bluuebunny/crossref_metadata_2025 huggingface dataset:
Scientific Jobs – an industrial perspective
Authors: Concha Nestor Omar
Published: June 2021

We all have been trained in the methods and tools of scientific research in academic laboratories because, truthfully, there is no better place, but it does not follow that academic research institutions are the only places where intellectually challenging science is being done and that only academic labs have the interesting problems to be tackled. In the broadest sense, the employment market is a collection of diverse opportunities, but there is no such thing as research in academia and research in industry. Quite a bit of introspection is called for to determine one's personal long term goals, free from considerations such as “I'm selling out”, or industry is the “evil empire”. Instead, be aware that you are also interviewing your prospective boss, the company, the work they do, and the way they work. Ultimately, an outstanding team of colleagues and a clear sense of the purpose of your endeavors are the key components of a rewarding and successful career.

Science in the pharmaceutical industry and biotechs is mission driven: the unmet medical needs of the patients, in delivering what is of benefit to society. Under these parameters, performance and deliverables are measure against goals and milestones. The opportunity to publish important scientific contributions is encouraged.

In general, industry jobs have a favorable work‐life balance. Internships, co‐op and summer studentships provide terrific opportunities offered by many organizations to allow both students and prospective employers to know each other, to understand the sense of fitting into the larger whole, and being valued for that.
Proteinase‐activated receptors, PAR1 & PAR2, regulate porcine coronary contractility via tyrosine kinase‐MAPKinase signaling involving a cyclooxygenase (COX)‐1 product
Authors: El‐Daly Mahmoud , Saifeddine Mahmoud , Triggle Christopher R. , Hollenberg Morley D.
Published: June 2021

Objective

PARs 1 and 2 regulate vascular tension and also trigger both tyrosine kinase (PYK) and MAPKinase signals, in part via EGF receptor (EGFR) transactivation. We hypothesized that PAR‐triggered MAPK and PYK activities regulate porcine coronary artery (PCA) tension via EGFR activation.

Methods

Contractions of L‐NAME‐treated PCA rings ± functional endothelium stimulated by PAR1/2‐selective activating peptides (PAR‐APs), EGF, angiotensin‐II (AngII) and PGF2α were monitored in the absence and presence of the signal pathway inhibitors: PP1 (Src kinase), AG1478 (EGFR kinase), U0126 (MAPK), PD98059 (MAPK), and the COX inhibitors, indomethacin (INDO, COX1,2), SC560 (COX1), & NS398 (COX2).

Results

Endothelium‐independent contractions by PAR1/2‐APs, Ang‐II, and EGF (but not by PGF2α) were inhibited by PP1, U0126, & PD98059. AG1478 blocked mainly PAR2‐AP and EGF contractions, Further, PAR‐AP & Ang‐II contractions, blocked by INDO & SC560, were mimicked by arachidonate. In contrast, PGF2α‐induced contraction was not affected by any inhibitor used.

Conclusions

PARs 1 & 2, like AngII, regulate coronary tension via a PYK‐MAPK pathway involving a COX1 metabolite, but by distinct GPCR mechanisms that differentially transactivate the EGFR. Support: CIHR‐Canada, Qatar Foundation NPPR and the Heart & Stroke Foundation of Canada.
The mechanism of the marine β‐carboline thromboxane B2 inhibitor Manzamine A: role of p90 ribosomal S6 kinase 1
Authors: Chandrasena Kevin , Hall Mary L , Mayer Alejandro M
Published: June 2021

Manzamine A (MZA) inhibited LPS‐treated rat microglia (BMΦ) TXB2 (IC50<0.016 μM) and O2− (IC50=0.1 μM) generation, (BMC Pharmacology 5(1) 6, 2005), LPS‐treated human BMΦ TXB2 (apparent IC50<0.7 μM) (Inflam Res. 53(S3): S217, 2004) and rat p90 ribosomal S6 kinase 1 (RSK1) (IC50 =15.01 μM) (Program 545.5, Society of Neuroscience, 2009). RSK1 is expressed in LPS‐treated rat microglia (BMΦ) (Faseb Journal 26: 1124.1, 2012). We hypothesized that in BMΦ MZA might inhibit RSK1 phosphorylation induced at residues S380 and T573 by kinases activated by PMA, a PKC activator. In order to test our hypothesis, we determined the presence of constitutive and phosphorylated RSK1 proteins in murine BV‐2 BMΦ cell line. Briefly, we prepared protein extracts from untreated, LPS (100 ng/mL)‐treated, LPS + PMA (1μM)‐treated BV‐2 BMΦ in the presence or absence of DMSO and MZA (3, 10 and 30 μM), and probed by western blot analysis using constitutive RSK1 (Cell Signaling) and phosphorylated RSK1 (pRSK1) antibodies against S380 (Epitomics) and T573 (Cell Signaling).

Results

1) Constitutive RSK1 and pRSK1 proteins were present in all BV‐2 BMΦ extracts tested; 2) PMA enhanced pRSK1 protein in LPS‐treated BV‐2 BMΦ; 3) MZA did not inhibit pRSK1 protein on either S380 and T573 in LPS + PMA‐treated BV‐2 BMΦ. Future studies will determine 1) whether RSK1 in primary rat microglia show a similar response to MZA, and 2) evaluate the effect of MZA on phosphorylation of RSK1 at residues S221, S154, T359, S363, S732. Support to KC by the Office of Research, Midwestern University is gratefully acknowledged.
EVALUATION OF RENAL FUNCTION IN ISOLATED PERFUSED KIDNEY AFTER AT1 RECEPTOR BLOCKADE IN RATS SUBMMITED TO HYPERLIPIDIC DIET.
Authors: Muller Cynthia Rodrigues , De Oliveira Leite Ana Paula , Américo Anna Laura Viacava , Fonteles Manassés Claudino , Farah Vera , Fiorino Patricia
Published: June 2021

