bluuebunny/crossref_metadata_2025 huggingface dataset:
The Repressor Element‐1 Silencing Transcription Factor (REST) regulates the Hypoxia Inducible Factor (HIF) network through a novel negative feedback loop
Authors: Cavadas Miguel Santos , Taylor Cormac , Cheong Alex
Published: June 2021

Hypoxia induces a number of metabolic changes with profound consequences for cell physiology and pathophysiology. Our understanding of the complex response to hypoxia has been focused on the Hypoxia‐Inducible Factor (HIF) and its regulation by prolyl‐hydroxylases (PHD). In solid tumours, both intra‐tumoral hypoxia and mutations in tumour suppressor genes can lead to the overexpression of HIF, a feature that has been associated with poor prognosis.

The Repressor Element‐1 Silencing Transcription Factor (REST), is best characterized as a transcriptional repressor of neuronal genes in non‐neuronal tissues. More recently, REST was found to be affected by ischemia and to be down regulated in breast and colon cancer. This lead us to investigate if REST was an hypoxic sensitive transcription factor and if it interacts with the HIF pathway.

Hypoxia induced an increase in REST translocation to the nucleus. siRNA knockdown of REST increased HIF, PHD2 and PHD3 protein expression. REST was found to be recruited to the HIF promoter, and HIF was recruited to the REST promoter in a hypoxia dependent way, indicating a novel negative feedback loop involving REST and HIF. In conclusion, REST was identified as a novel hypoxia‐sensitive transcriptional repressor that negatively regulates HIF, PHD2/3, acting as a novel negative feedback loop in the hypoxia response.
Time course of changes of FosB immunoreactivity in nucleus of solitary tract (NTS) during chronic intermittent hypoxia (CIH)
Authors: Wu Qiong , Mifflin Steve
Published: June 2021

FosB is a member of the transcription factor Activator Protein–1 (AP‐1) family. FosB is not constitutively expressed in the CNS and is induced following chronic stimulation. A previous study from our group found that the number of FosB immunoreactive neurons in the NTS is increased after 7 days of exposure to CIH (alternating 3min periods of 10% O2 with 3min 21% O2 from 8am–4pm). To determine the time course of the onset of FosB expression we measured the number of FosB immunoreactive neurons in male Sprague Dawley rats exposed to either room air (control) or to CIH for 1 day, 3 days, 5 days or 7 days (n= 6 for each CIH group and 14 for control group). Rats were perfused with paraformaldehyde and brain processed for FosB immunohistochemistry using an antibody (Santa Cruz) that does not distinguish FosB from ΔFosB. The number of FosB immunoreactive neurons in caudal NTS in the control group was 11± 2 cells/section (c/s). after 1 day CIH 40± 11 c/s, 3 days CIH 35±8 c/s, 5 days CIH 36±5 c/s and 7 days CIH 29±4 c/s. The differences between the control group and the CIH groups were significant. Our previous work demonstrated elevated FosB 1 day after the last exposure to 7 days CIH. 3 days after the last exposure to 7 days CIH the number of FosB immunoreactive neurons in NTS had returned to 10 ± 3 c/s (n=2). This study indicates that FosB expression develops rapidly during exposure to CIH and remains elevated during a 7 day exposure. Supported by HL 088052
Neonatal stress disrupts laryngeal chemoreflex function in anesthetized rat pups
Authors: Baldy Cécile , Kinkead Richard
Published: June 2021

In preterm infants, the presence of liquids or solids in the airways stimulates the laryngeal chemoreflex (LCR) that results in apneas with potentially life threatening consequences. Because neonatal stress interferes with respiratory control development, we tested the hypothesis that neonatal maternal separation (NMS) compromises LCR function in rat pups. Experiments were performed on 10 to 15 days old male pups that were undisturbed (controls) or subjected to NMS (3h/day each day beginning at P3 to P13). Pups were anesthetized (chloralose, 20 mg/kg + urethane, 1 mg/kg) and maintained at 35°C. Following tracheotomy, a water filled catheter was placed near the larynx. Breathing was monitored with intercostal EMG electrode. O2 saturation and heart rate were monitored with pulse oxymetry. Following baseline measurements, each pup received 10 μL injection of water near the larynx. This procedure was repeated 3 times with a 5 min recovery period between injections. In NMS rats, apnea duration tended to increase following each stimulation; this was not observed in controls. LCR‐related O2 desaturations and bradycardias were more important in NMS pups than controls. The mortality rate observed in NMS pups (83%) was greater than controls (33%). We conclude that neonatal stress compromises LCR function in young rats and could contribute to respiratory disorders in infants. This research was supported by CIHR.
Klotho Protects Lung Epithelial Cells against Oxidative DNA Damage
Authors: Ravikumar Priya , Smith Blaine , Hsia Connie C.W. , Moe Orson W.
Published: June 2021

Klotho is an anti‐aging protein through pleiotropic actions. We postulate that Klotho can protect the lung against O2 and Pi toxicity and tested the direct effects of Klotho in vitro using A549 lung epithelial cells exposed to hyperoxia (95–5% O2‐CO2) and increasing Pi (1 vs. 5 mM). Soluble or transmembrane full length Klotho was over‐expressed by transfection or recombinant soluble Klotho was added to the medium. DNA damage was assessed by ELISA assay of 8‐hydroxydeoxyguanosine (8‐OHdG) (OxiSelect™ Cell BioLabs Inc). Triplicate experiments were performed. The amount of 8‐OHdG (ng/ml, mean±SD) under each condition was compared by two‐way ANOVA. P ≤ 0.05 was considered significant.

