bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA Regulation of Hsp70.3 Protein Expression in Late Ischemic Preconditioning
Authors: Helsley Robert Nathaniel , Tranter Michael , Paulding Waltke , Ren Xiaoping , McGuinness Myc , Jones Keith
Published: June 2021

Introduction

We showed that the Hsp70.3 isoform is induced by NF‐κB after late ischemic preconditioning (IPC) and contributes to cardioprotection. However, the nearly identical Hsp70.1 isoform does not contribute to IPC induced cardioprotection. We propose that the functional differences between the twoHSP70s are due to post‐transcriptional regulation involving the 3' UTRs, which are divergent.

Results

Though NF‐κB inhibition completely reduces protein expression, it only partially reduces Hsp70.3 mRNA levels, showing that Hsp70.3 is regulated by both pre‐ and post‐transcriptional mechanisms after IPC. We hypothesized that an IPC stimulus reduces expression of Hsp70.3‐suppressing miRNA(s) allowing for post‐transcriptional stabilization of Hsp70.3 mRNA and increased Hsp70.3 protein levels. We assessed in vivo levels of 356 miRNAs in a mouse model of IPC, to identify miRNAs that are; 1) downregulated after IPC, and 2) predicted to target Hsp70.3. We identified miR‐378* as the only miRNA of those surveyed to meet these criteria and demonstrated functional inhibition of Hsp70.3 by miR‐378*.

Discussion

This work demonstrates post‐transcriptional (via miRNA) regulation of Hsp70.3. It appears that coordinated transcriptional upregulation (promoter) as well as suppression of targeting miRNAs are involved in increasing Hsp70.3 protein expression after IPC. This work was supported by the American Society of Pharmacology and Experimental Therapeutics (ASPET) with funds generously provided by ASPET and the University of Cincinnati.
Dual Allosteric Modulators of Neuronal Nicotinic‐Acetylcholine and GABA‐A Receptors
Authors: Gee Kelvin W , Johnstone Timothy , Hogenkamp Derk J , Yoshimura Ryan , Whittemore Edward R , Gu Zhenglin , Yakel Jerrel
Published: June 2021

Chemically induced LTP recorded in the CA1 hippocampal region can be induced by simultaneous activation of α7 nACh and inhibition of GABAA α5 receptors. This effect can be replicated by co‐applying molecules that exhibit similar activities at either receptor but not when each is applied alone. Our lab has designed an allosteric modulator that simultaneously modulates γ‐aminobutyric acid‐A (GABAA) α5 AND α7 neuronal nicotinic‐acetylcholine receptors (nAChRs). This molecule designated 522‐054 is a proposed lead molecule for treating Alzheimer's disease with putative cognition enhancing properties derived from modulation of both GABAA and nAChRs. 522‐054 is a positive allosteric modulator of α7 nAChRs and negative allosteric modulator of GABAA α5 receptors. 522‐054 enhances CA1 hippocampal output after mossy fiber stimulation and enhances CA1 EPSCs to the point of LTP like that observed following high frequency tetanic stimulation. 522‐054 evoked LTP in CA1 region is differentially blocked by α7 nAChR or GABAA α5 receptor antagonism. In the radial arm maze, the compound enhances cognition as measured by working memory errors and % correct performance.
RACK1 regulates PP2A‐mediated de‐phosphorylation of threonine 38 of the human nuclear xenobiotic receptor CAR
Authors: Mutoh Shingo , Osabe Makoto , Moore Rickie , Vinal Kellie , Sueyoshi Tatsuya , Negishi Masahiko
Published: June 2021

Before activation by therapeutics, the human nuclear xenobiotic receptor hCAR is sequestered in the cytoplasm, by phosphorylating threonine 38 (T38) within its DNA binding domain. Phenobarbital, the classic hCAR activator, de‐phosphorylates T38 to translocate hCAR into the nucleus (Mutoh, et al., J. Biol. Chem. in press). Given that T 38 is the target of de‐phopshorylation, we are now investigating the molecular mechanism by which T38 can be de‐phosphorylated. Subsequently, receptor for activated C kinase 1 (RACK1) has been identified as a protein that specifically interacts with the phosphorylated form of hCAR (T38D mutant of hCAR); by employing yeast two hybrid screening and co‐immunoprecipitation assays. T38 of recombinant hCAR protein was in vitro phosphorylated by pure protein kinase C and was used as the substrate for an in vitro de‐phosphorylation assay by pure protein phosphatase 2A (PP2A). PP2A was then found to de‐phosphorylate T38 in the presence of RACK1. To substantiate these roles for RACK1 and PP2A, whole cell extracts, prepared from Huh7 cells treated with siRNA RACK1 and siRNA PP2Ac, were incubated with recombinant hCAR proteins as the enzyme sources for phosphorylation. Both knock downs of RACK1 and of PP2Ac resulted in an increased phosphorylation of T38. These results indicate that PP2A de‐phosphorylates T38 and RACK1 regulates this PP2A‐mediated de‐phosphorylation.
A Novel Role for Ser25 in Regulating Class A Scavenger Receptor Trafficking
Authors: Nikolic Dejan Miodrag , Cholewa Jill , Post Steven R
Published: June 2021