The aim of this study was to evaluate the Angiotensin II effect on renal function. Male Wistar rats (n=8/group) were divided in Control Group (CG), commercial rat feeding, and Hyperlipidic Group (HG) by addition of 30% unsalted butter in commercial diet to rats during 8 weeks, with starts immediately after weaning. At the end of the protocol, the renal function was evaluated in isolated right kidney perfused with Krebs‐Henseleit solution containing 6g of previously dialyzed bovine serum albumin. The kidney was connected in the system and after 30 minutes (basal period) were injected Losartan (10mg/100 ml) and it was analyzed 30 minutes after losartan. The parameters evaluated were renal vascular resistance (RVR), urinary flow (UF), perfusion pressure (PP), renal plasmatic flow (RPF), glomerular filtration rate (GFR), sodium tubular transport (%TNa+) and potassium tubular transport (%TK+). In HG during the basal period there was an increase in RVR (HG:7.2 ± 0.85 vs CG:4.83 ± 0.49, mmHg.mL.g‐1.min‐1) and in PP (HG: 130±6.7 vs CG:111.5±2.9, mmHg). However, there was no change in GFR, %TNa+ and %TK+. After losartan, the HG showed a reduction in PP (21%) and RVR (44%), and increase RPF (44%), without changes in the FU and GFR, when compared with basal period. In conclusion, the increased of RVR and PP observed in HG is probably associated with increase of local Angiotensin II. Financial Support: Mackpesquisa (059/2011).
Subcellular Oxidative Stress Changes Induced by Cisplatin are reduced by Resveratrol In Vitro in Renal Tissue.
Authors: Valentovic Monica A , Ball John G , Wolfe Jacob , Van Meter Stephanie , Miller Rikki , Brown Rebekkah , Ramsey Derek
Published: June 2021

Cisplatin, a cancer chemotherapeutic agent, can induced nephrotoxicity. Resveratrol (RES) is a natural compound found in grapes, cranberries and nuts. This study investigated whether RES reduced cisplatin renal in vitro toxicity and focused on differences between cytosolic and mitochondrial oxidative stress mediated by cisplatin. Male Fischer 344 rats (200–250 g) were anesthetized, with isoflurane and the kidneys were isolated. Renal cortical slices were prepared and pre‐incubated with 30 ul ethanol (VEH) or 30 ug/ml resveratrol (RES, final concentration) for 30 min at 37 °C and incubated 120 min with 0–150 ug/mL cisplatin. Loss of membrane integrity was evaluated as leakage of lactate dehydrogenase (LDH). Oxidative stress and nitrosative stress were assessed in mitochondria and cytosol. LDH leakage required a 120 min exposure to cisplatin. An increase in protein carbonyls as detected by Oxyblot, was increased by cisplatin and totally prevented by RES. RES also decreased protein carbonyls in tissue not exposed to cisplatin. Our findings showed that a 30 min RES pre‐incubation diminished cisplatin renal toxicity and that protein carbonyl and 3‐ nitrotyrosine and early changes in oxidative stress prior to the onset of loss of membrane integrity. (Supported by NIH Grants INBRE 3P20RR016477–09S4; 5P20RR016477 and 8P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence).
The Acute Antinociceptive Effect of Hyperbaric Oxygen (HBO2) Is Not Accompanied by an Increase in Markers of Oxidative Stress
Authors: Liu Shulin , Zylstra Carlyn C. , Shirachi Donald Y. , Quock Raymond M.
Published: June 2021

Exposure to 3.5 ATA HBO2 causes antinociception in mice (Ohgami et al., NeuroReport 20:1325, 2010). However, breathing O2 at an elevated pressure can potentially cause oxygen toxicity. The aim of this study was to identify the determinants of HBO2 antinociception and the toxicity profile of HBO2. Male NIH Swiss mice were assessed for acute antinociceptive responsiveness under room air or 100% O2 at 1.0 or 3.5 ATA, using the acetic acid‐induced abdominal constriction test. Only the combination of 100% O2 and 3.5 ATA caused significant antinociception. The antinociceptive effect of 100% O2 was pressure‐dependent up to 3.5 ATA. Oxygen toxicity was assessed by analyzing the levels of two oxidative stress markers, MDA (malondialdehyde) and protein carbonyl, in brain, spinal cord and lung after HBO2 exposure. The results showed no significant increase of either marker in the tested tissues, indicating an absence of significant oxidative stress caused by an 11‐min HBO2 treatment. In fact, even a 60‐min HBO2 treatment failed to produce any significant changes. It is concluded that HBO2 at 3.5 ATA is able to produce significant antinociception without causing oxygen toxicity in mice. (This research was supported by NIH Grant AT‐007222 and the Allen I. White Distinguished Professorship at Washington State University.)
The Organic Cation Transporter 3 (OCT3) Facilitates Fetal Disposition of Metformin during Pregnancy
Authors: Lee Nora , Hebert Mary F , Easterling Thomas R , Wang Joanne
Published: June 2021

Metformin used in the treatment of gestational diabetes is transported by multiple organic cation transporters. Metformin crosses the placental barrier, but the underlying transport mechanisms are unknown. The goal of this study was to determine the role of organic cation transporter 3 (OCT3) in metformin transport across the placenta. The expression of OCT3 and other organic cation transporters were quantified in mouse and human placentas by real‐time PCR. The membrane localization of OCT3 in human term placenta was determined by immunofluorescence microscopy. Finally, the in vivo impact of Oct3 in fetal disposition of metformin was investigated in pregnant wild type and Oct3 knockout mice. Our results showed that both human and mouse placentas predominantly express OCT3 with minimal expression of OCT1/2, MATE1/2 and PMAT. OCT3 protein was localized to the basal membrane of syncytiotrophoblasts facing the fetus. After oral dosing of [14C] metformin, the overall drug exposure (AUC0–480 min) in maternal plasma was comparable between wild type and Oct3−/− pregnant mice. However, fetal AUC0–480 min of metformin in Oct3−/− pregnant mice was about 40% less than that in the wild type pregnant mice. These results suggest that OCT3 mediates transport of metformin from the placental cells into the fetus and plays an important role in the transport of metformin and other organic cations at the maternal‐fetal interface.
Detection of Phase‐1 Drug‐Metabolizing Epoxide Hydrolases in Glioblastoma Cells
Authors: Paris Shere Lynnette , Shivachar Amruthesh C
Published: June 2021