O2
21%
95%

Klotho
−
+
−
+

Transmembrane Klotho

Pi 1mM
0.093±0.003
0.086±0.009
0.172±0.005*
0.076±0.006 †

Pi 5mM
0.326±0.009
0.300±0.010†
0.563±0.013*
0.323±0.013 †

Soluble Klotho

Pi 1mM
0.093±0.003
0.082±0.002†
0.166±0.005*
0.072±0.001 * †

Pi 5mM
0.322±0.011
0.292±0.005†
0.546±0.012*
0.324±0.011 * †

Conditioned Medium

Pi 1mM
0.089±0.002
0.079±0.002†
0.164±0.004*
0.070±0.004 * †

Pi 5mM
0.317±0.007
0.299±0.005 †
0.531±0.012*
0.303±0.014 * †

95% vs. 21% O2;

Klotho vs. control at the same Pi and O2 concentrations.

All differences between Pi conditions were significant.

Increasing either O2 or Pi concentration increased 8‐OHdG production, indicating DNA damage; the effects are synergistic. In pulmonary epithelial cells, either Klotho expression or addition ameliorates hyperoxia‐induced DNA damage but has only mild effects on Pi‐induced DNA damage. These findings suggest that increasing Klotho concentration in the lung may be beneficial in the prevention of oxidant DNA damage.

Support

F32 HL103043, RO1 DK091392, DK092461, UO1 HL111146
Hypercholesterolemia does not affect pulmonary surfactant or the development of acute lung injury in rats
Authors: Banaschewski Brandon John Harrison , Hiansen Joshua Qua , McCaig Lynda , Yamashita Cory , Lewis James , Veldhuizen Ruud
Published: June 2021

Pulmonary surfactant is a lipoprotein complex necessary for lung function. The importance of surfactant is evident in Acute lung injury (ALI) in which the dysfunction of surfactant is thought to contribute to the hypoxemia characterizing the disease. Recent studies have specifically implicated elevated levels of cholesterol within surfactant as a mechanism for its dysfunction. It is not known if this effect is dependent on serum cholesterol. It was hypothesized that diet‐induced serum hypercholesterolemia affects surfactant composition and will cause development of more severe ALI. Wistar rats were fed a standard laboratory diet or high cholesterol diet prior to enrollment into one of three models of ALI: a ventilator induced lung injury model, a surfactant depleted lung injury model and an acid aspiration lung injury model. For each model measurements of lung injury, such as arterial oxygenation, as well as surfactant composition were measured. Results of this study indicated there were no significant differences in arterial oxygenation or surfactant composition between rats fed the standard diet versus the high cholesterol diet in any of the models of lung injury. In conclusion, these three complementary models of ALI suggest that diet‐induced serum hypercholesterolemia does not affect surfactant composition or the development of ALI. Funding was provided by the Canadian Institutes for Health Research.
PRMT1 directly methylates eNOS and thereby prevents Akt‐dependent serine1177 phosphorylation leading to decreased NO production
Authors: Chicoine Louis G , Pope Arthur J , Han Zhaosheng , Druhan Lawrence J , Nelin Leif D , Cardounel Arturo J
Published: June 2021

Diminished nitric oxide (NO) biosynthesis is implicated in the pathogenesis of cardiopulmonary disease. Much attention has focused on the production of endogenous eNOS inhibitors by protein‐arginine methyl transferase's (PRMT's). Our recent studies demonstrate that in addition to generating free methylarginines, PRMT's can directly methylate eNOS protein. We hypothesized that direct methylation of eNOS would decrease NO production by altering serine1177 phosphorylation. Silencing of PRMT1 in bovine pulmonary arterial endothelial cells (bPAEC) resulted in greater nitrite production than with scramble siRNA treatment. The levels of methylated eNOS by western blotting were substantially lower than in the control cells. Treatment of bPAEC with the PRMT1 siRNA resulted in greater levels of serine1177 phosphorylation and lower levels of Threonine495 phosphorylation of eNOS compared with scramble siRNA treatment. In studies using purified human eNOS addition of PRMT1 decreased NO production while addition of a biologically active Akt increased NO production compared to control. The addition of both PRMT1 and biologically active Akt resulted in NO production that was not different from control. Results from these studies implicate PRMT‐dependent methylation of eNOS as a novel post‐translational modification regulating eNOS activity through Akt‐dependent phosphorylation of serine1177.
The chaperone heat shock protein 90 (Hsp90) participates in the endothelial store operated calcium entry heterocomplex
Authors: Kadeba Pierre I , Scammell Jonathan G , Cioffi Donna L
Published: June 2021

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in protein complexes that participate in various cellular signaling events. FK506‐binding proteins (FKBPs) are regulators of store operated calcium (SOC) entry. The association of Hsp90 with FKBPs has been shown to be responsible for many of their effects. However, it is not known whether Hsp90 participates in FKBP action on SOC entry. Here we show that membrane‐cytoskeleton preparations of pulmonary endothelial cells resolve Hsp90 in plasma membrane fractions where the calcium‐selective Isoc subunits, TRPC1/4, are found. Hsp90 co‐immunoprecipitates with TRPC4. Endothelial cells treated with the Hsp90 inhibitor geldanamycin show decreased global SOC entry compared to untreated controls. Collectively, these data reveal the participation of Hsp90 in the endothelial SOC entry heterocomplex. Supported by 5R00HL089361.
Glutathione regulation of epithelial sodium channels (ENaC) in alveolar type1 and type 2 cells.
Authors: Kreiner Lisa , Downs Charles A. , Brown Lou Ann , Helms My N.
Published: June 2021