Class A scavenger receptors (SRA) are cell‐surface macrophage proteins that mediate ligand internalization and cell adhesion. Previous studies indicate that SRA activates and is regulated by PI3‐kinase (PI3K). The importance of specific structural determinants of SRA has not been well defined. In this study, we investigated the role of putative phosphorylation sites in the cytoplasmic tail of SRA in regulating SRA function (internalization and adhesion). Putative phosphorylation sites at S25 (PKC), S46 (CK‐II and PKC) and S53 (PKC) were identified, substituted with alanine, and the function and regulation of mutant receptors studied in transfected HEK cells. Changing S46 or S53 had no discernible effect on the function or regulation of SRA. Similarly, changing S25 did not alter ligand internalization, PI3K activation, or cell adhesion. However, mutating S25 abolished the PI3K‐sensitive regulation of surface SRA localization. We next investigated whether inhibiting PKC mimicked the effect of mutating S25. Indeed, treatment with a selective PKC inhibitor (Bis I) abrogated the PI3K dependency of SRA surface localization in both HEK cells expressing wild type SRA and in peritoneal macrophages, which endogenously express SRA. Overall, our results indicate that phosphorylation of S25 by PKC modulates the requirement for PI3K in the translocation of SRA to the cell surface.
Gender Differences in Cardiac Responses to Catecholamine Stress in Caloric Restricted Mice
Authors: Yan Lin , Gao Shumin , Lai Lo , Ge Hui , Hong Chull , Vatner Dorothy E. , Vatner Stephen F.
Published: June 2021

Caloric restriction (CR) is the most extensively studied intervention that extends longevity and protects against stress. Generally, women show greater longevity and stress protection than men. We examined gender differences in response to the stress of chronic isoproterenol (ISO) infusion (60mg/kg/day for 1 week), delivered by subcutaneous mini‐osmotic pumps, in young male and female mice (3–6 months old, n=13/group) subjected to 6 weeks of CR. LV ejection fraction (LVEF) was similar in female and male mice with CR (70%±2% vs 71%±2%). Surprisingly, at 1 week after ISO, LVEF in CR female mice was lower than that in CR male mice (37%±4% vs 50%±3%, p<0.05), and the females began to develop heart failure, as reflected by higher lung/body weight (8.6±0.4) compared with males (7.9±0.1). Furthermore, ISO‐induced apoptosis was much greater, p<0.05, in CR females (1.67±0.23 positive cells/mm2) than in CR males (0.84±0.17 positive cells/mm2). In contrast to the well established cardiac protection in pre‐menopausal females, our results indicate that CR female hearts demonstrated less tolerance to chronic catecholamine stress than CR male hearts.
Apolipoprotein Mimetic Peptide (L‐4F) Regulation of Adiposity via Increases in Estradiol/Testosterone Ratio in Obese Female Mice
Authors: Burgess Angela Patricia Haynes , Li Ming , Kim Dong Hyun , Vanella Luca , Palladino Leah , Angevine Kristine , Mensah‐Osman Edith , Abraham Nader
Published: June 2021

During obesity, women are more resistant to weight loss, have more inflammation, and are at a higher potential risk for cardiovascular disease than men. Our hypothesis is these differences are due to abnormalities in estradiol/testosterone ratio, adiponectin, and inflammatory cytokines generated by adipocytes. Obese (ob) male and female and wildtype (wt) male and female mice were administered apolipoprotein mimetic peptide (L‐4F) (50 mg/kg daily) or vehicle for 6 weeks. L‐4F is an inducer of HO‐1, and serum levels of estradiol, testosterone, adiponectin, TNFα, and IL‐6 and vascular levels of HO‐1 were measured. Wt female had increased HO‐1 protein and HO activity compared to wt male. However, ob female had lower HO‐1 protein than male ob which was associated with decreased adiponectin and three‐fold higher levels of IL‐6. These findings were ameliorated with L‐4F treatment. L‐4F led to a reduction of visceral and subcutaneous fat, and decreased weight in ob males, but not in ob females. Low plasma estradiol levels in ob females were increased after L‐4F treatment. These novel findings indicated that females were more resistant to weight loss than males due to pre‐existing highly inflammatory environment and reduced estradiol/testosterone ratio. Additionally, L‐4F can positively regulate hormonal and metabolic changes associated with complications of diabetes and metabolic syndrome. Funding support include NIH grants DK‐068134, HL‐55601, and HL‐34300.
T‐lymphocytes contribute to angiotensin II‐mediated thrombosis in cremaster muscle arterioles
Authors: Senchenkova Elena Y. , Granger D. Neil
Published: June 2021

While there is mounting evidence that chronic arterial hypertension is associated with altered hemostatic, inflammatory and immune systems, the nature of the interaction between these systems remain poorly understood. The aim of this study was to assess the contribution of T cells and cytokines (TNF‐α, IFN‐γ, IL‐6) to the arteriolar thrombosis induced by AngII infusion (2 wks, 1 ug/kg/min). Intravital microscopy was used to quantify the time to thrombus initiation and to flow cessation in murine cremaster muscle arterioles exposed to light/dye activation. AngII‐enhanced thrombus development was evaluated in the following experimental groups: WT mice, immunodeficient Rag‐1−/−mice, Rag‐1−/−reconstituted with T cells from WT donors, and in the following cytokine‐deficient mice: TNF‐α −/−, IFN‐γ −/− and IL‐6−/−mice. While WT mice exhibited accelerated thrombosis in response to AngII infusion, this was not observed in Rag–1−/− mice. Adoptive transfer of WT T cells into Rag‐1−/−mice restored the AngII‐induced thrombus response. IFN‐γ −/− mice exhibited a thrombosis response similar to WT mice. The time to thrombus initiation was extended in TNF‐α −/−mice. However, AngII did not induce an acceleration of either phase of thrombus formation in IL‐6−/− mice. Our study provides novel evidence implicating both T‐lymphocytes and IL‐6 in the accelerated thrombosis associated with AngII‐induced hypertension. (HL26441).
Early Pattering in the Zebrafish Inner Ear
Authors: Riley Bruce B , Padanad Mahesh
Published: June 2021

It is well established that Fgf signaling is essential for otic placode induction, but downstream mechanisms are still poorly characterized. In zebrafish, pax8 expression marks an early response to Fgf signaling. We show that pax8 mediates two key functions. First, it is necessary and sufficient to activate expression of fgf24 in newly specified otic cells. This appears to expand the otic field through ongoing recruitment. Second, pax8 cell‐autonomously represses foxi1, one of the co‐factors initially required to activate pax8. In addition to activation pax8, foxi1 plus Fgf also activate sox3, though in broader domain including both otic and epibranchial placodes. We find that failure to down‐regulate Foxi1 in prospective otic cells causes them to transfate to epibranchial fate. Thus, pax8 simultaneously maintains otic fate by blocking epibranchial fate and expands the otic territory by augmenting Fgf signaling.