Glioblastoma Multiforme (GBM) is a devastating prognosis for human brain tumors known for rapid proliferation, tumor invasiveness, cell migration, and drug resistance. Angiogenesis appears to be the main contributor of Glioblastoma's aggressive tumor growth. Epoxide Hydrolases (EH's) constitute an important class of drug metabolizing enzymes mainly distributed in liver and other extrahepatic tissues, including the brain. SEH substrates are responsible for vasoactive proliferation which may be an important contributor to angiogenesis and drug resistance. Previous studies have used sEH inhibitors to detect sEH activity in glioma cell lines U251 and U87 by fluorescence assay. In this Pilot study we report the expression of mEH and sEH's in GBM‐derived U118MG cell lines. The expression of mEH and sEH was determined by immunocytochemical staining ad western blotting analyses. Our results show that U118MG cells stained positive for both EH's and these results were confirmed by western blot analysis. These results demonstrate that mEH and sEH are coexpressed in GBM U118MG cells. Future goal is to further elucidate the possible roles of sEH in tumor cell proliferation by use of sEH inhibitors to reuce Glioblastoma's resistance to drug therapy.
A novel peptide to induce production of antibodies for detection of equine CC10
Authors: Prayaga Sudhirdas , Venugopal Changaram
Published: June 2021

Recurrent airway obstruction (RAO) in horses is a type‐1 immediate hypersensitivity allergic response involving reaginic antibodies mainly IgE and some IgG subclasses. Antigen antibody reaction leads to degranulation of mast cells, resulting in release of inflammatory mediators. The overall goal of the study was to establish a direct function correlation between Clara Cell proteins (CC10) and RAO in horses. The specific aims were: 1) to develop a rabbit polyclonal antibody to the “C” terminal 21 amino acid peptide of equine CC10 protein, 2) to validate this antibody in an antibody capture endpoint ELISA with peptide as the capture antigen and peroxidase conjugated goat anti‐rabbit IgG as detection antibody, 3) to detect the peptide using a competitive ELISA, 4) to determine CC10 specific IgG titer in clinically healthy and RAO‐affected horses using the competitive ELISA system. The results showed that the newly developed peptide with 21 amino acids produced polyclonal antibodies in rabbits. An antibody titer of >;100,000 was detected at a dilution between 1000 and 10000. A concentration‐dependent inhibition of antibody binding to peptide‐coated plate by the peptide was observed in competitive inhibition assay. Clinically healthy horses had significantly higher IgG titer suggesting the peptide's homology to common environmental antigens. The peptide has potential to induce antibody production against allergens. Supported by Equine Health Studies Program, LSU School of Veterinary Medicine.
Rhamnan sulfate enhances the endothelial glycocalyx and decreases the LDL permeability of human coronary artery endothelial cells in vitro
Authors: Cancel Limary Melissa , Tarbell John M
Published: June 2021

Human coronary artery endothelial cell monolayers were incubated with two rhamnan sulfate isoforms (RS1 and RS2) for 24 hrs before the permeability of low density lipoprotein (LDL) was measured. In addition, some monolayers were induced to undergo apoptosis with tumor necrosis factor‐α/cycloheximide (TNF‐α/CHX ) in the presence or absence of RS1 or RS2 and the permeability of LDL was measured. Both RS1 and RS2 significantly reduced the LDL permeability of control monolayers by 5‐fold. When the monolayers were induced to undergo apoptosis to increase their permeability, incubation with both RS1 and RS2 completely abolished the TNF‐α/CHX‐induced increase. Neither RS1 nor RS2 had any effect on the apoptosis rate of control or TNF‐α/CHX‐treated monolayers indicating that their effects were not mediated by apoptosis inhibition. Immunostaining of RS1 and RS2‐treated monolayers for a major gycocalyx component, heparan sulfate (HS), showed a significant increase in the coverage of HS compared to control monolayers. This suggests that enhanced HS synthesis may mediate the reduction in LDL permeability associated with RS1 and RS2. We believe that the enhanced glycocalyx provides added resistance to the leaky junction pathway that is the main pathway for LDL transport across the endothelium. Research supported by NIH HL094889 and Daniels Health Sciences.
Measurement of retinal blood flow rate in diabetic rats: disparity between techniques due to shunting of capillary flow
Authors: Leskova Wendy , Watts Megan N. , Eshaq Randa , Carter Patsy R. , Harris Norman R.
Published: June 2021

The objective of this study was to clarify whether retinal blood flow (RBF) rates are altered in rats during streptozotocin (STZ)‐induced diabetes. Reports of this possibility in the literature are mixed, possibly due to the different techniques used to measure RBF. STZ (or vehicle alone, in controls) was injected into the rats (30 mg/kg for 3 days) to induce a model of type I diabetes. Eight weeks later, RBF rates were measured in the anesthetized rats using intravital microscopy and a fluorescent tracer (FITC‐dextran) in a mean circulation (transit) time index of flow. In separate rats, RBF was measured using a microsphere infusion technique. The induction of diabetes resulted in a 30–35% decrease in RBF (***p<0.001) in the STZ rats as determined by the microsphere measurement. However, in a striking contrast to these results, the mean transit times of injected FITC‐dextran were ~3× faster in the STZ rats compared with the controls (**p<0.01). We hypothesize that this discrepancy is due to shunting of RBF away from the deep retinal capillary layers in the STZ rats, with the altered flow pattern allowing the rapid transit of tracer from the arterioles into the venules. Funded by NIH EY017599 (NRH).
Effects of anti‐hypertensive interventions on the inflammatory response in the spontaneously hypertensive rat
Authors: Marvar Paul J , McBryde Fiona D , Paton Julian F.R.
Published: June 2021