We used single channel patch clamp recordings to examine the effect of oxidative stress, via H2O2 accumulation, and alteration of the glutathione redox potential on the activity of ENaC. We found that addition of 20 μM H2O2 significantly increased ENaC open probability (Po) from 0.34+0.16 to 0.470+0.16 in alveolar type 1 (AT1) and 0.17+0.05 to 0.44+0.07 in type 2 (AT2) cells; P<0.05. We also found a linear decrease in ENaC activity as the GSH/GSSG redox potential became less negative (n=19; P<0.05). Sequentially treating cells with H2O2 and then GSH resulted in significant decreases in ENaC activity. Likewise, singular treatment of GSSG significnatly decreased ENaC Po from 0.39 + 0.06 to 0.13 + 0.05 and decreased the number of active channels in the membrane, thus implicating GSSG as an important pro‐injury signaling molecule in edematous lung disorders. Fluorescent maleimide labeling and co‐immunoprecipitation studies show that α‐ENaC subunit is glutathionylated. Real time X‐ray imaging studies show that GSH and GSSG are important determinants of alveolar fluid clearance in vivo. From these results we conclude that ENaC is regulated by changes in the glutathione redox potential. Research was supported by R00HL09226 awarded to MNH; Children's Center for Development in Lung Biology Grant awarded to MNH
21‐benzylidene digoxin effect on tight junctions proteins
Authors: Rocha Sayonarah C , Rincon‐Heredia Ruth , Gomes Natalia LS , Moreira Otacilio C , Contreras Gerardo R , Barbosa Leandro A
Published: June 2021

The importance of Na,K‐ATPase for the formation of junctional complexes is well established. The aim of this work is to characterize the biological effect of a digoxin derivative, 21‐benzylidene digoxin (21‐BD) on tight junctions. MDCK cells are treated with 5, 10 and 50 μM 21‐BD and the immunofluorescence of tight junctions (TJs) proteins α1 Na,K‐ATPase and the transepithelial electrical resistance (TEER) were performed. Treatment with 50 μM 21‐BD induces a sustained increase of the TEER for, at least, 87 h and that is dependent on the doses. As expected, TEER change is associated to alterations of the expression of the integral plasma membrane proteins claudin‐2 and 4, as well as the membrane peripheral protein ZO‐1 after 50μM 21‐BD treatment. We have observed the characteristic “chicken fence pattern” that corresponds to the localization of these proteins in the TJs. Also, 21‐BD treatment caused the increase of the membrane localization and α1 Na,K‐ATPase RNAm expression levels. Treatment with several cardiac steroids causes different effects on cell junctions. The cardiac steroids treatment causes disruption of TJs and only ouabain in low concentrations (10 nM) increases the sealing of TJs and accelerates cell polarity. The effect of 21‐BD on TJs have demonstrated that cardiac steroids other than ouabain can act as TJs enhancer and that it can has implications on cancer metastasis. Supported: CNPq, Fapemig, CAPES
Arachidonic acid‐induced apoptosis in human renal proximal tubular epithelial cells
Authors: Alexander Larry D , Hamzeh Mona T
Published: June 2021

Arachidonic acid can induced several injury effects on renal tubular cells. The present study was aimed to investigate the effects of arachidonic acid on cellular apoptosis in human proximal tubular epithelial cells (HK‐2) and its signaling mechanisms. Treatment with various doses of arachidonic acid resulted in dose‐dependent decreases of cell viability and increases of reactive oxygen species (ROS). Arachidonic acid induced the phosphorylation of p38 MAPK, extracellular signal‐regulated kinase (ERK1/2), and c‐Jun N‐terminal kinase (JNK), the nuclear translocation of NF‐κB, and IκB‐degradation. Cell death detection assay revealed arachidonic acid induced apoptotic cell death of HK‐2 cells. Arachidonic acid‐induced apoptosis was significantly attenuated by inhibition of the NF‐κB and mitogen‐activated protein kinase (MAPK) signaling pathways. Moreover, the antioxidant N‐acetyl‐L‐cysteine (NAC), significantly inhibited arachidonic acid‐induced apoptosis and NF‐κB and MAPK activation. Taken together, these results indicate that arachidonic acid induced apoptosis mainly by stimulating ROS production and through NF‐κB and MAPK signaling pathways in human renal proximal tubular cells.
Advanced oxidation protein products induce cardiomyocyte death via Nox2/Rac1/superoxide‐dependent TRAF3IP2/JNK signaling
Authors: Valente Anthony J. , Siebenlist Ulrich , Chandrasekar Bysani
Published: June 2021

Advanced oxidation protein products (AOPPs) are formed during chronic oxidative stress as a result of reactions between plasma proteins and chlorinated oxidants. Their levels are elevated during various cardiovascular diseases. Since elevated AOPPs serve as independent risk factors for ischemic heart disease (IHD), and cardiomyocyte (CaM) death is a hallmark of IHD, we hypothesized that AOPPs will induce CaM death. AOPPs‐modified mouse serum albumin (AOPPs‐MSA) induced significant death of neonatal mouse CaM (NMCM) that was attenuated by RAGE, but not CD36 knockdown. Notably, TRAF3IP2 (aka CIKS/Act1) knockdown blunted its pro‐apoptotic effects. AOPPs‐MSA stimulated Nox2/Rac1‐dependent superoxide generation, TRAF3IP2 expression, and TRAF3IP2‐dependent JNK activation. The superoxide anion generating xanthine/xanthine oxidase system and hydrogen peroxide both induced TRAF3IP2 expression. Further, AOPPs‐MSA suppressed anti‐apoptotic Bcl2 and Bcl‐xL expression, an effect reversed by TRAF3IP2 knockdown, or forced expression of mutant JNK, WT Bcl2 or Bcl‐xL. AOPPs‐MSA also induced adult CaM death in a TRAF3IP2‐dependent manner. These results indicate for the first time that AOPPs induce CaM death via Nox2/Rac1/superoxide‐dependent TRAF3IP2/JNK activation in vitro, and suggest that AOPPs may contribute to myocardial injury in vivo. Thus TRAF3IP2 is a potential therapeutic target in IHD.
Osteopontin Stimulates Cardiac Myocyte Apoptosis via the Involvement of ER Stress and Mitochondrial Death Pathway
Authors: Dalal Suman , Zha Qinqin , Daniels Christopher R. , Steagall Rebecca J. , Das Bhudev C. , Gadeau Alain‐Pierre , Joyner William L. , Singh Mahipal , Singh Krishna
Published: June 2021

