and are sensitive to Rho kinase (ROCK) inhibition. We tested (1.) if renal nerves or paracrine acting ET‐1 are physiological regulators of UUT contractions, (2.) if the RhoA/ROCK pathway mediates NE‐ or ET‐1‐induced UUT contractions, and (3.) if NE or ET‐1 stimulate contractions in human UUT. Renal nerves were removed by kidney transplantation or unilateral denervation in rats. Collecting duct (CD)‐specific ET‐1 k.o. mice served as a model with low renal ET‐1. In vivo, UUT contraction frequency was similar in denervated and control kidneys. In vitro, denervated rat UUTs had a significantly higher spontaneous contraction frequency than controls but did not show NE or ET‐1 supersensitivity. UUTs of CD‐specific ET‐1 k.o. mice and their controls showed similar in vivo and in vitro contractile activity, NE and ET‐1 sensitivity. RhoA GTP formation and MYPT1 phosphorylation was stimulated by ET‐1 but not by NE in rat UUTs. NE and ET‐1 elicited rhythmic and tonic contractions in isolated human UUT tissue. We conclude that renal innervation regulates UUT pacemaker activity and that the RhoA/ROCK pathway mediates ET‐1‐induced UUT contractions. We suggest that rodent data on UUT function can be at least in part translated to humans.
Intermittent hypoxia in vitro reduces TRPC4 and neuropeptide expression in 4B cells
Authors: Nedungadi T.Prashant , Bachelor Martha , Cunningham J.Thomas
Published: June 2021

Patients with obstructive sleep apneas (OSA) are at risk of developing several co‐morbidities including metabolic disturbances. However, the mechanisms underlying the initiation of metabolic dysfunctions in OSA patients are unclear. Anorexigenic hypothalamic peptides are depolarized through the activation of Canonical Transient Receptor Potential Channel 4 (TRPC4). We used parvocellular paraventricular nucleus hypothalamic 4B cells, to address an existing gap in our understanding of the molecular mechanisms by which hypoxia alters neuropeptide and TRPC4 function. Serum starved 4B cells were either exposed to normoxia (NO) or were subjected to either 24 or 60 cycles of intermittent hypoxia (IH) [1.5% O2 for 30s followed by 4m of 21% O2], by placing the cells in a Biospheric Oxycycler. Trypan blue extraction cell viability test showed similar numbers of viable cells for both groups. Real time RT‐PCR showed significant reduction in Neuropeptide Y (NPY) gene expression (NO: 1.1 ± 0.2 vs IH: 0.4± 0.01) after 24 cycles. This reduction was further exaggerated (p=0.01) after 60 cycles of IH, in addition to reduction in leptin and TRPC4 mRNA (p=0.001). There were no differences in mRNA levels of c‐fos, corticosterone releasing hormone and vasopressin after 24 cycles of IH. Alterations in the activity of neuropeptides in 4B cells during IH could be mediated by blunted TRPC4 expression. P01 HL88052.
Carotid body ablation improves survival, breathing disorders and autonomic control in heart failure rats
Authors: DEL RIO RODRIGO , Marcus Noah J , Schultz Harold D
Published: June 2021