Clinical and pre‐clinical studies have found inflammation to be a contributing factor in the genesis of various forms hypertension. Current clinical interventions, such as renal denervation, target the autonomic nervous system to treat hypertension, however the effects of these blood pressure lowering strategies on the immune system are unknown. The current study examined immune cell activation and tissue infiltration following autonomic denervation procedures in an animal model of neurogenic hypertension. As determined by flow cytometry, the spontaneously hypertensive rat (SHR) exhibited a significant increase in the percentage of T lymphocytes (CD3+) in both the brainstem (3.3 ± 0.4 vs 1.6 ± 0.2; p=0.003) and aortic tissue (17.2 ± 1.6 vs 9.9 ± 2.1; p=0.01) compared to the normotensive control. These animals then underwent either renal denervation (RD) or carotid sinus denervation (CSD). Blood pressure in the SHR was significantly lowered following both RD and CSD. Compared to non‐denervated SHR, the total circulating leukocytes (CD45+) and CD3+ cells were attenuated following blood pressure lowering in the RD and CSD groups (p<0.001). These data suggest that reducing blood pressure with procedures targeting the autonomic nervous system is paralleled by reductions in tissue inflammation associated with hypertension. Supported by NIH‐HL107675–01, Marie Curie‐IIF – 276147 and British Heart Foundation.
Maternal Separation (MS) enhances angiotensin II (Ang II)‐induced hypertension in Dahl rats fed a high salt diet
Authors: Loria Analia Silvina , Pollock David M , Pollock Jennifer S
Published: June 2021

Previously, we showed that MS, a model of early life stress, enhances the response to hypertensive stimuli in a non‐salt sensitive rat strain. The aims of this study were to test whether MS in Dahl salt‐sensitive (DS) rats exaggerates: 1) the acute or chronic behavioral stress response, or, 2) AngII and/or high salt diet‐induced hypertension. MS was performed in male DS rats by separating about half of the pups from the dam 3 hr a day from days 2–14 after birth. Undisturbed littermates served as controls (C). Rats were implanted with telemetry transmitters (10 weeks of age, n=4–6) and allowed to recover. Mean arterial pressure (MAP) was not different between C and MS rats at baseline (118±1 vs. 120±2 mmHg). Acute air jet stress‐induced pressor response measured as area under the curve (AUC) was not different in C and MS rats. AUC due to chronic restraint stress was not different in C and MS rats as well. Separate groups of rats were fed either a high salt diet (HSD, 4%NaCl), infused with AngII (200 ng/kg/min) or infused with AngII + HSD for one week. MAP was not different in C and MS rats fed a HSD (176±5 vs. 163±7 mmHg). AngII‐induced hypertension was not different in C and MS rats (146±4 vs. 150±1 mmHg). AngII+HSD increased MAP in MS rats compared to C rats (167±5 vs. 152±2 mmHg, respectively, ptime=<0.05, pseparation= <0.05 and pinteraction= <0.05). We conclude that MS exacerbates AngII‐induced hypertension in the presence of HSD in DS rats.
Heme oxygenase‐1 induction attenuates TNF‐α induced hypertension in pregnant rats
Authors: George Eric M , Arany Marietta , Cockrell Kathy , Stec David E , Granger Joey P
Published: June 2021

Preeclampsia (PE) is a hypertensive disorder of pregnancy initiated by placental insufficiency and chronic ischemia. In response, several pathways activated in the placenta are responsible for the maternal syndrome, including increased production of the anti‐angiogenic protein sFlt‐1 and inflammatory cytokines, especially TNF‐α. Previous studies demonstrated that heme oxygenase‐1 (HO‐1) induction can block TNF‐α pathways in vitro and attenuate placental ischemia‐induced sFlt‐1 in vivo. Here, we investigated whether HO‐1 induction could attenuate TNF‐α induced hypertension in pregnant rats. In response to TNF‐α infusion (100ng/day i.p.), maternal mean arterial pressure increased vs control animals (120±4 vs 103±3 mmHg). HO‐1 induction had no effect in control animals, but significantly decreased MAP in TNF‐α infused animals (109±5 mmHg). Placental sFlt‐1 was increased by TNF‐α infusion (909±46 vs 758±45 pg/mg), which was significantly reduced by HO‐1 induction (842±102 pg/mg), suggesting a therapeutic role for HO‐1 induction in PE. Research supported by NIH grants HL105324–01, HL51971, HL088421, HL088421‐S1, and AHA grant 11POST7840039.
SPAK‐independent activation of NCC in salt‐sensitive hypertension
Authors: Terker Andrew Scott , Yang Chao‐Ling , McCormick James A. , Rogers Shaunessy , Ellison David Hoadley
Published: June 2021

Background

Recently, it was reported that adrenergic stimulation activates the thiazide sensitive Na‐Cl cotransporter (NCC) and causes salt‐sensitive hypertension. Here, we tested whether NCC activation is required.

Results

Norepinephrine (NE) infusion increased blood pressure (BP) after one week, an effect enhanced by salt loading. Total and phosphorylated NCC (pNCC, a marker of activation) were increased after three weeks. The salt‐dependent BP rise was greatly attenuated in NCC knockout (KO) mice. Kidneys from mice acutely treated with NE showed increased pNCC (2.83 fold increase, P<0.01) within 30 minutes, while total NCC remained unchanged. This effect was not abrogated in AT1aR KO mice. To determine the adrenergic receptor subtype involved, phenylephrine, isoproterenol, or both were administered. The combination increased pNCC more than either alone. NE stimulation did not change SPAK abundance in the DCT, so we speculated that OSR1 might be involved. NE increased pNCC abundance even in SPAK KO mice. Further, acute NE increased the apical OSR1 in the DCT of wild‐type and SPAK KO mice.