Increased osteopontin (OPN; matricellular protein) expression associates with myocyte apoptosis and myocardial dysfunction. The objective of this study was to identify OPN receptor, and understand the mechanism by which OPN induces myocyte apoptosis. Adult rat ventricular myocytes (ARVM) and transgenic mice expressing OPN in a myocyte‐specific manner were used for in vitro and in vivo studies. Treatment with purified OPN (20 nM) protein or adenoviral‐mediated OPN expression induced apoptosis in ARVM. OPN co‐immunoprecipitated with CD44 receptor. Neutralizing anti‐CD44 antibodies inhibited OPN‐stimulated apoptosis. OPN activated JNKs, and increased expression of Bax and levels of cytosolic cytochrome c suggesting involvement of mitochondrial death pathway. OPN increased endoplasmic reticulum (ER) stress, as evidenced by increased expression of Gadd153 and activation of caspase‐12. Inhibition of JNKs using SP600125 or ER stress using salubrinal or caspase‐12 inhibitor significantly reduced OPN‐stimulated apoptosis. Expression of OPN in adult mouse heart associated with increased myocyte apoptosis and LV dysfunction. In the heart, OPN expression increased JNKs and caspase‐12 activities, and expression of Bax and Gadd153. OPN, acting via CD44 receptors, induces apoptosis in myocytes via the involvement of ER stress and mitochondrial death pathway.
Bioluminescent imaging of hepatic caspase‐3 activity in mice
Authors: Ozaki Michitaka , Haga Sanae , Ozawa Takeaki
Published: June 2021

Apoptosis‐mediated damage of cells and organs is a serious concern in various pathological situations. Here, we demonstrate caspase‐3 activity by the luciferase‐based optical probe of live liver cells challenged by various stimuli, and also in vivo imaging of caspase‐3 activity of the post‐ischemic liver.

METHODS

We developed a novel probe (pcFluc‐DEVD) of cyclic luciferase reflecting caspase‐3 activity. By transfecting an adenovirus cording for pcFluc‐DEVD (AdpcFluc‐DEVD), we investigated whether this probe monitors apoptosis in live AML12 liver cells by staurosporine (STS), Fas‐ligand (FasL) or hypoxia/reoxygenation (H/R), and in the post‐ischemic liver in mice.

RESULTS

STS and FasL activate caspase‐3 in caspase‐8‐independent and dependent manner, respectively. First, we measured caspase‐3 activity in STS‐ or FasL‐treated AML12 cells transiently transfected with AdpcFluc‐DEVD. In live AML12 cells, the signals from the probe started to increase immediately and peaked at 2–4 hr after 1 μM STS and 2 μg/ml Jo2 administration, respectively. In both stimuli, caspase‐3 activity was paralleled with apoptotic cell death. Next, we measured caspase‐3 activity in post‐hypoxic AML12 cells by this probe. During hypoxia, caspase‐3 activity dropped to the bottom level. However, it rapidly recovered to the pre‐hypoxic level after reoxygenation and started to rise until 4 – 6 hr post‐H/R.

Then, we tried to evaluate in vivo apoptosis by measuring caspase‐3 activity in mouse hepatic ischemia/reperfusion (I/R). 60min of hepatic ischemia caused great increase of capase‐3 activity at 6 – 9 hr post‐I/R, whereas 30 min of liver ischemia caused small increase at 2 hr post‐I/R (Figure).

CONCLUSIONS

The caspase3 probe successfully illustrated apoptotic damage in vitro and in vivo. Visualizing apoptotic damage of cells/organs may provide important insights into various physio‐pathological conditions.
Cyclic AMP‐Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2Cadrenoceptors through the actin binding protein filamin‐2
Authors: Motawea Hanaa KB , Jeyaraj Selvi C , Pawlowski Marcin , Kloczkowski Andrzej , Chotani Maqsood A.
Published: June 2021

The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac‐Rap1A and RhoA‐ROCK‐F‐actin signaling in arteriolar smooth muscle cells to increase expression and cell surface translocation of functional α2Cadrenoceptors (α2C‐ARs) that mediate vasoconstriction. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We have identified a novel protein‐protein association between α2C‐AR C‐terminus 14 amino acids and the actin‐binding protein filamin‐2, which was confirmed by site‐directed mutagenesis and co‐immunoprecipitation, co‐localization, and siRNA knock‐down approaches. Computational modeling and docking predictions further supported the specificity of this interaction. The cAMP‐Rap1‐ Rho‐ROCK signaling facilitated receptor translocation via phosphorylation of filamin‐2 Ser‐2113. The α2C‐AR 14 amino acids region, when used as decoy peptide, inhibited endogenous receptor surface translocation. Together, these studies show a vital role of the C‐terminus in receptor translocation, and provide a rationale for designing protein‐protein interaction small molecule inhibitor as a novel approach to target intracellular α2C‐ARs.
Cyclic nucleotide dependent regulation of fibroblast migration by multi drug resistant protein 4
Authors: Sinha Chandrima , Ren Aixia , Arora Kavisha , Moon Chang‐Suk , Yarlagadda Sunitha , Zhang Weiqiang , Cheepala Satish , Schuetz John D , Naren Anjaparavanda P
Published: June 2021