Sympathetic hyperactivity and breathing instability contribute to decompensation during chronic heart failure (CHF). Enhanced carotid body (CB) chemoreflex drive has been related to increased sympathetic outflow and breathing disorders in CHF. We hypothesized that ablation of CB afferents (CBA) improves cardiorespiratory function and survival in CHF. Selective CBA was performed on male Sprague‐Dawley rats after 16 weeks (wk) of coronary artery ligation (EF < 45%). Resting breathing and ventilatory responses to acute hypoxia (FiO2‐10%) were measured by plethysmography prior to and one wk after CBA/sham surgery. Heart rate (HRV) and systolic blood pressure variability (BPV) and spontaneous baroreflex gain (BG) were similarly determined at rest. Neuronal activation was assessed in the rostral ventrolateral medulla (RVLM) at the end of the study using Fra‐1 expression. CHF rats compared to sham exhibited elevated apnea incidence, hypoxic ventilatory responses, LF/HF HRV, LF‐BPV, and RVLM Fra‐1 expression and decreased BG. CBA in CHF rats decreased (pre‐ vs. post‐CBA, P < .05), LF/HF HRV (2.1±0.3 vs. 0.9±0.2), LF‐BPV (18.4±4.5 vs. 7.2±2.0 mmHg2), apnea frequency (16.8±1.8 vs. 10.6±0.6 events/h), RVLM Fra‐1 expression (1.5±0.1 vs. 0.9±0.1 a.u.), and increased BG (1.0±0.1 vs. 1.9±0.3 ms/mmHg) to levels comparable to sham rats. Mortality rate at 16 wk post infarct was elevated in CHF compared to sham (33% vs. 7%). CBA at 2 wk post infarct reduced the mortality rate in CHF rats at 16 wk to 15%. Our results show that CBA reduces hyperactivation of the RVLM and improves cardiorespiratory control and survival in CHF rats. Supported by NIH PO1‐HL62222.
The Influence of inlet boundary conditions upon hemodynamic analysis of human cerebral aneurysms with computational fluid dynamics technique
Authors: Fukuda Shunichi , Shimogonya Yuji , Tsukahara Tetsuya , Imoto Yasuhide , Morikawa Susumu , Fukuda Miyuki
Published: June 2021

We previously suggested that arise in wall shear stress (WSS) plays a central role in cerebral aneurysm formation due to asymmetric shape of the circle of Willis. Several models have recently been reported details of hemodynamics in human aneurysms with computational fluid dynamics analyses, carried out in most cases using assumed flow rates as inlet boundary conditions. However, measured values derived from physiological data of individual patients may have to be used since a shift in hemodynamic stress is caused by disturbed hemodynamics in the circle of Willis. Thus, we examined the influence of inlet boundary conditions between theoretically assumed and actually measured values on hemodynamics in human aneurysms of the anterior communicating artery where bilateral anterior cerebral arteries (ACA) converge. There is a significant difference in flow rates between assumed and measured values. Locations of high WSS, high gradient oscillatory number, and enhanced flow disturbance inside aneurysms are observed more frequently using measured values, caused by a difference in flow rates between bilateral ACA. The results illustrate that measured flow rates should be used as inlet boundary conditions.
Signal transduction in cerebral arteries after subarachnoid hemorrhage ‐ a phosphoproteomic approach
Authors: Povlsen Gro Klitgaard , Parker Benjamin , Larsen Martin Røssel , Edvinsson Lars
Published: June 2021

Following subarachnoid hemorrhage (SAH), cerebral vasculopathy contribute to delayed ischemia and poor outcome. We hypothesise that this is triggered by early SAH‐induced intracellular signals in cerebral arteries. To reveal these, protein extracts of rat cerebral arteries isolated 1h post‐SAH were digested, and labelled phosphopeptides were quantified by high resolution tandem mass spectrometry. Selected phosphorylations were validated by immunoblotting and monitored over a time‐course and pharmacological inhibition of key components was performed. We observed major SAH‐induced phosphorylations on focal adhesion complexes, extracellular regulated kinase 1/2 (ERK1/2), calcium calmodulin‐dependent kinase II, signal transducer and activator of transcription (STAT3) and c‐Jun, the latter two downstream of ERK1/2. Inhibition of ERK1/2 starting 6h post‐SAH prevented later increases in cerebrovascular constrictor receptors, matrix metalloprotease‐9 and wall thickness and improved neurological outcome. STAT3 inhibition partially prevented the vasculopathy but had no effect on neurological outcome. In conclusion, quantitative mass spectrometry is a strong approach to study in vivo signalling in vascular tissue. ERK1/2 inhibition after SAH prevents pathological cerebrovascular changes several days later and improves outcome.
Population Differences in Uropathic Bacteria in Diabetic and Meprin Deficient Mouse Models
Authors: Whitaker Jasmine , Conley Sabena , Harrison Scott H , Ongeri Elimelda , Hurley Steven L , Han Jian
Published: June 2021