Supported by NIH‐NIDCD R01‐DC03806.
Angiotensin II Induced Cerebral Microvascular Inflammation and Increased Blood‐Brain Barrier Permeability via Oxidative Stress
Authors: Zhang Ming , Mao Yingying , Tuma Ronald F , Chabrashvili Tinatin
Published: June 2021

Although angiotensin II (Ang II) has been shown to cause endothelial dysfunction, its role in inflammatory responses, especially in brain, remains unclear. The goal of this study is to investigate the mechanisms through which Ang II mediates cerebral microvascular inflammation. C57BL/6 male mice were subjected to slow pressor dose of Ang II infusion (Alzet mini‐pump) ata rate of 400ng/kg/min for 14 days. Intravital microscopic observation was used to investigate the leukocyte/endothelial interactions in pial microvessels in vivo. A superoxide scavenger 4‐hydroxy‐TEMPO (Tempol) was added to drinking water (2mmol) on day 4th following Ang II infusion. Blood‐brain barrier (BBB) permeability was evaluated by Evans Blue extravasation ex vivo. Ang II increased (p<0.01) the leukocyte adhesion on day 4th of infusion compared to vehicle. This effect was maintained on day 14th (p<0.05) and also was accompanied with an increase of leukocyte rolling (p<0.01). Ang II increased the BBB permeability on day 14th (p<0.05). Tempol attenuated the increased leukocyte/endothelial interactions and protected the BBB integrity (p<0.01). In conclusion, Ang II causes cerebral microvasculature inflammation, which leads to the increased BBB permeability. This effect of Ang II is, at least in part mediated via oxidative stress since Tempol reverses the leukocyte rolling and adhesion, and prevents the increased BBB permeability.
Inhibition of protein kinase C beta II attenuates local hyperglycemia‐induced leukocyte‐endothelial interactions
Authors: Chen Qian , Iames Edward , Torino Daniel , Perkins Kerry‐Anne , Rueter Brian , Prince Catherine , Young Lindon Howard
Published: June 2021

Hyperglycemia is causally related to the vascular complications of diabetes. This patho‐physiological process may be mediated by the activation of protein kinase C (PKC) beta II resulting in an increased oxidative stress and inflammatory response. However, the role of a PKC beta II inhibitor regulating hyperglycemia‐induced endothelial‐leukocyte interactions has not been evaluated in vivo in real‐time. The effect of PKC beta II peptide inhibitor on leukocyte‐endothelial interactions in rat mesenteric postcapillary venule circulation was determined by using intravital microscopy. We found that intraperitoneal injection of 25 mM D‐glucose (2.5 ml, n=6) caused intraperitoneal hyperglycemia and exhibited significantly higher leukocyte rolling, adherence, and transmigration compared to control rats which were intraperitoneal injection of saline (2.5 ml, n=4, P<0.05). By contrast, intraperitoneal injection of D‐glucose with 10 mM PKC beta II inhibitor (12.5 μl, n=3/25.0 μl, n=6) induced the similar intraperitoneal hyperglycemia, but showed significantly less leukocyte‐endothelial interactions compared to D‐glucose injection (P<0.05). In summary, the PKC beta II inhibitor attenuates local hyperglycemia‐induced leukocyte‐endothelial interactions, which may be related to the attenuation of oxidative stress in blood. This study was supported by Center for Chronic Disorders of Aging at PCOM.
TNFα compromises the inner ear microcirculation in a sphingosine kinase 1/sphingosine‐1‐phosphate dependent manner ‐ a novel mechanism for sudden hearing loss (SHL)
Authors: Scherer Elias Q. , Yang Jingli , Ivanov Karolina , Diehl Christian David , Reimann Katrin , Wangemann Philine , Pohl Ulrich , Canis Martin , Strieth Sebastian , Voigtlaender‐Bolz Julia , Lidington Darcy , Bolz Steffen‐Sebastian
Published: June 2021

This study establishes a link between inflammation and inner ear vascular dysfunction. Recovery of auditory function in of SHL patients treated with a TNFα inhibitor were consistent with a vascular origin. We investigated the inner ear microcirculation using (1) an in vitro model of the spiral modiolar artery (SMA), the end artery feeding the inner ear, (2) intra vital microscopy of stria vascularis (SV) perfusion, and (3) in vitro measurement of cochlear lateral wall capillary (LWC) constriction. We demonstrate that in all parts of the cochlear microcirculation (SMA, SV and LWC), TNFα induces a proconstrictive state via activation of sphingosine‐1‐phosphate (S1P). Analysis of the molecular signalling pathway identified the phosphorylation of sphingosine kinase 1 (the S1P generating enzyme activated by TNFα) as a new therapeutic target for SHL. We conclude that any pathology linked to the release of TNFα has the potential to reduce cochlear blood flow and cause SHL. The present study integrates SHL into the family of cardiovascular pathologies, with immediate implications related to risk stratification, diagnosis and treatment. CIHR‐MOP‐84402, CFI‐11810, ORF‐11810, CSN, KRICRF‐TUM‐8758155, HSFO‐NI, HSFO‐CI, SFUT, Boehringer Ingelheim, DAA, NIH‐R01‐DC04280.
Caveolae/lipid raft modulates TNF‐alpha‐induced upregulation of cell adhesion molecules in human brain endothelial cells
Authors: Eum Sung Yong , Andras Ibolya E , Choi Yean Jung , Hennig Bernhard , Toborek Michal
Published: June 2021

