bluuebunny/crossref_metadata_2025 huggingface dataset:
Programmed ribosomal frameshifting occurs at multiple sites and by multiple paths (109.3)
Authors: Tinoco Ignacio , Yan Shannon , Kim Hee‐Kyung
Published: June 2021

Programmed frameshifting is used by prokaryotes and eukaryotes to synthesize two or more proteins from the same messenger RNA. We have studied minus‐one frameshifting in the dnaX gene in E. coli, whose mRNA contains the usual frameshifting signals: an internal Shine Dalgarno sequence, a slippery sequence (AAAAAAG), and a stem‐loop. We used bulk mass spectrometry, single‐molecule laser tweezers, and single‐molecule FRET in our studies. We found that minus‐one frameshifting occurs at the Lys codons in the slippery sequence, but also at codons on either side. Furthermore, the minus‐one frameshift occurs by slips of the ribosome of ‐1, +2, or even ‐4 nucleotides. The translation trajectories show step‐by‐step progression as each codon is translated, but at the slippery sequence large‐scale fluctuations in position of the ribosome are seen. This agrees with the mass spectrometry results showing multiple sites and multiple paths of frameshifting.

After peptide bond formation the tRNAs undergo a classic‐hybrid equilibrium before the elongation factor, EF‐G•GTP, catalyzes the translocation. Our single‐molecule FRET studies of fluorophore‐labeled ribosome and tRNA found that the presence of the stem‐loop stabilizes the ribosome in the hybrid state before translocation. The longer time spent in this state allows the ribosome to sample other states, and thus may favor sites and paths in addition to a zero‐frame move.
Spinal protein phosphatase 1 constrains respiratory plasticity after sustained hypoxia (1091.2)
Authors: Huxtable Adrianne , Peterson Timothy , Kopp Elizabeth , Mitchell Gordon
Published: June 2021

Plasticity is an important aspect of the neural control of breathing. One well‐studied form of respiratory plasticity is phrenic long‐term facilitation (pLTF) induced by acute intermittent, but not sustained hypoxia. pLTF pattern sensitivity partially reflects differential regulation by okadaic acid‐sensitive protein phosphatases (PP) during intermittent vs. sustained hypoxia (Wilkerson et al., J Neurosci, 2008), although the relevant phosphatase has not been identified. Here, we show that pLTF is revealed after sustained hypoxia (25 min, 10.5% O2) in rats pre‐treated with bilateral intrapleural injections of small interfering RNAs (siRNAs, Accell‐modified, Thermoscientific; 30 μl/side, 3.33 μM, 3 days) targeting PP1 mRNA (40±14% baseline, n=4). siRNAs targeting PP2A (10±13%, n=3), PP5 (12±13%, n=4) and non‐targeting siRNAs (‐1±11%, n=5) had no effect. siRNAs had no effect on the response during hypoxia. Preliminary immunohistochemistry confirms a small knockdown of targeted phosphatases within the phrenic motor nucleus. PP1 appears to be the relevant okadaic acid‐sensitive phosphatase regulating pLTF.

Grant Funding Source: Supported by: HL111598, HL080209, Francis Family Foundation (AGH).
Spike frequency adaptation in avian intrapulmonary chemoreceptors does not rely on Na+/H+ exchange (1092.9)
Authors: Sheridan Krystal , Hempleman Steven , Lonjaret Jean‐Guillaume , Meehan Shane , Bassett Peter
Published: June 2021

IPC are essential for avian breath‐by‐breath respiratory control and are inversely sensitive to lung PCO2. Evidence suggests IPC detect intracellular pH rather than molecular CO2, and that pHi reflects a dynamic interaction of intracellular CO2 hydration/dehydration, buffering, and transmembrane acid/base exchange. We used the Na+/H+ exchange (NHE) blocker dimethyl amiloride (DMA) to test NHE’s role in single‐unit IPC response to combined phasic and tonic CO2 step stimuli in anesthetized mallards (n=9). DMA depressed mean (tonic) IPC discharge response to CO2 step stimuli by 15%, 31%, 51%, and 58% at 1,3,8, and 13 µmol/kg, (p<0.05), but did not affect phasic discharge (spike frequency adaptation index, SFA). Depression of tonic CO2 response confirms earlier reports, and the new result that NHE has no effect on the phasic part of IPC step response demonstrates that other mechanisms must underlie SFA.