Meprins, metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in urinary tract infections (UTIs) and diabetic nephropathy. Women with UTIs have been shown to have high levels of meprin A in their urine. This study evaluated for the presence of uropathic bacteria in the urine of two strains of mice, C57BL/6 (n=17) and C3H/He (n=14), with normal and deficient levels of meprin A respectively. Streptozotocin (STZ) was used to induce type 1 diabetes in 8 week old female mice. Urine samples were collected at 8 weeks post‐STZ injection, and population differences in bacteria were surveyed through differential media (CHROMagar Orientation). Both the overall quantity and the range of different types of uropathic bacteria were significant in response to the interacting effect of meprin A and diabetes status (two‐way ANOVA, P<0.05). For the C3H/He group, there was an increase of 20% to 40% of specimens reporting more than one variety of uropathic bacteria from control to diabetes, and for the C57BL/6 group, 0% to 30%. These results indicate that diabetes and levels of meprin A affect variety and quantity of bacteria in the urinary bladder.
Steviol retards cyst growth through inhibition of ERK and wnt/β‐catenin pathways
Authors: Yuajit Chaowalit , Nantavishit Jiriporn , Muanprasat Chatchai , Chatsudthipong Varanuj
Published: June 2021

Cystogenesis in polycystic kidney disease (PKD) involves multiple pathways. One of them is cAMP‐stimulated epithelial cell proliferation and cyst enlargement. Previously, we found that steviol could inhibit cyst enlargement in part by inhibition of CFTR function. The present study was aimed to investigate the detailed mechanisms of steviol on cyst growth using Madin Darby canine kidney (MDCK) cyst models. We found that steviol (100 μM) reversibly inhibited MDCK cyst growth by 38.3%. Steviol at the dose up to 200 μM had no cytotoxicity on MDCK cells. Western blot analysis of cysts extracted from collagen demonstrated that steviol reduced ERK 1/2 phosphorylation. In addition, treatment with 100 μM of steviol for 24 h significantly abolished ß‐catenin protein expression in MDCK cells by 33.6%. These findings suggest that steviol retards MDCK cyst enlargement, in part, by retarding cell proliferation through inhibition of ERK signaling and wnt/ß‐catenin pathways. Thus, steviol represents a promising natural plant‐based drug candidate for polycystic kidney disease due to its multiple targets.

This work was supported by the Thailand Research Fund, the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative and Faculty of Science, Mahidol University, Thailand.
Lymphocyte‐specific adaptor protein, LNK, inhibits angiotensin II‐induced hypertension and inflammation
Authors: Saleh Mohamed A. , Harrison David G , Madhur Meena S
Published: June 2021

Several large‐scale genome wide association studies have identified a number of loci associated with human hypertension. One such locus encodes the gene LNK, lymphocyte‐specific adaptor protein. The mechanism by which LNK contributes to blood pressure regulation is unknown. The objective of our study is to examine the role of LNK in angiotensin II (Ang)‐induced hypertension and inflammation. Using mice deficient in LNK (LNK−/−), we found that loss of LNK results in an increase in tail cuff blood pressure after two weeks of Ang infusion (213±3.4 mmHg vs 177±6.1 mmHg, p<0.001). Renal inflammation was examined by staining for CD3, a marker of T cells. At baseline, LNK−/− exhibited slightly higher numbers of T cells in the cortex and medulla compared to wild type C57 mice (WT) and this was markedly increased in response to 2 weeks of Ang infusion. Cytokine analysis performed on the culture supernatants of splenic T cells demonstrated that baseline levels of interleukin (IL) 17A and F and tumor necrosis factor alpha were higher in LNK−/− mice compared to WT mice. The pro‐inflammatory cytokine, IL17A, was further increased in LNK−/− mice in response to Ang infusion while IL17F levels were decreased by Ang infusion. Taken together, these data demonstrate that LNK is a negative regulator of renal and systemic inflammation and inhibits Ang induced hypertension.
Superoxide and Isoketal formation in Dendritic Cells from Hypertensive mice activate T cells and promote Hypertension
Authors: Kirabo Annet , Galindo Cristi , Wu Jing , Thabet Salim , Bikineyeva Alfiya , Dikalov Sergey , Amarnath Venkataraman , Davies Sean , Roberts Jackson L , Harrison David Glenn
Published: June 2021