The blood‐brain barrier (BBB) is a physical and metabolic barrier separating the microenvironment of the central nervous system (CNS) from the peripheral circulation. Accumulating evidence suggests that TNF‐α plays a pivotal role in the activation of brain endothelium leading to the disruption of BBB and neuroinflammation. Exposure of human brain endothelial cells to TNF‐α increased expression of ICAM‐1 and VCAM‐1as well as elevated adhesion of brain endothelial cells to leukocytes. These effects were blocked by disruption of caveolae/lipid raft structure by cholesterol depleting agents or silencing of caveolin‐1. In addition, TNF‐α‐induced upregulation of CAMs was impeded by inhibitors to caveolae/lipid raft‐associated signaling molecules, such as Src kinase, NADPH oxidases (NOX), and nitric oxide synthases. The results in this study suggest that TNF‐alpha induces CAMs expression in human brain endothelium through NOX‐mediated generation of reactive oxygen species in caveolae/lipid raft domain. Therefore, selective targeting of lipid raft/caveolae‐mediated NOX in TNF‐alpha‐dependent brain endothelial inflammation may lessen adverse effects of neuroinflammation as well as neurodegenerative diseases. Supported in part by NIH grants (P42 ES 07380, MH63022, MH072567, and NS39254) and the American Heart Association (09SDG2300037).
AT1 receptor‐ independent oxidative stress in angiotensin II (Ang II) infused Male Spontaneously Hypertensive Rats (SHR)
Authors: Bhatia Kanchan , Sullivan Jennifer C
Published: June 2021

The AT1‐receptor blocker, Candesartan lowers Ang II induced hypertension and oxidative stress. We hypothesize that Ang II/AT1 receptor induces oxidative stress via NADPH oxidase. 12 week old rats were divided into 4 groups control, Ang II‐infused (200ng/kg/min, 14 days minipump), Ang II+Candesartan (10 mg/kg/day, 14 days in drinking water), Ang II+Tempol (30 mg/kg/day, 14 days in drinking water). The renal cortex was isolated to measure NADPH oxidase and O2.‐ levels via lucigenin. Ang II‐infusion significantly increased NADPH activity compared to control (733±26 vs 260±13, p<0.001). Tempol lowered NADPH activity (401±20, p<0.01) but Candesartan was comparable in AngII SHR (727±38). Ang II increased basal O2.‐ in SHR (10.±1, p<0.05). Co‐treatment with Candesartan and Tempol lowered O2.‐ levels (5.4±0.5,p<0.01; 1.0±0.9,p<0.01). Cortical TBARS levels were comparable in all groups. 8‐isoprostane levels increased with Ang II (68±6 vs 35±4., p<0.01) and was comparable in Candesartan and Tempol groups. Western blot analysis showed Ang II decreased Nox 4 expression (4.6±0.3 rdu vs 3.5±0.2 rdu, p<0.05) whereas rest of the groups were comparable. Subunits of NADPH oxidase, catalase and SOD were comparable in all groups. We conclude that Ang II induces oxidative stress independent of AT1 receptor via NADPH oxidase suggesting an alternate mechanism. Funded by NIH.
Physiological regulation of ENaC activity in the distal nephron by local bradykinin signaling
Authors: Pochynyuk Oleh
Published: June 2021

ENaC activity in the aldosterone‐sensitive distal nephron sets sodium plasma levels to control blood pressure in response to negative feedback regulation by the renin‐angiotensin‐aldosterone system. In complement to this, paracrine factors intrinsic to the collecting duct, including bradykinin (BK), may also have an important role in controlling ENaC‐mediated sodium reabsorption. Patch clamp electrophysiology on freshly isolated murine collecting ducts (CD) was used to test whether ENaC is a proximal target for bradykinin signaling. Bradykinin acutely and reversibly decreases ENaC open probability via B2 receptors: Inhibition of B2 receptors with HOE‐140 prevents BK‐mediated decreases in ENaC Po. Disruption of this paracrine signaling in B2 −/− mice abolishes BK actions on ENaC and results in elevated resting ENaC Po. We propose that sodium retention due to increased basal ENaC activity contributes, in part, to the salt‐sensitive hypertension in B2 −/− mice. B2 receptors couple to both Gq/11‐PLC, and Gαi‐mediating decreases in adenylyl cyclase activity. Inhibition of PLC with U73122 but not saturation of intracellular [cAMP] with 8‐cpt‐cAMP disrupts regulation of ENaC by BK. Rapid decreases in apical PI(4,5)P2 levels in response to BK stimulation parallel decreases in ENaC activity. Inhibition of Ca2+ release from the ER does not prevent decreases in ENaC activity in response to bradykinin. This is consistent with decreases in PI(4,5)P2 levels but not subsequent increases in intracellular Ca2+ account for BK‐mediated decreases in ENaC activity in response to stimulation of B2‐Gq/11‐PLC.
Role of the cAMP/PKA signaling cascade in vasopressin‐induced trafficking of TRPC3 channels in principal cells of the collecting duct
Authors: Goel Monu , Zuo Cheng‐Di , Schilling William P.
Published: June 2021

The transient receptor potential channels TRPC3 and TRPC6 are abundantly expressed in principal cells of the collecting duct (CD) along with the water channel, aquaporin‐2 (AQP2). All three channels localize to intracellular vesicles, but upon stimulation with arginine‐vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined in principal cells of the CD. Translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino‐Cys1, D‐Arg8]‐vasopressin (dDAVP), a specific V2‐receptor agonist, and blocked by [adamantaneacetyl1, O‐Et‐D‐Tyr2, Val4, Aminobutyryl6, Arg8,9]‐vasopressin (AEAVP), a specific V2 receptor antagonist. Translocation was also activated by forskolin, a direct activator of AC, or by di‐butyryl‐cAMP, a membrane‐permeable cAMP analog. AVP‐, dDAVP‐, and forskolin‐induced translocation was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by di‐butyryl‐cAMP was unaffected by AEAVP, but could be blocked by H89. Taken together, these results demonstrate that AVP stimulation of V2‐receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade. Supported in part by HL097355.
Caveolin‐1 mediates constitutive NKCC2 endocytosis in thick ascending limbs
Authors: Ares Gustavo Rosier , Ortiz Pablo A.
Published: June 2021