Grant Funding Source: Supported by: NIH R15 HL087269‐02 and R25 GM056931‐15.
Role of CFTR in embryonic stem cell programming (1094.4)
Authors: Liu Zhenqing , Yuan Ping , Jiang Xiaohua , Chan Hsiao Chang
Published: June 2021

Embryonicstem cells (ESC) are unique cell types characterized by their capacity of self‐renewal and potential of differentiation. The balance between self‐renewal and differentiation in ESC is controlled by a complex interplay.Cystic fibrosis transmembrane conductanceregulator (CFTR) is a cAMP‐regulated anion channel which is predominantly expressed in a wide range of epithelial tissues. Mutations in the gene encoding CFTR cause cystic fibrosis. Recently, we have found that CFTR is functionally expressed in both human and mouse ESC. To further understand CFTR function in ESC pluripotency and differentiation, we derived CFTR mutated (DF508) ESC with C57BL6 background. We found that DF508 ESC proliferate faster than wild type cells, and had increased expression of self‐renewal markers such as Oct4, Nanog and Sox2. Wnt/β‐catenin pathway is an important pathway that controls mouse ESC pluripotency. Intriguingly, our western Blot analysis revealed that Cyclin D2, a downstream target gene of β‐catenin, was significantly upregulated in DF508 ES cells. Taken together, our results suggest CFTR may play a role in regulation of ESC pluripotency and differentiation.
Decreased expression of activated placental heat shock protein 27 in human and ovine intrauterine growth restriction (1095.15)
Authors: Merill Daniel , Bahr Brigham , Arroyo Juan
Published: June 2021

Intrauterine growth restriction (IUGR) has been documented to have increased placental apoptosis at term. Previous reports from our lab showed apoptosis as an early event in an ovine model of IUGR. Our objective was to determine the expression of active HSP27 in human IUGR placentas near term and to compare its expression to those found in the placenta on an ovine hyperthermia induced model of IUGR. Human placenta tissue samples were collected at term for active HSP27 determination. Pregnant sheep were placed in hyperthermic (HT) conditions to induce IUGR. Compared to controls, IUGR pregnancies showed: 1) reduced activation (1.7‐fold, p<0.009) of HSP27 in the human placenta; 2) smaller placentas (349±21g v. 169±22g; p<0.03) and fetal (2914±201g v. 1718±433g; p<0.03) weights in the ovine IUGR model; 3) Increased placental apoptosis in this model; 4) a 1.8‐fold increase (p<0.005) in active HSP27 in the ovine placenta near term; and 5) localization of active HSP27 to the trophoblast cells of the ovine placenta.

We conclude that decreased active HSP27 at term is present when placental apoptosis is increased during IUGR. This could be a factor leading to the decreased placental weight observed during this disease (Supported by NIH grant 7K99HD055054‐02).

Grant Funding Source: 7K99HD055054‐02
Quercetin attenuates induction of COX‐2 in rats subjected to acute unilateral ureteral obstruction (1096.8)
Authors: Carlsen Inge , Nørregaard Rikke
Published: June 2021

In response to acute unilateral ureteral obstruction (UUO) inflammatory processes initiates. The aim of this study is to evaluate whether quercetin, a natural occurring bioflavonoid, have anti‐inflammatory and protective functions in the kidney during acute UUO.