Conclusion

NE activates NCC acutely to cause salt‐sensitive hypertension. NCC can be activated independent of SPAK, likely by OSR1. These results identify a novel role for OSR1 and NCC in salt‐sensitive hypertension. Supported by 1RO1DK095841, RO1DK51496, and by a VA Merit Review.
Adenylyl cyclase VI is necessary for AVP stimulation of ENaC
Authors: Bugay Vladislav , Mironova Elena , Roos Karl , Ramkumar Nirupama , Kohan Donald , Stockand James
Published: June 2021

Vasopressin is an important physiological modulator of renal sodium excretion, which is fine‐tuned in the collecting duct. This action of vasopressin is mediated in part by adenylyl cyclase‐stimulated cAMP regulation of the epithelial Na+ channel (ENaC). Multiple adenylyl cyclase isoforms are expressed in the collecting duct. The adenylyl cyclase isoform that is most important to vasopressin‐regulation of ENaC in the collecting duct is obscure. To assess this, the basal activity of ENaC and the response of this channel to desmopressin and water‐deprivation were compared in wild type mice and mice having collecting duct principal cell‐specific knockout of adenylyl cyclase type VI. Patch clamp analysis of split‐open cortical collecting ducts revealed no difference in basal ENaC activity; however, the stimulatory effects of water‐deprivation and desmopressin on ENaC were absent in knockout mice. These results demonstrate that adenylyl cyclase type VI is necessary for vasopressin stimulation of ENaC in the collecting duct but that this isoform is not necessary for basal channel activity under normal conditions.
Role of Rab11a Positive Compartments in Endocytic Traffic in Primary Proximal Tubule Cells
Authors: Raghavan Venkatesan , Mattila Polly E. , Rbaibi Youssef , Weisz Ora A.
Published: June 2021

The proximal tubule (PT) reabsorbs ~ 70% of sodium, bicarbonate, chloride ions, phosphate, glucose, water, and the majority of the plasma proteins from the glomerular filtrate. Despite the critical importance of endocytosis for PT cell function, the organization of the endocytic pathway in these cells remains poorly understood. We have used live‐cell imaging to dissect the itinerary of apically internalized fluid and membrane cargo in polarized primary cultures of PT cells isolated from mice. Cells from the S1 segment could be distinguished from more distal segments by their robust uptake of albumin and low expression of γ‐glutamyltranspeptidase. Rab11a in these cells is localized to numerous dynamic spherical compartments that resemble the apical vacuoles found in electron microscopy analysis of cultured PT cells. These structures are highly dynamic and receive both membrane and fluid phase cargo. In contrast, Rab11a in immortalized kidney cell lines is localized to a condensed and largely tubular apical recycling compartment through which recycling membrane proteins (but not fluid phase cargoes) transit. The unusual morphology of Rab11a‐positive compartments in primary PT cells may reflect the demands of handling a high endocytic load and suggests a possible role for Rab11a in early sorting steps of fluid and membrane proteins in these cells. This research was supported by NIH R01 DK54407 and P30 DK079307.
Human cyst fluid from autosomal dominant polycystic kidney disease (ADPKD) patients stimulates two interacting chloride channels
Authors: Flaig Stephanie M. , Blazer‐Yost Bonnie L.
Published: June 2021

In ADPKD renal cysts enlarge slowly over a patient's lifetime, but renal function remains intact until midlife. Once renal function begins to decline, renal failure occurs rapidly. Studies were performed to determine the factors that may be involved in the cyst expansion and renal decline during the last stage of ADPKD. We used electrophysiology techniques to study ion transport of a mouse principal cells of the cortical collecting duct (mpkCCD) cell line after exposure to human cyst fluid. It was previously shown that basolateral addition of cyst fluid stimulated a Cl− secretory response and that the active component of cyst fluid is lysophosphatidic acid (LPA). Inhibitor studies indicate that the Clsecretory response is due to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) as well as a Ca2+‐ activated Cl− channel (CaCC), with the characteristics of TMEM16A. Interestingly, the LPA stimulated Cl− secretory response is not accompanied by increased cAMP and is substantially inhibited by Bapta‐AM, a chelator of intracellular Ca2+. These results suggest a key role for Ca2+ in the LPA‐stimulated Cl− secretory response as well as an interaction between CFTR and CaCC.
The lectin‐like domain of TNF directly increases ENaC activity
Authors: Czikora Istvan , Alli Abdel A , Bao Hui‐Fang , White Richard , Shabbir Waheed , Lemmens‐Gruber Rosa , Tzotzos Susan , Fischer Bernhard , Matthay Michael A , Eaton Douglas C , Lucas Rudolf
Published: June 2021

Rationale

Aims of this study were to investigate whether the TIP peptide, mimicking the alveolar liquid clearance (ALC)‐promoting lectin‐like domain of TNF, is able to activate amiloride‐sensitive Na+ transport in H441 cells and to directly interact with specific ENaC domains.

Methods

Peptide effects on Na+‐uptake in cells pretreated or not with N‐glycosidase F, were assessed using perforated patch or single channel measurements. Peptide‐ENaC interactions were assessed upon incubating either a) biotinylated TIP or mutant TIP peptide‐coupled or b) GST‐ENaC‐α domain‐coupled beads with lysates from ENaC‐α overexpressing H441 cells or peptides, respectively.

Results

TIP, but not mutant TIP peptide activates amiloride‐sensitive Na+ currents and single ENaC channels in glycosylated, but not de‐glycosyated H441 cells. Binding to human ENaC‐α subunit occurs within the regulatory carboxy‐terminal, but not the extracellular loop or N‐terminal domains of the recombinant protein.

Conclusions

These data suggest for the first time that the lectin‐like domain of TNF, upon internalization, directly activates ENaC, by binding to the carboxy‐terminal domain of the channel's α‐subunit. This provides a rationale for the observed therapeutically promising ALC‐promoting activities of the peptide in vivo.
High salt diet stimulates ENaC in Dahl salt‐sensitive rats
Authors: Bao Hui‐Fang , Yue Qiang , Eaton Douglas , Zhang Zhi‐Ren , Ma He‐Ping
Published: June 2021