The Na/K/2Cl cotransporter (NKCC2) undergoes endocytosis in thick ascending limbs (TALs) in the absence of hormonal stimulation (defined as constitutive endocytosis). The caveolin‐1 (Cav‐1)‐mediated endocytic pathway is important in protein trafficking in other cells. We hypothesized that constitutive NKCC2 endocytosis in TALs is mediated in part by Cav‐1. To test our hypothesis we measured: localization of Cav‐1, the effect of Cav‐1 siRNA (chronic inhibition) and filipin III (acute inhibition) on constitutive NKCC2 endocytosis. Endocytosis was measured by biotinylation and Western blot. We found that Cav‐1 as well as NKCC2 are expressed in the apical membrane and sub‐apical space of rat TALs. Cav‐1 siRNA decreased Cav‐1 expression in TALs by 60 ± 12% at 3 days. In control TALs transduced with a scramble siRNA, constitutive NKCC2 endocytosis averaged 1.05 ± 0.08 a.u/min. However, in TALs transduced with Cav‐1 siRNA, NKCC2 endocytosis was reduced to 0.75 ± 0.05 a.u/min (p < 0.05 vs scramble), a 30% decrease. Moreover, in the absence of filipin III, NKCC2 endocytosis averaged 1.02 ± 0.23 a.u/min. In TALs incubated with filipin III, the rate of NKCC2 endocytosis was blocked to 0.07 ± 0.03 a.u/min (p < 0.05 vs vehicle) a 93% decrease. Neither Cav‐1 siRNA nor filipin III affected the total NKCC2 pool. We concluded that constitutive NKCC2 endocytosis in THALs is mediated in part by a Cav‐1 dependent process.
Hypertension Resistance Polymorphisms in ROMK Alter Channel Function by Different Mechanisms
Authors: Fang Liang , Li Dimin , Welling Paul A
Published: June 2021

The ROMK potassium channel plays a critical role in renal sodium handling. Genome sequencing efforts in the Framingham Heart Study cohort recently revealed an association between suspected loss‐of‐function polymorphisms in ROMK and resistance to hypertension, suggesting that ROMK activity is also a determinant of blood pressure in the general population. Here, we examine whether these sequence variants do, in fact, alter ROMK function and explore the mechanisms. As assessed by two‐microelectrode voltage clamp in Xenopus Oocytes, 3/5 of the variants (R193P, H251Y and T313FS) displayed an almost complete attenuation of whole‐cell ROMK channel activity. Surface antibody binding to external epitope tagged channels and analysis of glycosylation‐state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of ER retention. The other variants (P166S, R169H) had no obvious effect on the basal channel activity or surface expression but, instead, displayed a regulated‐gating defect. Apparent PIP2 binding affinity of the variants was reduced, causing channels to become susceptible to inhibition by GPCR‐mediated stimulation of PIP2 hydrolysis. In summary, hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in blood pressure control.
Kcnmb4−/− mice have decreased tubular dilation under high urinary flow
Authors: Holtzclaw J David , Grimm P Richard , Sansom Steven C.
Published: June 2021

Our laboratory has shown that with volume expansion, flow through the tubules increased by 10‐fold as Na and water reabsorption was inhibited. How the renal tubules accommodate the very large increases in tubular flow has never been answered. We propose that during high tubular flow, tubular cross‐sectional area (CA) is increased by reducing the size of the cells lining the nephron. The large‐conductance, calcium‐activated K channels (BK) can play a role in cell volume regulation. Furthermore, BK are expressed in several nephron segments including the thick ascending limb (TAL), distal convoluted tubule (DCT), connecting tubule (CNT), and cortical collecting duct (CCD). We placed wild type (WT) and BK‐β4−/− (Kcnmb4−/−) mice on a normal (0.6% K) or K‐adapted (5% K or KA) diet for 10 days and measured tubular CA of the proximal tubule (PCT), TAL, CNT, and CCD. Immunohistochemical analysis with segment specific antibodies revealed that KA mice had significantly increased CA of all tubular segments compared to phenotype matched controls on a normal diet. However, the CA of the TAL, CNT, and CCD of the KA Kcnmb4−/− were significantly less than that of the KA WT mice (TAL: 174.4 vs 203.3; CNT: 278.1 vs 444.0; CCD: 901.6 vs 1401.0 μm2) indicating an inability of the Kcnmb4−/− tubules to fully dilate. No edema was present in the KA kidneys. These results reveal that increased flow leads to tubular dilation by a BK‐β4 dependent mechanism. Supported by NIH/NIDDK grant R01DK71014 (SCS).
Role of GlyT2 expressing neurons in the Bötzinger Complex for respiratory rhythm and pattern generation
Authors: Abdala Ana Paula Lima , Liu Beihui H. , Rybak Ilya A. , Smith Jeffrey C. , Paton Julian F. R.
Published: June 2021

Computational modeling of the respiratory network and experimental data both suggest that reciprocal inhibition between post‐inspiatory (post‐I) and augmenting expiratory neurons is essential for normal breathing: e.g. global blockade of glycinergic receptors abolishes post‐I activity in motor outflow. Here, we found a way to selectively inactivate glycinergic neurons in the Bötzinger Complex (BötC). For that, we generated a novel lentiviral vector to specifically express allatostatin receptors (AstR) under the control of the glycine transporter‐2 (GlyT2) gene promoter, which is a reliable marker for glycinergic neurons. Binding of exogenously applied allatostatin (AL) to the AlstR inhibits the target neurons. The lentivirus was injected bilaterally (109 pfu) into the BötC of Wistar rats. After 10 days, we simultaneously recorded phrenic (PN), hypoglossal (HN) and central vagal (cVN) nerves in these rats in situ. We applied AL (1 μM) systemically to selectively inhibit glycinergic neurons in the BötC, which markedly reduced PN amplitude changing its pattern from a ramp to a ‘square‐wave’ or ‘bell’ shape. AL also reduced the post‐I component in the cVN and increased respiratory rate. These effects were reversed by washing out AL. We conclude that GlyT2 expressing neurons in the BötC are critical for generation of an eupneic‐like respiratory pattern with the normally expressed post‐I activity. Support NIH R01 NS057815.
Oxygen Uptake Efficiency Plateau during Cardiopulmonary Exercise Testing Is Best Predictor of Early Death in Heart Failure Patients
Authors: Sun Xingguo , Hansen James E
Published: June 2021

Our goal was to identify better predictors of early death in patients with chronic left ventricular heart failure (CHF). Potential predictors, derived from cardiopulmonary exercise tests (CPX), were compared with other commonly used cardiovascular measurements. The prediction of early death in CHF patients remains challenging.