Rats were subjected to 24 hour UUO and quercetin was administrated IP at a dose of 50mg/kg/day, and functional parameters as well as immunoblotting and QPCR was performed for inflammatory markers, including COX‐2 and IL‐1β and the oxidative stress marker heme‐oxygenase‐1 (HO‐1).

Kidney weight as well as plasma creatinine and urea were increased in response to 24h UUO. Quercetin administration reduces the increased plasma urea level, while kidney weight and plasma creatinine were not affected. COX‐2 and HO‐1 expression were increased in inner medulla in rats subjected to UUO, quercetin attenuates this induction. IL‐1β was induced in UUO rats, however, treatment with quercetin further increase IL‐1β mRNA level.

Taken together, data demonstrated that quercetin play a role for the regulation of COX‐2 in response to acute UUO.
Dynamic measurements of [IP3]i in cardiac differentiated P19 (CD‐P19) cells with a novel quantum dots (QD) and gold nanoparticles (AuNP) nanobiosensor, QD‐IP3‐AuNP. (1097.4)
Authors: Mao Hua , Wong Lid
Published: June 2021

We reported the first measurements of the [IP3]i in intact CD‐P19 cells with a novel nanobiosensor for IP3, QD‐IP3‐AuNP. QD‐IP3‐AuNP was fabricated with QD580 (donor) and AuNP (acceptor); an IP3 receptor peptide (RP) was conjugated between them. QD fluorescence increases proportionally to [IP3] bound to the RP. Fluorescence microscope measurements (Ex=488; Em=535 and 580) were made in CD‐P19 cells loaded with (i) QD‐IP3‐AuNP, (ii) Fluo‐3, and (iii) both QD‐IP3‐AuNP and Fluo‐3. Differentiation of P19 cells was induced with 1% DMSO. The responses of [IP3]i and [Ca2+]i were interrogated with two agonists, McN343 (M1) and UTP (P2Y2); and three antagonists, pirenzipine(M1), suramine (P2), and thapsigargin (Ca‐ATPase). In each sample (n=5), cumulative doses (µM) of 0, 0.1, 1, 10, of either agonist were added, following with the applications of 10 µM of one of the antagonists and the repeated cumulative doses of the respective agonist, all at 6 min apart. The dose dependent increases of [IP3]i and [Ca2+]i by both agonists were inhibited by their respective receptor antagonists. For both agonists, thapsigargin had no effect on the induced increases of [IP3]i but further the induced increases in [Ca2+]i. These data suggest that [IP3]i measurements with QD‐IP3‐AuNP in CD‐P19 cells could be a robust platform for cardiac drug discovery and toxicology.
Decreased plasma CO2 induces redistribution of the V‐ATPase in the choroid plexus epithelium: involvement in regulation of cerebrospinal fluid pH? (1098.4)
Authors: Christensen Henriette , Damkier Helle , Paunescu Teodor , Brown Dennis , Praetorius Jeppe
Published: June 2021

Little is known about the mechanisms behind cerebrospinal fluid (CSF) pH control. The CSF is produced by the choroid plexus epithelium (CPE), located in the ventricular system of the brain. It is believed that CSF pH is regulated by the CPE but the mechanism is not yet understood.The CPE expresses several acid/base transporting proteins. One of these is the V‐ATPase, which is found in nearly all cell types where it acidifies intracellular organelles. However, in some cell types the protein acidifies the extracellular milieu upon translocation to the plasma membrane. In this study we have investigated the subcellular localization of the V‐ATPase in the CPE at normal and low levels of pCO2. Mice were subjected to acute hyperthermia, causing hyperventilation and a decrease in blood pCO2. Immunohistochemical analysis of the CPE was carried out on the paraffin embedded brain sections by fluorescence‐ and electron microscopy using an antibody against the A subunit of the V‐ATPase.Under control conditions, the V‐ATPase was located in numerous vesicles that were evenly distributed throughout the cytoplasm. However, during hyperthermia, a statistically significant redistribution of the V‐ATPase towards both the luminal and basolateral plasma membrane domains of CPE cells was found. This indicates that the V‐ATPase in the CPE responds to systemic acid/base conditions and may help guard the CSF against large changes in pH.
Interactions between activators of the larval bullfrog epithelial cation channel (1099.3)
Authors: Holbird Doyle
Published: June 2021