Role of cyclic nucleotides and cyclic nucleotide‐dependent signaling molecules in cell migration has long been known. However, the concept that it is not just the absence or presence, but a highly coordinated balance between cyclic nucleotides that regulates cell migration, is new and revolutionary. In this study, we used multi‐drug resistance protein 4 (MRP4)‐expressing cell lines and MRP4 knockout mice as model systems and wound healing assays as the experimental system to explore this unique and emerging concept. MRP4, an ATP binding cassette transporter protein, localizes to the plasma membrane and functions as a nucleotide efflux transporter and plays a role in the regulation of intracellular cyclic nucleotide levels. Here we demonstrate that mouse embryonic fibroblasts (MEFs) isolated from Mrp4−/− mice have higher intracellular cyclic nucleotide levels and migrate faster compared to MEFs from Mrp4+/+ mice. In contrast to these moderate increases seen in the absence of MRP4, a robust increase in cAMP levels was observed following treatment with forskolin and IBMX, which inhibits fibroblasts migration. In response to externally added cell‐permeant cyclic nucleotides (cpt‐cAMP and cpt‐cGMP), MEF migration appears to be biphasic. Altogether our data provide the first experimental evidence supporting the novel concept that balance between cyclic nucleotides is critical for fine‐tuned cell migration.
An oxychlorine solution changes the time‐course of peritoneal cytokine production associated with surgical adhesion formation
Authors: Zavala Manuel , Kross Robert David , Rojas Saul , Bobadilla Rosa Amalia , Campos Rafael , Carrasco‐Yepez Maricela , Villanueva Cleva
Published: June 2021

The goal of the study was to determine the time‐course of peritoneal inflammatory (IL‐1 beta, IL‐6 and TNF‐alpha) and antiinflammatory (IL‐4 and IL‐10) cytokines in male Wistar rats (12–14 week‐old) subjected to an experimental model of surgical adhesions, with or without a peritoneal treatment with a proprietary anti‐adhesion oxychlorine solution (OS, 100 ppm). All the cytokines but IL‐1 beta increased significantly within the first hour of surgery returning to the basal concentration 11 hours later. IL‐1 beta had a delayed increase (12 h), which returned to basal concentrations 12 h later. OS significantly reduced the production of TNF‐alpha, IL‐1 beta, IL‐10 and IL‐6. The strongest effect was on IL‐10. IL‐4 increased with OS (first hour). The early production of IL‐6, TNF‐alpha, IL‐10 and IL‐4 indicates that macrophages 1 M1 (inflammatory) and M2 (repairing) are simultaneously activated from the beginning of the surgery and both probably play a role in adhesion formation. OS could be interfering with the early stimuli that activate M1 and M2 macrophages, which could explain its anti‐adhesion suppression effect (supported by grant SIP20121039).
Caveolin‐1 Supports the P2Y2 Receptor Signaling
Authors: Martinez Namyr Antonio , Ayala Alondra Mali , Martinez Magdiel , Quiñones Monica , Miranda Jorge D. , Silva Walter I.
Published: June 2021

Damage to the CNS cells’ can cause a differential spatiotemporal release of multiple factors into the extracellular space. Among such factors, nucleotides, and their interaction with the P2Y2 nucleotide receptors (P2Y2Rs) have gained prominence as putative modulators of gliotic responses after CNS injury. Yet, the molecular mechanisms underlying these interactions and responses remain to be explored. Discontinuous sucrose density gradient separation of 1321N1 cells (expressing recombinant hP2Y2R) homogenates revealed co‐fractionation of P2Y2Rs in cav‐1 (+) membrane‐raft fractions. Likewise, confocal microscopy revealed that a significant percent of P2Y2Rs co‐localized with cav‐1 in its sub‐cellular distribution. Blocking cav‐1 expression in hP2Y2‐1321N1 cells elicited abnormal intracellular Ca2+ mobilization responses and uncharacteristic patterns of AKT, ERK1/2 and p38 phosphorylation levels when stimulated with nucleotide agonists, as determined by microfluorometric calcium imaging analyses and immunoblotting, respectively. Our findings suggest that P2Y2Rs reside in membrane caveolae of non‐stimulated 1321N1 cells and that this sub‐cellular compartment may couple its downstream signaling machinery. We hypothesize that P2Y2Rs’ signaling cascades are linked to their expression in cav‐1 (+) micro‐domains. NAM is supported by the NIGMS‐NIH award number R25GM061838 at the UPR‐MSC.
pH Regulatory Transporters in Pancreatic Ductal Adenocarcinoma (PDAC)
Authors: Kong Su Chii , Christensen Sandra , Hoffmann Else Kay , Novak Ivana , Pedersen Stine Falsig
Published: June 2021

PDAC is among the most aggressive human malignacies, with a 5‐ year survival rate of 1–4%. Elucidation of specific molecular alterations in PDAC is urgently needed to devise new treatment strategies. Malignant tumors with rapid progression such as PDAC often undergo extensive metabolic changes resulting in increased acid production. We and others have shown in other cancers that this leads to the upregulation of acid extruding transporters, which in turn favors further cancer progression. The aim of our studies is to elucidate the regulation and roles of pH regulatory ion transporters in PDAC. We used qPCR to screen a panel of 4 human PDAC cell lines (AsPC‐1, BxPC‐3, Panc‐1, MIAPaCa‐2) against a normal human pancreatic ductal cell line (HPDE). The Na+/H+ exchanger 2 (NHE2) was upregulated in 3 of 4 cell lines, whereas NHE1 was upregulated only in BxPC3 cells; the V‐type H+‐ATPase subunit a3 was upregulated in all PDAC cell lines; all cell lines except BxPC3 exhibited upregulation of either AE2 or AE3 Cl−/HCO3−exchangers, and also the Na+‐HCO3− transporters SLC4A7 and –A8 tended to be upregulated in PDAC; surprisingly, the H+/lactate cotransporter MCT1 was downregulated in all PDAC cell lines and a similar trend was seen for MCT4. In conclusion, PDAC cell lines exhibit dramatic changes in the mRNA expression level of pH‐regulatory transporters. Ongoing experiments are addressing the functional relevance of this in PDAC.