METHODS & RESULTS

508 patients with CHF due to systolic dysfunction had resting cardiovascular measurements, six‐minute walking tests and CPX. The peak O2 uptake (VO2), peak O2 pulse, anaerobic threshold (AT), ratio of ventilation to CO2 output (VE/VCO2), VE‐vs‐VCO2 slope, and oxygen uptake efficiency plateau (OUEP, i.e. Highest VO2/VE) were ascertained. Outcomes were six‐month mortality (all cause death). During six‐month follow‐up, 19 patients died and 489 patients survived. The single best predictor of early mortality was the decreased OUEP (<65%pred)(OR=12.8). The lowest VE/VCO2, VE‐vs‐VCO2 slope, peak VO2, AT and peak O2 pulse alone were strong predictors, their best OR values were 9.4, 7.6, 6.0, 5.1 and 4.3 respectively (p<0.001). No non‐exercise measurements (i.e. NYHA, QRS duration, ejection fraction, blood pressure, cardiac output and stroke volume) discriminated mortality.

CONCLUSIONS

CPX parameters are powerful prognosticators of early mortality in CHF patients, especially the reduced OUEP.
Orthostatic Tolerance and Breast High Compression Garments
Authors: Evans Joyce M , Schneider Rebecca S , Platts Steven H , Brown Angela K , Stenger Michael B , Knapp Charles F
Published: June 2021

Orthostatic intolerance (OI) is observed in ~25% of astronauts after spaceflight. Although astronauts wear an antigravity suit (AGS) during re‐entry, OI is still a concern after this suit is removed. NASA recently determined that thigh high compression stockings were effective against OI, but to a lesser degree than the AGS. We evaluated stockings with added abdominal compression as a countermeasure to post‐spaceflight OI.

METHODS

Ten healthy volunteers (6M, 4F) participated in three 80° head‐up tilts on separate days: (1) normovolemic, (2) hypovolemic with breast‐high compression stockings (BCS), or (3) hypovolemic without BCS. To simulate plasma volume (PV) changes seen in space flight, hypovolemia was induced by IV infusion of furosemide (0.5 mg/kg) and 48 hr of a low‐salt diet. Hypovolemic testing occurred 24 and 48 h after furosemide. One‐way repeated measures ANOVA was used to test for differences in blood pressure (BP) and heart rate (HR) responses to head‐up tilt. Time to presyncope was compared with Kaplan‐Meyer survival analysis.

RESULTS

Hypovolemia reduced PV by 8%. BCS significantly prevented tilt‐induced presyncope (p=0.015), excessive decreases in BP (p<0.04) and increases in HR (p=0.02) in hypovolemic test subjects.

CONCLUSION

BCS is an effective countermeasure to OI in this hypovolemic model of space flight. These stockings are readily available, inexpensive, and can be worn for days after spaceflight as astronauts readapt to Earth gravity. Supported by KY NASA EPSCoR.
Weightbearing In Simulated 1/6th and 1/3rd Gravity Does Not Prevent Cancellous Bone Loss
Authors: Macias Brandon Richard , Swift J M , Lima F , Greene E S , Allen M R , Bloomfield S A
Published: June 2021

The effect of partial gravity (G) (as on the Lunar surface) on weight bearing bone remains undefined; a new model (the partial G mouse) provides for graded reductions in weight bearing. We hypothesized that mice exposed to 1/6th G and 1/3rd G will experience significant reductions in cancellous bone mass and altered microarchitecture as compared to ambulatory control animals but that the magnitude of these changes would be less than in 0 G mice.

Methods

Forty‐three female BALB/cByJ mice were randomly assigned to cage control (1G), “zero‐gravity” hindlimb unloaded (0G), 1/6th gravity (G/6), or 1/3rd gravity (G/3) groups for a 21‐day suspension protocol. In vivo peripheral quantitative computed tomography (pQCT) scans were conducted at the proximal tibia metaphysis on days 0 and 21 to measure volumetric bone mineral density (vBMD). Ex vivo micro‐CT scans were conducted at the distal femur to quantify bone volume (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N).

Results

Body mass decreased significantly (7.6%) in 0G mice and non‐significantly in the G/3 or G/6 groups (−3.9%). In vivo total vBMD at the proximal tibia decreased significantly in the 0G (20%), G/6 (14%), and G/3 (14%) groups compared to 1G mice (−3%). Relative to the 1G group, distal femur BV/TV, Tb.Th, and Tb.N in the 0G, G/6 and G/3 groups were lower (24%, 12%, 14%, respectively). Mineral density gray scale of distal femur cancellous bone was significantly lower only in 0G (−2.6%) vs. 1G mice.