It has long been suggested that the renal epithelial sodium channel (ENaC) contributes to the development of salt‐sensitive hypertension. However, it remains unclear whether high salt diet can stimulate ENaC in Dahl salt‐sensitive (DS) rats (a model of salt‐sensitive hypertension). Our confocal microscopy data showed that high salt diet decreased ENaC expression in the cortical collecting ducts (CCD) of control rats, but increased ENaC expression in the CCD of DS rats. Interestingly, high salt diet also altered ENaC plasticity along the renal tubules by inducing ENaC expression in the thick ascending limb of DS rats, but not of control rats. Our cell‐attached patch‐clamp data showed that high salt diet increased ENaC open probability in the split‐open CCD of DS rats, but not of control rats. Since it is known that high salt diet elevates reactive oxygen species (ROS) in the kidneys of DS rats and our recent studies have shown that hydrogen peroxide (one form of ROS) stimulates ENaC in cultured distal nephron cells, high salt diet may stimulate ENaC in CCD and induce ENaC expression in the thick ascending limb by causing oxidative stress in the kidneys of DS rats (supported by NIH 5R01‐DK067110 to HPM).
Mechanisms of intracellular calcium elevation in human airway epithelial cells by an airway disease‐relevant extract of hog‐barn dust
Authors: Toews Myron L. , Baker Jonathan I. , Schulte Nancy A. , Heires Art J. , Bidasee Keshore R. , Romberger Debra J.
Published: June 2021

A subset of workers in swine confinement facilities develop chronic inflammatory lung disease, and an aqueous hog‐barn dust extract (HDE) induces diverse inflammation‐related responses in cultured BEAS‐2B human airway epithelial cells, including release of inflammatory cytokines. These responses require the calcium (Ca)‐dependent protein kinase C‐alpha isozyme, and we showed previously that HDE stimulates Ca elevation in these cells. Two distinct fractions of HDE stimulate Ca responses, so we hypothesized that two different Ca regulatory pathways would be involved. BEAS‐2B cells were loaded with Fluo‐4 and treated with agents to alter various Ca signaling pathways, and the effects of these agents on HDE modulation of intracellular Ca were then assessed by Flex Station assays. Pretreatment with pertussis toxin, the phospholipase C (PLC) inhibitor U73122 but not its inactive analog U73343, and the IP3 receptor Ca channel inhibitors 2‐APB and xestospongin C all markedly reduced the HDE Ca response. TrpV1 receptor Ca channels mediate some responses to inhaled pollutants, and the TrpV1 inhibitor capsazepine and TrpV1 inactivation by pretreatment with resiniferatoxin both markedly reduced HDE Ca responses also. These data suggest that a Gi/o‐mediated PLC pathway to IP3 receptor activation and TrpV1 channel activation both contribute to HDE Ca responses. Supported by NIOSH 2R01‐OH008539.
Myocardial Activation of p38 alpha MAPK Results in Cardiac Insulin Resistance by Chronic Insulin Signaling
Authors: Qi Yajuan , Dostal Davide , Guo Shaodong
Published: June 2021

Diabetic cardiomyopathy is widely present in diabetic patients and the major course of its death is heart failure. The molecular mechanism that links diabetes to cardiomyopathy is incompletely understood. Insulin resistance is a hallmark of type 2 diabetes and hyperinsulinemia is characteristics of insulin resistance. However, whether insulin itself is a determinant factor leading to cardiac insulin resistance and dysfunction is uncertain. Our previous studies demonstrated that insulin receptor substrate protein 1 and 2 (IRS2 and IRS2) are major mediators of insulin action. Using rat neonatal cardiomyoctes, we analyzed insulin signaling components, including IRS1, IRS2, PI3Kinase and MAPKinase, and assessed their contribution to insulin action and resistance in regulation metabolic gene expression. We found that acute insulin stimulation for 0.5 hour activated Akt and Foxo1 phosphorylation, while chronic insulin stimulation for 24 hours sustained p38á MAPK activation accompanied with reduction in both IRS1 and IRS2, whereas inhibiting p38á largely rescued the decrease of IRS1 and IRS2 and expression of p38á MAPKinase by adenovirus infection degraded IRS1 and IRS2 proteins. Taken together, these data suggest that chronic insulin exposure can result in insulin resistance, impair energy metabolism, and likely contributes to heart failure in type 2 diabetes, involving in activation of p38á MAPK.
NF‐kB–dependent IL‐6 expression, induced by electrical stimulation, is reduced in dystrophic mdx skeletal muscle cells.
Authors: Jaimovich Enrique , Henríquez Carlos , Altamirano Francisco
Published: June 2021

In skeletal muscle cells, membrane depolarization increases reactive oxygen species (ROS) due to NOX2 activation; this is related with changes in gene expression. Our aim was to study IL‐6 gene in wt and mdx muscle cells, and its relationship with NOX2 and NF‐kB activity. Differentiated wt and mdx myotubes were stimulated with 250p, 0.5 ms at 20 Hz. ROS production was assessed with CM‐DCFA. NOX2 was assessed using apocynin and DPI. NF‐kB transcriptional activity was studied with a luciferase reporter, p65 phosphorylation by western blot and gene expression by real time PCR.

NOX2 subunits (gp91 and p47) were upregulated in mdx myotubes. After stimulation, ROS production was higher in mdx compared to wt. NF‐kB activity after both electrical and ROS stimulation increased in wt myotubes, but not in mdx. NOX2 inhibition reduced NF‐kB activation. IL‐6 gene expression induced by both electrical and ROS stimulation was higher in wt compared to mdx muscle cells.

ROS production and NF‐kB activity are linked in normal cells. Altered NF‐kB, excess ROS production and differential gene expression could explain oxidative stress and progressive damage reported in DMD.