Funding: Marie Curie Actions (ITN IonTraC)
His‐183 is a Critical Structural Determinant of NH3/NH4+ Transport by Rhbg
Authors: Nakhoul Nazih L , Abdulnour‐Nakhoul Solange M , Le Trang , Rabon Edd , Lee Hamm L.
Published: June 2021

Renal Rhbg is localized to the basolateral membrane of intercalated cells and is involved in NH3/NH4+ transport. The structure of Rhbg is not yet resolved however a high resolution crystal structure of AmtB, a bacterial homologue of Rh, has been determined. We used bi‐dimensional hydrophobic cluster analysis to align the sequence of Rhbg to AmtB & identify important sites of Rhbg that may affect function. Our model positioned a conserved His‐183 within the hydrophobic pore where it presumably serves to control NH3 transport. Three mutations of His‐183 (to Ala, Leu or Asp) were generated and expressed in oocytes. To determine function, we measured NH3/NH4+ & methyl amine/ammonium (MA/MA+) ‐ induced currents and pHi in oocytes expressing the mutants and compared them to measurements in control oocytes or those expressing wild‐type Rhbg. In all mutants, except H183L, NH4+ current was smaller than that in oocytes expressing WT Rhbg. However MA+‐induced current was completely abolished in all mutants. In oocytes expressing H183L mutants, NH3/NH4+ caused significantly smaller changes in pHi & Vm than in Rhbg and MA/MA+ did not cause any change in pHi or Vm. The NH3/NH4+ and MA/MA+ ‐ induced pHi & Vm changes in the mutant were not different from those in H2O‐injected oocytes. These data indicate complete loss of function upon substituting His‐183. The loss of function indicates that this site is critical for transport by Rhbg Supported by VA Merit grant and by Tulane Institutional Fund
A novel plate reader based assay for inhibitor studies of slc4a10 protein and other acid/base transporters
Authors: Nguyen An Tuyet , Praetorius Jeppe , Damkier Helle Hasager
Published: June 2021

The function of members of the slc4a family of HCO3− transporters is commonly determined using fluorescence‐microscopy of tissue loaded with pHi sensitive dyes. The cells are subjected to an acidification induced by NH4Cl and the Na+ dependent pHi recovery is determined in the presence of CO2/HCO3−.

The purpose of the present study was to implement the method in a plate‐reader based assay to increase the throughput of the method by measuring an entire cell population, and performing multiple experiments in parallel, thus improving stability and reliability of data.

A 3T3 NIH‐fibroblast cell line was stably transfected with the Na+/HCO3− transporter encoded by slc4a10. pHi of cells was measured in 8 wells of 96‐well plates in an Enspire® platereader using the fluorescent pHi indicator BCECF. Control experiments included DIDS, an inhibitor of bicarbonate transporters.

The Na+ dependent pHi recovery in slc4a10 transfected cells was 17.8±5.35 ×10−4 pH units/s (n=12). 200 μM DIDS inhibited the recovery by 65% (6.16±3.35 ×10−4 pH units/s, n=12). The endogenous Na+/H+ exchange in the cells was similarly reduced to 66% by 10 μM EIPA in the absence of CO2/HCO3−.

In conclusion, this method is a novel and valuable tool for high throughput screening of pharmacological agents inhibiting the acid/base transporters.
Rheogenic amino acid transport by Drosophila CG4991 of the Amino Acid Auxin Permease (AAAP) transporter family
Authors: Conlon Nichola J , Edwards Noel , Anderson Catriona MH , Cheek Timothy R , Thwaites David T
Published: June 2021

To increase our understanding of how differences in structure determine membrane transporter function, a member of the Amino Acid Auxin Permease (AAAP) family from Drosophila was investigated. CG4991 is an orthologue of the mammalian SLC36 proton‐coupled amino acid transporter family showing 32% identity in amino acid sequence with human PAT1 (SLC36A1). Transport characteristics were compared with those for mammalian PAT1 and PAT2 (SLC36A2). CG4991 was characterised functionally, following expression in Xenopus laevis oocytes, by radiotracer uptake, competition experiments, and measurements of rheogenic transport using the two‐electrode voltage‐clamp technique. As observed with mammalian PAT1 and PAT2, CG4991 functions as a pH‐dependent dipolar amino acid transporter with optimal taurine uptake at extracellular pH 5.0–5.5. Taurine uptake was independent of sodium at both pH 5.5 and 7.5. CG4991 demonstrates both similarities and differences in substrate selectivity when compared to PAT1 and PAT2. For example, CG4991, like PAT1, transports proline, glycine, beta‐alanine, betaine and MeAIB but unlike PAT1 transports serine. In conclusion, the commonality of CG4991, PAT1 and PAT2 transporter characteristics, suggests conservation of function despite relatively low levels of sequence identity. Supported by the Medical Research Council (MRC).
Variations in oxytocin receptor (OTR) density in term trophoblast depend on the contactile activity of the uterus
Authors: Szukiewicz Dariusz , Szewczyk Grzegorz , Pyzlak Michal
Published: June 2021