Conclusion

These data suggest that partial weight bearing (as high as 1/3rd G) does not provide enough mechanical loading to prevent the significant deterioration of cancellous bone observed in the 0G non‐weight bearing condition. Funded by NSBRI via NASA Cooperative Agreement NCC 9‐58.
IL‐6 and Delayed Onset Muscle Soreness Do Not Vary During the Menstrual Cycle
Authors: Chaffin Morgan E , Berg Kris E , Meendering Jessica R , Trehearn Tamra L , French Jeffrey A , Davis Jeremy E
Published: June 2021

The purpose of this study was to determine if a difference in interleukin‐6 (IL‐6) and delayed onset muscles soreness (DOMS) exists in the early follicular (EF) and mid‐luteal (ML) phases of the menstrual cycle in a group of competitive female distance runners following exhaustive running. Nine well‐trained, female endurance runners whose mean age was 26.8 ± 4.3 yr participated in this study. Each subject performed one 75 min high intensity interval running session during the EF phase (low estrogen and progesterone) and once during the ML phase (elevated estrogen and progesterone) of the menstrual cycle. Venous blood was collected before and after exercise to determine the IL‐6 concentration. Pre‐exercise plasma was assayed for estrogen and progesterone concentrations. Estrogen and progesterone levels were significantly reduced in the EF phase (68.44 ± 28.73 pg/ml and 8.70 ± 3.42 ng/ml, respectively) when compared to the ML phase (85.78 ± 14.75 pg/ml and 13.73 ± 2.54 ng/ml, respectively) of the menstrual cycle (p < 0.05). Plasma IL‐6 levels increased from pre to post exercise in the EF and ML phases of the menstrual cycle (p < 0.001). However, IL‐6 and DOMS were not higher in the EF phase than in the ML phase. Furthermore, there was no relationship between the level of IL‐6 and DOMS. The results suggest that menstruating female runners need not vary training throughout the month in order to reduce DOMS.
Exercise and endogenous antioxidant enzyme activity in perfused and ischemic cardiac tissue
Authors: Quindry John C , Schreiber Lindsey E , McGinnis Graham R , Irwin J Megan , Landram Michael , Urbiztondo Zea , Allums Lacey
Published: June 2021

Endogenous antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPx), and the superoxide dismutase isoforms (SOD) prevent ischemia reperfusion (IR)‐generated oxidative stress during IR. The contribution of these antioxidant enzymes to redox balance in perfused and ischemic tissues from exercised hearts is unknown. This investigation, used 4 month old male SD rats, to examine CAT, GPx, and SOD in perfused and ischemic regions of exercised and sedentary hearts. Treadmill exercise (3×60m/day) was used to precondition against IR injury. Animals were exposed to either a short duration (20mI/30mR) or long duration (50mI/120R) IR challenge. Evans blue infusions denoted perfused and ischemic tissue. ECG analysis and TTC staining indicated significant IR injury in sedentary hearts. Exercise partially prevented IR‐generated ECG arrhythmias and necrosis. MnSOD activity was elevated by exercise in both perfused and ischemic tissue exposed to short duration IR, but only in perfused tissue exposed to long duration IR. Cardiac protein carbonyl content was elevated in ischemic tissue from sedentary but not exercised hearts. These data demonstrate that MnSOD remains elevated in both ischemic and perfused cardiac tissue during short duration IR, but is depleted by longer duration IR. (JQ ‐ HL087256)
An Anatomic Study of Coronoid Cartilage Thickness with Special Reference to Fractures
Authors: Rafehi Samah , King Graham , Athwal George
Published: June 2021

Current classification systems of coronoid fractures are based on plain radiographic and/or CT measurements where cartilage is radiolucent and therefore not taken into account. Hence, during surgery, coronoid fracture fragments are much larger than anticipated for when viewed on CT. The main goal of this research is to determine the ulnar coronoid cartilage thickness in order to aid surgeons and clinicians in the proper classification and operation of coronoid bone fractures. Thirty ulnas have been dissected from embalmed cadaveric upper extremities and placed into a 30% diluted hypaque cartilage contrasting agent prior to CT. Using OsiriX (3.6–64 bit), 3‐D models for each ulna were constructed. Using the OsiriX point tool, 12 identifiable landmarks were placed onto the ulnar coronoid process, and then translated onto 2D orientation slices. This serves the purpose of measuring cartilage thickness from coronal, sagittal, and axial coronoid CT slices. Knowing the cartilage thickness will not only allow for proper classification of ulnar coronoid fractures by surgeons/clinicians, but will also allow for the accurate engineering of coronoid head prostheses in cases of irreparable coronoid process fractures. Moreover, discrepancies between biomechanical/anatomical studies and clinical outcome studies could then be resolved.

Grant Funding Source: Internal
The effect of astragalus polysaccharide on the expression of VEGF in Müller cell cultured in high level of glucose
Authors: Ke Min , Dai Bo , Wang Lin
Published: June 2021

Objective

To investigate the molecular mechanism of diabetes mellitus retinal injury and the protective role of astragalus polysaccharide ( APS ) on it.

Methods

Separated the retinal tissue from 5 days Wistar rat using trypsinase digest methods and cultured in 1640 with 20% FBS, at 37°C in humidified air with 5% CO2. The cells were identified by glial fibrillary acid protein (GFAP) using immuocytochemistry methods. The second generation, GFAP positive Müller cells were divided into 4 groups: 1) control group: cultured in the normal culture medium; 2) APS group: cultured in the normal culture medium with 400ug/ml APS; 3) high glucose group: cultured in the normal culture medium with 20mmol/L glucose; 4) high glucose APS group: cultured in the normal culture medium with 400ug/ml APS and 20mmol/L glucose. Three days later, the expression of vascular endothelial cell growth factor (VEGF) in Müller cells were detected by immunohistochemistry and reverse transcription‐polymerase chain reaction (RT‐PCR) methods respectively.

Results

The expression of VEGF in Müller cells of high glucose APS group was significantly decreased comparing with that of high glucose group. There is no significantly difference between other groups.

Conclusion

APS may prevent and treat diabetic retinopathy by reduced the expression of VEGF in Müller cells.
A 10 base pair element that is essential for autonomous plasmid replication in procyclic trypanosomes is dispensable in bloodstream‐forms
Authors: Urban John , Sauchelli Mark , Patnaik Pradeep
Published: June 2021

Trypanosoma brucei is a unicellular digenetic parasitic protozoan that alternates between the bloodstream of its mammalian host (bloodstream–forms) and the mid‐gut and salivary glands of its insect vector (procyclic and metacyclic forms respectively). These stages are bio‐chemically distinct.