The epithelium of the larval bullfrog Rana Catesbeiana contains an apically located non‐selective cation channel. This channel has been shown to be activated by a variety of compounds including acetylcholine (ACh), amiloride and ATP. The purpose of this present study was to further characterize the tadpole channel in terms of interactions between these three ligands at their sites of activation. Tadpole skins were mounted in Ussing chambers with NaCl Ringers bathing the basolateral side. Perfusion of ligands in Ca++ free Ringers was done alone and in various combinations to test for interactions such as potentiation and suppression of ligand effects. ATP was found to potentiate the effects of amiloride and ACh at low concentrations, yet at high concentrations the responses were not additive as would be expected if two different populations of channels were being activated. ACh and amiloride perfusion resulted in a suppression of the ACh response. The addition of two ligands had little effect upon the desensitization time constant. Each of the ligands largly cross‐desensitized the other ligands. These results are consistent with a scenario in which all three ligands activate the same response.
Got my first faculty job ‐ now what? (11.1)
Authors: McGee Richard
Published: June 2021

Although all career steps and transitions are challenging on the road to an academic career (or any career these days襃), the one that is often most difficult and least discussed is the last one when you have actually gotten the job. So much attention is placed on trying to GET a faculty position that what to do when you get one is often lost in the shuffle.This session will draw on a career‐full of experiences doing research and leading research training that have led to a novel position as a ‘faculty coach’ at Northwestern University Feinberg School of Medicine. In the current environment, it is no longer realistic to try to “make it on my own” when starting a new faculty position. Frameworks will be provided on how to: seek out the right kind of guidance during the first years of a faculty position; balance individual research directions with collaborations; decide on the types of research funding mechanism to seek when; approach the complex process of grant conceiving and writing as skills to consciously learn from expert teachers. Online tools and novel approaches to grant writing we have developed to assist early stage grant writers will be introduced. The session will be useful for both those who have obtained a faculty position and others who want to plan ahead for when they get one. This systematic approach is generalizable to other types of careers with appropriate modifications based on the expectations of those careers.
ICAM‐1: a novel mechanism by which the inflammatory response augments myogenesis (1102.10)
Authors: Goh Qingnian , Dearth Christopher , Awadia Sahezeel , Garcia‐Mata Rafael , Corbett Jacob , Pizza Francis
Published: June 2021

Recent investigations by our laboratory demonstrated that skeletal muscle cell expression of intercellular adhesion molecule‐1 (ICAM‐1), an important protein of the inflammatory response, augmented events of myogenesis in which myotubes are forming, adding nuclei, aligning, fusing, synthesizing proteins, and hypertrophying. Our current work extended these findings by incorporating pharmacological tools with an in vitro approach to discern underlying mechanisms associated with ICAM‐1 mediated myogenesis. Antibody neutralization revealed the extracellular domain of ICAM‐1 to be fundamentally important in augmenting homotypic adhesion of myoblasts, leading to myotube formation and myonuclear accretion. In contrast, inhibition of the cytoplasmic domain of ICAM‐1 with a cell permeable peptide revealed its critical role in augmenting both myotube formation and myonuclear accretion, as well as myotube alignment and fusion, rates of protein synthesis, and overall myotube size. Peptide inhibition further demonstrated that the signal transducing function of the ICAM‐1 cytoplasmic domain augmented events of myogenesis through mechanisms involving increased Rho GTPase (Rac1 and cdc42) and Akt/p70s6k activity. Taken together, our findings extend knowledge of the immunobiology of skeletal muscle cells by revealing a novel mechanism through which the inflammatory response facilitates myogenesis.