Oxytocin (OT) stimulates uterine contractions during labor. Before initiation of labor, increased sensitivity to OT must be accompanied by a strong upregulation of OTR in the myometrium and decidua. Despite OTR is present in the placental trophoblast cells, its expression and role in the context of uterine contractions or placental separation have not been studied thoroughly. Here we examined comparatively term placentae (Group I; N = 12) obtained after cesarean sections performed without spontaneously induced uterine contractions and placentae delivered after normal labors (Group II; N = 12). Mean OT concentrations was measured by ELISA in the placental samples taken from maternal surface. Next, the cytotrophoblast cells were isolated using Kliman's method based on trypsin, DNase and a 5–70% Percoll gradient centrifugation, and cultured in normoxia. After 5 days the cultures were fixed and immunostained for OTR. Quantitative morphometry was applied for evaluation of OTR expression. There were no significant differences in the mean OT concentration between the groups (p >; 0.05), whereas 11.2‐fold increase in the expression of OTR was observed in trophoblast cultures established in Group I. Thus, induction of labor with normal uterine contractions involves upregulation of OTR in placental region contiguous to the uterine wall. It may be crucial for the course of third stage of labor.
Regulation of basal cell plasticity by epidermal growth factor (EGF) and c‐Src in vivo in the mouse epididymis
Authors: Roy Jeremy , Ruan Ye Chun , Hill Eric , Visconti Pablo , Krapf Dario , Vinegoni Claudio , Breton Sylvie
Published: June 2021

Epididymal basal cells (BCs) sense the luminal environment by sending cell body projections that cross the tight‐junction barrier. BCs express the EGF receptor and its associated tyrosine kinase, c‐ Src, and we examined their role in their luminal sensing ability. BCs were visualized by in vivo multiphoton microscopy in our newly generated transgenic mice (KR5‐tdTomato), which express tdTomato exclusively in BCs. We found that BCs periodically elongate and retract their luminal‐reaching projections. Laser scanning confocal microscopy followed by 3D modeling showed that in vivo treatment with EGF (1 μM) or the EGFR inhibitor, Gefitinib (200 mg/kg/day), increased and decreased the number of luminal‐reaching BCs (labeled for keratin 5), respectively. Local EGF injection induced an increase in ERK1/2 phosphorylation. BCs in c‐Src knockout mice, which are infertile, exhibited no luminal‐reaching projections. We propose that EGF/EGFR/ERK1/2, together with c‐Src, are positive regulators of the luminal‐reaching property of BCs. A better understanding of the novel sensory function of BCs in the epididymis will help define their role in male fertility. This study is supported by NIH grant RO1DK085715.
Thick ascending limb‐specific nuclear receptor ERRβ regulates renal ion transports
Authors: Crambert Gilles
Published: June 2021

Renal functions are regulated to cope with the demands of the organism through activation of different receptors. Among them, the family of the nuclear receptors (NR) participates at the long‐term processes. Identity of the NR expressed in the kidney is known, but their quantitative localization in this ultrastructured organ remained poorly described. Using qPCR on microdissected mouse renal nephronic segments, we established a quantitative expression map of NR along the nephron. This map can serve to identify NR with specific localization. Thus, we unexpectedly found that the estrogen‐related receptor β (ERRβ) is expressed predominantly in the Henle loop (TAL). In vivo treatment with an ERR inverse agonist (diethylstilbestrol) showed a link between this receptor family and the expression of NKCC2, and resulted in phenotype presenting some similarities with the Bartter syndrom (hypokalemia, urinary Na+ loss and volume contraction). We then used a mouse TAL cell line to dissect the mechanisms by which NKCC2 and other transporters are modulated after stimulation of ERRβ with a selective agonist (GSK4716) using CHiP experiments.

The TAL segments being involved in the regulation of extracellular volume, blood pressure, Ca2+ and Mg2+ homeostasis, identification of ERRβ as a new regulator of ion transport in this particular segments may contribute to better understand dysfunctions of these parameters.
Relationship between the length of class and student success in a comparative vertebrate zoology course: a case study
Authors: Zinkevich Natalya S
Published: June 2021

Numerous studies have demonstrated that the attention span of a typical student is about 10 to 15 minutes, while most university classes last at least 50 minutes, arguing against long lectures as a pedagogical strategy. Active learning approaches have been shown to engage students and improve the retention of material learned; however it is unclear what the optimal length of the class should be. Here, we compare student success in a comparative vertebrate zoology class that met once per week (total class length: 3 hours 30 minutes with 10 minute breaks after 50 minutes intervals; 7 students) and one that met four times a week (class length: 50 minutes; 16 students). The class was taught during the spring and fall semesters, 2012, by the same instructor with a comparable variety of active learning approaches. Final and intermediate course grades will be compared to assess student success. Student achievement in the laboratory portion of the course, which had a comparable structure in the two semesters (class length: 3 hours 50 minutes) will be used as a control. Qualitative results from student evaluations will also be presented.
Evolution of Vertebrate Limb Morphology
Authors: Tabin Clifford James
Published: June 2021

The vertebrate limb is an extraordinarily flexible structure that has evolved to accomplish a wide range of tasks in different taxa, from the grasping human hand, to the wing of the bat, the digging limb of the mole or the running leg of the horse. To understand the developmental basis for such diversity, we are focusing on the diverged morphology of the hindlimbs of jerboas, bipedal Asian rodents closely related to mice. These hindlimbs show a number of derived characteristics, including greatly increased size, altered allometric growth of individual elements (such as the metatarsals which are extended to a length similar to that of the femur or tibia); fusion of elements, and reduction in number of digits. We have found that changes in relative growth of the skeletal elements are achieved, to a large extent, by modulating the size of the individual hypertrophic cells in the developing growth plate, a multi‐phasic process involving cell swelling as well as proportional cell growth. It is the final phase of proportional mass increase, an IGF1‐dependent step, that appears to be modulated evolutionarily as skeletal element size is varied. Decrease in digit number has also been investigated in these animals. The normal set of five digit primordia appear to be specified in the jerboa hindlimbs as they are in the mouse. However, the lateral digits are lost through an expansion of cell death, regulated by Msx2 and Bmp4. A similar mechanism appears to have been used in the evolution of the single central digit of the horse.
TRAINING 3‐YEAR STUDENT ON EVIDENCE‐BASED MEDICINE
Authors: Tuleshova Gulnara , Abduldayeva Aigul , Derbissalina Gulmira , Delellis Nailya , Êàrp Leonid
Published: June 2021