Chromosomal DNA replication is an unexplored aspect of trypanosome biology. We are using an autonomously replicating plasmid as a model system to identify sequences that regulate this fundamental biological process in the parasite. One such element maps to a developmentally modulated procyclin promoter that drives reporter gene transcription on this episome. Linker substitution of a 10 base‐pair segment within Domain III of this promoter reduces reporter gene expression by ~10 fold, but abolishes the ability of this plasmid to replicate in procyclic trypanosomes. The close association of replication and transcription control elements and the interplay between these two processes as the parasite shifts from one developmental stage to another is of interest to us.

We now demonstrate that the requirements for the replication of these episomes differ between the two developmental stages of this parasite as the 10 base pair Domain III element is dispensable in bloodstream‐forms. In addition, our results suggest that the procyclin‐promoter on this plasmid does not support reporter gene expression in bloodstream forms. Since there are no other known T. brucei promoters on this episome, it would appear that transcription (and possibly replication) initiates adventitiously on this plasmid in the blood‐stage of this parasite. Promiscuous initiation of transcription and replication may be an interesting feature of trypanosome biology.

Funded by William Paterson University SURP and NIH
Innovative technology for DNA blood stabilization at room temperature
Authors: de Rozieres Sohela , Doedt Thomas , Kist Rita , Stassinopoulos Angela , Heckel Dirk , Oelmuller Uwe , Muller‐Cohn Judy , Muller Rolf
Published: June 2021

Long term storage, shipping and biobanking of patient blood DNA for future analyses are on the rise. Conventional storage and shipment of blood poses space, cost and energy problems. Biomatrica, Inc. has developed a novel technology to store, ship and archive blood samples at room temperature based on the natural principles of anhydrobiosis (“life without water”), a biological mechanism employed by some multicellular organisms that enables their survival in a dry state for extended periods. QIAsafe DNA Blood (QSB), a product developed in collaboration with QIAGEN, allows the dry storage and shipment of whole blood at room temperature for long periods until isolation of DNA is necessary. Adding water to the QSB‐stored blood DNA releases the matrix shell protection, which can then be followed by DNA isolation/extraction. DNA yields and integrity from QSB‐stored blood are comparable to frozen controls (−20°C or −80°C). Our data demonstrates that blood stored for 9 months at room temperature or 45°C (equivalent to approx. 3 years of room temperature storage) allows recovery of high molecular DNA that is in quality and amount of recovery equivalent to freezer stored material. The isolated DNA can be used in a broad range of downstream applications including PCR, qPCR, genotyping, and DNA sequencing experiments. QBS eliminates the need of blood storage in freezers reducing cost and eliminating freeze/thaw degradation.
Innovative technology to stabilize DNA at room temperature in tissue and cells
Authors: Wilkinson Steven , Law Billy , Whitney Scott , Coulon Laurent , Clement Omo , Shireen Lutfunnessa , Stassinopoulos Angela , Muller‐Cohn Judy , Muller Rolf
Published: June 2021

Current methods for storing and shipping human and animal tissue or cells for clinical, forensic and biomedical research needs are costly and can be insufficient for reliable molecular diagnostics requiring preservation of high‐quality genomic DNA. Animal tissues are commonly shipped on dry‐ice or in liquid nitrogen which are costly and often not practical for the collection of samples in the field. Another common method of tissue preservation, formalin fixation followed by paraffin embedding (FFPE), is also impractical for field collection and often results in damage to nucleic acids. We have combined synthetic chemistry with the natural principles of anhydrobiosis (the capacity of some organisms to protect their molecular integrity and survive extreme desiccation for hundreds of years) to stabilize genomic DNA in animal tissue and mammalian tissue culture cells at room temperature. DNAgard™, a novel liquid formulation preserves the integrity of genomic DNA in tissue and cells stored for at least 60 days at room temperature. DNA yield is comparable to frozen controls. The data demonstrates that genomic DNA integrity is preserved in samples stabilized with DNAgard during extended exposure to extreme heat (50°C), with DNA remaining intact for 1 month in tissue samples and for two months in cultured cell samples. Tests were designed to explore the capacity of this product as a shipping application, and results demonstrate that DNAgard™ preserves the integrity of genomic DNA in animal tissue and cell samples when exposed to extreme fluctuations in temperature.
The effect of targeted silencing of ferritin in the yellow fever mosquito, Aedes aegypti
Authors: Bhakta Janki B , Geiser Dawn L , Kohlhepp Edwin A , Zhou Guoli , Winzerling Joy J
Published: June 2021

Mosquitoes are vectors of human pathogens that cause disease, such as malaria, yellow fever and dengue fever. Pathogen transmission occurs when the female mosquito takes a bloodmeal from a human host. The female mosquito must bloodfeed for egg production, or oogenesis. An essential and concentrated nutrient in the blood meal is iron, which is required for optimal egg development and viable offspring. Ferritin is a major iron‐storage and transport protein expressed in mosquitoes. The protein increases following a blood meal in the female ovaries, midgut, fat body and hemolymph. Insect ferritin consists of 24 subunits of the heavy chain homologue (HCH), which contains the site for ferroxidase activity, and the light chain homologue (LCH), which is used for iron storage. This study evaluates the importance of these subunits for iron transport and storage within mosquitoes by suppressing them using RNA interference (RNAi) techniques. We suppressed ferritin expression by injecting the animals with double stranded RNA (dsRNA) and collecting whole animals, their tissues and eggs following blood feeding to examine the effect of message knockdown and protein expression. Interfering with iron delivery and usage in mosquitoes may be a strategy to reduce the number of eggs laid and pathogen transmission rates. Research support from NIH (GM056812), the Undergraduate Biology Research Program and a grant from the Honors College from the University of Arizona.