Fondecyt 1110467 (EJ), AT‐24100066 (FA).
Oxidative stress effect before fermentation to bioethanol and alcoholic beverages production
Authors: Arellano‐Plaza Melchor , Noriega‐Cisneros Ruth , Gschaedler‐Mathis Anne , Montoya‐Pérez Rocío , Cortés‐Rojo Christian , González‐Hernández Juan Carlos , Saavedra‐Molina Alfredo
Published: June 2021

During ethanol and alcoholic beverages production, yeast cells are affected by several stress conditions that are adverse to growth (oxidative, osmotic and ethanol stress among others) and they should detect and respond to these conditions, otherwise alcoholic fermentation can be negatively affected. In this work we have analyzed the fermentative behavior of four commercial and non‐commercial strains in synthetic musts. The yeast used were two Saccharomyces cerevisiae and two Kluyveromyces marxianus, isolated from mezcal processes in México. According to the data, indicate that common patterns of stress response are found among all the strains and can been classified into three groups depending on whether or not alcoholic fermentation was completed. The oxidative stress with H2O2 decreased the specific growth rate during the fermentation more than the stress induced by menadione, but the ethanol production at the end of the fermentation, not showed a significant difference between the H2O2 and the menadione. Our results indicate a clear relationship between stress resistance and fermentative behavior and this opens up the possibility of using this information as a criterion for the future selection of bioethanol and alcoholic beverages yeasts.
Constitutively active phosphodiesterase type 4 controls large conductance Ca2+‐activated K+ channel activity in guinea pig urinary bladder smooth muscle
Authors: Xin Wenkuan , Cheng Qiuping , Li Ning , Petkov Georgi V.
Published: June 2021

Nonselective pharmacological inhibition of phosphodiesterases (PDE) elevates cellular cAMP levels, leading to activation of the large conductance Ca2+‐activated K+ (BK) channels and attenuation of guinea pig urinary bladder smooth muscle (UBSM) excitability and contractility. Here, we investigated the mechanism of selective inhibition of cAMP‐specific PDE4 on UBSM excitability and contractility using the perforated patch‐clamp technique on isolated UBSM cells and isometric tension recordings of UBSM isolated strips. Our data showed that selective PDE4‐inhibition with rolipram increased the spontaneous transient BK currents frequency and hyperpolarized the cell resting membrane potential (RMP). Blocking BK channels with paxilline reversed the hyperpolarization effect of rolipram and depolarized the RMP back to the control levels. PDE4‐inhibition did not cause RMP hyperpolarization in the presence of cAMP‐dependent protein kinase (PKA) inhibitor, H‐89. PDE4 inhibition reduced the UBSM spontaneous phasic and carbachol‐induced contraction frequency and amplitude, as well as the electrical‐field‐stimulation‐induced contraction amplitude. In conclusion, selective inhibition of the constitutively active PDE4 suppressed the guinea pig UBSM excitability and contractility via a BK channel‐dependent mechanism involving the PKA signaling pathway. Supported by NIH DK08424 to GVP.
TNFα receptor endocytosis balances inflammatory and apoptotic signaling in endothelial cells
Authors: Choi Hyehun , Lamb Fred S
Published: June 2021

Tumor necrosis factor‐α (TNFα) impairs endothelial cell (EC)‐dependent vasodilation. It is not known how endocytosis affects apoptotic vs. proliferative effects of TNFα. We hypothesized that receptor endocytosis modulates the response of ECs to TNFα and impaired vascular relaxation is mediated by MAPK. Mesenteric resistance arteries from C57BL/6 mice were treated with TNFα and selective inhibitors (Dynasore for endocytosis, SP600125 for JNK, SB203580 for p38) for 24hrs and vasodilation was assessed. TNFα impaired acetylcholine (ACh)‐induced relaxation and this effect is exacerbated by Dynasore (Fig. 1, n=6). These effects were JNK and p38‐dependent (Fig. 2, n=6). In cultured mesenteric ECs, TNFα induced JNK, p38, and ERK1/2 phosphorylation but only JNK and p38 activation were potentiated by Dynasore (p‐JNK, 2‐fold; p‐p38, 1.4‐fold; n=3). Thus TNFα‐induced endothelial dysfunction is endocytosis, JNK or p38‐dependent. TNFα receptor endocytosis balances pro‐inflammatory with pro‐apoptotic (JNK, p38) signaling in ECs.
Abnormal ER and mitochondrial communication underlies ER stress in cerebrovascular insulin resistance
Authors: Katakam Prasad V.G. , Rutkai Ibolya , Liu Dan , Grovenburg Samuel M , Gordon Angellica O , Busija David W
Published: June 2021

We previously demonstrated impaired mitochondria (mito)‐mediated vasodilation, abnormal mitochondrial Ca2+ uptake and vascular insulin resistance (IR) in cerebral arteries of Zucker obese rats (ZO). Mitofusin 2 (MFN2) tethers mitochondria to endoplasmic reticulum (ER) and creates Ca2+microdomains of interorganelle communication. Our objective is to elucidate the impact of IR on ER‐mito interaction. Expression levels of key mitochondrial proteins (MFN2 and VDAC) and ER stress markers (immunoglobin heavy chain binding protein (BiP), C/EB‐Phomologous protein (CHOP), and X‐box binding protein 1 (XBP1)) and phosphorylated neuronal nitric oxide synthase (p‐nNOS) was determined. Isolated cerebral arteries from ZO and their lean (ZL) controls were treated with vehicle or insulin (1mU/ml) under normal and oxygen‐glucose deprived (OGD) conditions for 4 hours. ZO arteries displayed decreased MFN2 and VDAC and increased CHOP, BiP, XBP1 and nNOS at baseline. Insulin+OGD treatment increased MFN2 and p‐nNOS but reduced BiP in ZL whereas it promoted decreased MFN2 and p‐nNOS but increased BiP in ZO. Thus, ZO arteries with IR display impaired mito‐ER communication at baseline which is further enhanced by insulin under OGD.
Habituation to repeated stress is reversed by systemic angiotensin II: a mechanism involving nitric oxide in the paraventricular nucleus of the hypothalamus
Authors: Anilovitch Julia Leor , Davern Pamela J , Burke Sandra L , Evans Roger G , Head Geoffrey A
Published: June 2021