Modernization of medical education in the Republic of Kazakhstan in the light of the Bologna process envisages the need for staff training of the new formation. In this regard, the ongoing reform of medical education in Kazakhstan focuses on the quality of professional graduates training of medical schools. In 2006, a new national state standard of medical education of the Republic of Kazakhstan was worked out, where the final goals of specialist training in the public health service at diverse education stages are clearly marked. The peculiarities of SES‐2006 are: the transition to 3‐level education, a focus on competence‐orient education, availability of components for the selection and the emergence of new disciplines, including such disciplines as “Evidence‐based medicine”, “Communication skills”, “Biostatistics” “Epidemiology”. The Knowledge of the issues of evidence ‐ based medicine at the present is necessary for each clinician. Medical students in training must learn to think and work in accordance with the principles of EBM.

The Purpose of training on “Basics of EBM” is the creation of knowledge bases of EM, the formation of skills that will permit to evaluate medical information critically for rational use in further practice. The objectives of the training are to acquaint students with the basic terms of EM, the main types of clinical research, usingmethods of EM database, to develop the ability.
Impact of evidence‐based innovative interventions on students’ performance in a Nigerian medical school
Authors: Anyaehie Ugochukwu Bond , Okeke Tochukwu
Published: June 2021

Need for periodic evaluation of students’ performance and observed initial findings of poor performance and students preference of peer‐tutoring to didactic lectures at the University of Nigeria Medical School, we implemented innovations at improving learning environments for Physiology in our institution. These innovations included securing the services of Faculty clinicians to augment existing number of lecturers, use of question‐driven format for assessments, evidence‐based change in tutorial format and post‐results interactive review of continuous assessment questions. Present study is a retrospective review of medical student's performance in 2nd MBBS examinations physiology pro and past the innovations. Data were collected and analyzed by descriptive and inferential statistics using the statistical software for social sciences (SPSS) version 15. Chi‐square test (x2) was used to determine the association between the student's performance before and after our interventions. Furthermore binary logistic regression was equally used to determine the association between student's performance before and after. The level of significance was set at p< 0.05. A total of 2152 students sat for the professional examination over the study period, and 1485 students passed the examination at first attempt giving an overall pass rate of 69%. The pass rate from 2008 when our interventions started was significantly higher than the pass rate before this reform (2004 – 2007) [(76.4%), (n =1646) vs 63.4% (n=1364); P<0.0001]. There was significant association also when the model was adjusted for after 2008 (OR, 1.901, 95% CI (1.566–2.307), P=O.001. Results support the call for institutional leadership by clinical investigators and PhD physiologists with strong translational interests, evidence‐based innovative alternatives to passive lecture formats and students involvement in program evaluation.
Contrasting aspects of university instructor/student e‐mail usage in teaching activities
Authors: El‐Sabban Farouk
Published: June 2021

Use of e‐mail is very common in every day life of many people, among which are those in the academic community. University students and instructors are proficient users of e‐mail and this aspect can prove beneficial in teaching activities and in cross communication. Data presented herein are based on 5 years of experience in using e‐mail in teaching science and non‐science courses at the College for Women, Kuwait University. The course instructor sets a separate e‐mail account for each course being taught with an easy password for students to open. Its main purpose is for the instructor to disseminate relevant course information, materials and announcements. Students are advised clearly not to use this e‐mail for communicating with the course instructor or for their own personal purposes, but use their personal e‐mail accounts to communicate with the course instructor – to avoid intersecting messages. Preliminary analysis of e‐mail messages show that main usage by instructor was to sent (%): handouts and syllabi (32), assignment‐related (25), reminders and notices (20), materials of interest (13). In contrast, received messages from students revealed that they were (%): assignment‐related (41), and other than about assignments (59). Assignment‐related messages from students were about (%): requesting postponement of deadline for assignment submission (39), sending assignments as attachments to messages (35), apology for late submission of assignments (17), and inquiries about assignments (9). Based on the instructor's experience, e‐mail usage in teaching achieved a satisfactory level and is encouraged. Also, its benefits and effectiveness are to be assessed through well‐planned studies that can enrich the emerging relevant literature.
Training Efficient Nutrition Counseling in Virtual Reality
Authors: Kohlmeier Martin , Adams Kelly , Morris Evan , Powell Margo
Published: June 2021

Healthcare providers are tasked with providing nutrition guidance to their patients, something they are neither taught adequately nor given an opportunity to improve their counseling skill under supervision. Unmentored learning on the job is unlikely to help them conduct effective and efficient sessions. We have created an online virtual reality (VR) environment where learners can practice how to counsel patients about lifestyle change that lowers their blood cholesterol levels.

Seamless patient video responds to user prompts. Input items support selection from a range of domains or sub domains, thus allowing the user to freely navigate through content space just like in a natural dialog. The patient responses depend both on the selected topic and the specific phrasing of the user's input. Diverse patient scenarios with differences in stage of change and attitude allow multiple sessions without repetition of the dialogs. The user can select a training mode of the application and get continuous visual feedback from a dashboard. The gauges indicate accuracy of the user's nutrition guidance, consideration of the patient's stage of change, and reliance on motivational interviewing techniques. The sessions are timed to emphasize the importance of efficient interactions. After each counseling session, the application shows a dialog transcript linked to a display of the continuously recorded performance metrics.