Oxidative injury and inflammation have been implicated in the genesis of hypertension but the mechanisms involved are not fully understood. We examined the hypothesis that oxidative stress in dendritic cells alters endogenous proteins via Isoketal‐modification leading to formation of neo‐antigens, T cell activation and a predisposition to blood pressure elevation. Dendritic cells isolated from mice with angiotensin II‐induced hypertension had a 5‐fold increase in NADPH oxidase‐dependent superoxide production when compared to sham‐treated mice (334.0±49.7 versus 65.8±4.5 pmol/mg protein). This was associated with an accumulation of Isoketals and activation of IL‐1b and IL‐6 production in these cells. Microarray analysis revealed a predominant alteration in expression of genes involved in cell‐mediated immunity and antigen processing in dendritic cells from hypertensive mice. Treatment with a potent Isoketal scavenger, 2‐hydroxybenzylamine, markedly attenuated angiotensin II‐induced hypertension (142.59 ± 8.98 mmHg versus 175.53 ± 5.19 mmHg in controls). Adoptive transfer of dendritic cells from hypertensive mice polarized T cells from naïve recipients to IL‐17 and IFN‐γ producing CD8+ cells and also primed development of hypertension in mice given a sub‐pressor dose of angiotensin II (157.45 ± 33.86 mmHg versus 119.90 ± 17.33 mmHg in controls). These studies show that angiotensin II‐induced hypertension activates dendritic cells, in large part by causing superoxide production and formation of Isoketals. We propose that Isoketal‐modified proteins can be presented as neoantigens by dendritic cells, which in turn trigger T cell activation leading to hypertension.
EFFECT OF RESISTANCE TRAINING ON CIRCULATING Th1 CYTOKINES IN HIV+ MEN RECOVERING FROM SUBSTANCE ABUSE
Authors: Vingren Jakob L , Curtis John H , Duplanty Anthony A , Lee Elaine C , Budnar Ronald G , McFarlin Brian K , Hill David W
Published: June 2021

Human immunodeficiency virus (HIV) and substance abuse (drug and/or alcohol) independently impair the immune system. INF‐γ and IL‐2 are Th1 cytokines involved in modulating immunity relevant to HIV. INF‐γ inhibits HIV replication in latently infected cells and low production of IFN‐γ is correlated to progression to AIDS. IL‐2 slows apoptosis of HIV‐infected cells. Thus, changes in Th1 cytokines could have important implications for HIV+ individuals.

Purpose

To examine the effect of resistance training on resting concentrations of circulating Th1 cytokines.

Methods

Sixteen men (42 ± 11 years, 180 ± 9 cm, 89 ± 21 kg) infected with HIV and enrolled in a 60‐day in‐patient substance abuse treatment program. Participants were assigned to either supervised resistance exercise 3 times per week (RT) or no exercise training (Control) for six weeks. Before (Pre) and after (Post) the 6‐week period, resting and fasted blood samples were obtained and analyzed for serum INF‐γ and IL‐2.

Results

The 6‐week intervention did not change INF‐γ (RT: Pre: 1.4 ± 2.1 pg·ml−1; Post: 1.2 ± 2.6 pg·ml−1; Control: Pre: 2.3 ± 3.9 pg·ml−1; Post: 2.2 ± 3.9 pg·ml−1) or IL‐2 (RT: Pre: 0.4 ± 0.5 pg·ml−1; Post: 0.2 ± 0.1 pg·ml−1; Control: Pre: 0.1 ± 0.1 pg·ml−1; Post: 0.2 ± 0.1 pg·ml−1)

Conclusion

A six‐week resistance training program does not elicit changes in the circulating Th1 cytokines INF‐γ and IL‐2 in HIV+ men undergoing substance abuse treatment.