Long‐term angiotensin II (Ang) induced hypertension leads to activation of hypothalamic neurons. In the present study, we determined whether this treatment might prevent the habituation to repeated stress (RS, 60 min airjet daily for 7 days). Rabbits were exposed to RS alone or combined with Ang (6 weeks, 20ng/kg/min) or neither (control). RS exposure reduced the renal sympathetic nerve activity (RSNA) response to acute stress and inhibited neuronal activation (Fos expression) in the hypothalamus. Ang treatment restored the RSNA response to acute stress and also increased the activation of the paraventricular nucleus (PVN, 16±3 vs 39±3, P<0.001) compared with RS, but not of the amygdala, dorsomedial hypothalamus, arcuate and supraoptic nuclei. The activated PVN neurons contained NADPH oxidase (11±3 v 18±5 P<0.01) and nitric oxide (NO) synthase (16±3 vs 45±4, P<0.001) but not vasopressin or corticotrophin releasing factor. Thus, superoxide and NO production in the PVN is associated with elevated sympathetic responses to RS exposure in Ang‐induced hypertensive rabbits. This study was supported by the National Health & Medical Research Council of Australia.
A role for Gαi2 proteins in the acute neural control of blood pressure.
Authors: Carmichael Casey Yumi , Wainford Richard D
Published: June 2021

Aim

We have demonstrated hypertension mediated by failure to upregulate hypothalamic paraventricular nucleus Gαi2 proteins in rats fed a high salt diet, but the role of this mechanism is unknown in acute settings. We examined the effect of central Gαi2 proteins in the neural control of blood pressure in response to an acute pharmacological and physiological challenge.

Methods

Naïve and 24hr ICV Gαi2 oligodeoxynucleotide (ODN; 25μg/5μl)‐pretreated conscious Sprague‐Dawley rats were continuously monitored for changes in HR and MAP in response to intravenous (IV) infusion of phenylephrine and sodium nitroprusside or 3M NaCl IV bolus (0.14 ml/100g). To determine cardiac baroreflex relationships, MAP was slowly raised to ~175 mmHg using phenylephrine and lowered to ~50 mmHg using sodium nitroprusside (N=8/gp).

Results

24h Gαi2 ODN pretreatment did not affect the cardiac baroreflex response. In response to IV sodium, peak changes in HR were significantly greater in naïve animals vs. Gαi2 treated animals (Naïve + 3M NaCl ΔHR=−79±15 bpm vs. Gαi2 + 3M NaCl ΔHR=−59±12 bpm; P<0.05). We observed no difference in peak MAP post‐IV NaCl between groups (Naïve + 3M NaCl MAP 147±4 mmHg vs. Gαi2 + 3M NaCl MAP 149±3 mmHg). In naïve rats, MAP returned to baseline by 100 min whereas Gαi2 animals remained significantly elevated for 120 min (P<0.05).

Conclusion

Endogenous Gαi2 proteins are involved in neural control of HR and MAP during an acute sodium challenge. These data suggest brain Gαi2 signal transduction is required to mediate sodium‐sensitive neural responses to maintain physiologically appropriate blood pressure regulation.
The Antifibrillatory Effects of Vagus Nerve Stimulation in the Rabbit Ventricle involves cGMP‐Dependent Mechanisms
Authors: Brack Kieran , Winter James , Ng G. Andre
Published: June 2021

We have previously shown that vagus nerve stimulation (VNS) increases effective refractory period (ERP) and ventricular fibrillation threshold (VFT) whilst reducing electrical restitution slope, via an acetycholine independent ‐ nitric oxide (NO) dependent mechanism. NO activates soluble guanylyl cyclase [sGC] to produce cyclic guanylyl monophosphate (cGMP) to modulate cardiac function. Our aim was to investigate if the effects of the VNS‐NO pathway on ERP, VFT and restitution slope were mediated via cGMP mechanisms. The innervated rabbit heart was used (n=5, 2.5–3kg) in constant flow Langendorff mode. Ventricular ERP was determined using a single extrastimulus protocol (300ms cycle length) and maximal restitution slope was measured. VFT was the minimum current needed to induce sustained VF with burst pacing (30×30ms). These were measured at baseline [BL] and during VNS [10Hz, 9V] in control (Ctrl) and following sGC inhibition with (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one [ODQ], 10μM). Data are mean±SEM, Student's T‐Test, P<0.05 * vs BL, ** vs Ctrl. VNS increased ERP and VFT whilst reducing restitution slope. During sGC inhibition, the vagal effect on restitution slope and VFT were reduced, whilst the effect on ERP was preserved. Cyclic GMP is involved in the protective effects of VNS‐NO pathway against VF but concurrent cGMP independent mechanisms could also be at work.

KEB is supported by a British Heart Foundation IBSR Fellowship

Table
ERP (ms)
Slope
VFT (mA)

Ctrl BL
131.0±3.3
1.48±0.2
3.6 ±0.7

Ctrl VNS
158.0±9.3*
0.99±0.21*
7.0±0.6*

Change
16.8±5.9
−0.5±0.1
3.4±0.2

ODQ BL
144.2±4.5
1.3±0.1
3.5±0.7

ODQ VNS
159.0±7.6*
1.2±0.1
5.2±0.8*

Change
12.0±3.0
−0.1±0.1**
1.7±0.1**
An avian model for exploring brain stem rhythmogenic behavior during early development
Authors: Vincen‐Brown Michael Alan , Still Katie D. , Quick Forrest , Pilarski Jason Q.
Published: June 2021

Brain‐related breathing problems are common in early life. Despite intensive investigations into mammalian breathing control, our understanding of respiratory rhythm generation is still unclear, especially during the prenatal period. We aim to develop the avian embryo as a more tractable prenatal model to study breathing related behaviors. Birds are highly active endotherms, like mammals, and provide access for manipulations that mammals cannot. In the rodent, automatic breathing behaviors have been identified in isolated transverse brainstem slices. We hypothesize that there is a similar complex within the avian brainstem. Thus, our goal is to identify the central pattern generator for breathing in the bird. We used an activity dependent dye, electrical recordings, and Nissl staining to generate a neuroanatomical brain stem map. We examined isolated brain stems from Corturnix japonica near internal pipping when breathing movements are present. Results suggest that birds possess a spontaneously generated rhythm in the ventral lateral medulla similar to mammals. We further show that in some preparations tonic activity became rhythmic when inhibitory neurotransmission was blocked. These results suggest critical differences between the prenatal development of central breathing control of bird and mammals, including the importance of GABA and glycine neurotransmission prior to parturition.