Grant Funding Source: This research was supported by the ACSM Foundation Doctoral Student Research Grant(Q Goh)
The effect of lipid concentration on myosin heavy chain isoform expression in cultured Weddell seal skeletal muscle cells (1102.26)
Authors: Garcia Teresa , Kanatous Shane
Published: June 2021

Prior studies have shown that myoglobin expression is increased when muscle cells are exposed to hypoxia and a second stimulus such as exercise. Aerobic muscle fibers expressing either Type I or IIA myosin heavy chains (MHC) have more myoglobin than other fiber types, and changes in the expression of myoglobin are usually attributed to a change in fiber type. Recently, De Miranda et al. (2012) showed that cultured Weddell seal skeletal muscle cells grown in normoxic conditions experienced an increase in myoglobin expression in response to increased lipid concentration in the culture media. The objective of this study was to determine if increasing lipid in the media would affect the expression of MHC that would underlie the increase in myoglobin expression. Weddell seal cells from primary culture were proliferated in normoxic conditions for seven days in growth media supplemented with either 2.5% or 5.0% lipid, and both treatments were differentiated for seven days in either hypoxic (0.5% oxygen) or normoxic (21% oxygen) environments. Cells were homogenized, and protein was separated via gel electrophoresis and stained with Coomassie. A difference in the presence of MHC isoforms was not detected between the 2.5% and 5.0% treatment groups, in either oxygen condition. We conclude that lipid may be acting independent of changes in MHC via another undescribed pathway to increase myoglobin expression.

Grant Funding Source: Supported by Colorado State University
Effect of 17β estradiol on statin‐induced apoptosis (1102.38)
Authors: Bielman Marie , Barlas Kyle , Hoffman Carl , Ball Karen
Published: June 2021

We have previously studied the effects of estrogen (E) on statin‐induced myotoxicity with E providing no apparent protection. In our studies E was added simultaneously with the statins, possibly explaining the lack of E effect. The anti‐apoptotic effect of E has been shown to be tightly linked to induction of hsp27. The purpose of this study was to establish the dependence of an anti‐apoptotic effect on prior E exposure. C2C12 cells were cultured in growth media (GM) and plated in 6‐well plates. After plates reached approximately 70% confluence, cells were exposed to one of six treatments: control (GM), E (10‐8 M 17β Estradiol), SS (10‐4 M Simvastatin), or three different treatments of E + SS (no E pretreatment; 45 min E pretreatment; or 45 minute E pretreatment with continued E+SS). After 24‐48 hours exposure, cells were DAPI‐stained and apoptotic nuclei were quantified. Data was analyzed by 1‐way ANOVA; p<0.05. As in previous studies, SS induced apoptosis with a significant increase in condensed nuclei (33.8% +/‐ 11.7% vs 6.2% +/‐ 2.6% in GM). Addition of E had no effect under any of the three E+SS conditions (45.4% +/‐ 15.6%, 34.9% +/‐ 14.5%, 35.5% +/‐ 15.4%). These results further support our earlier findings that question the ability of E to protect against statin‐induced toxicity. Pre‐treatment with E, known to induce hsp27, had no impact on the response to SS. Further studies are needed to explain the discrepancy between these data and E effects in other models of apoptosis.

Grant Funding Source: NSF STEP Grant
Phosphorylation of nNOSα promotes muscle cell differentiation (1102.40)
Authors: Kar Rekha , Masters Bettie , Roman Linda
Published: June 2021

Nitric Oxide (NO), produced by the nitric oxide synthases (NOS), regulates muscle cell structure and function. Neuronal NOS (nNOS) is the predominant isoform of NOS expressed in skeletal muscle, and exists as several splice variants, including nNOSα, µ and β. As opposed to myotubes, which mostly express nNOS µ, myoblasts predominantly express nNOSα. Phosphorylation of NOSs has been shown to regulate their activity, and studies from our group showed that nNOSµ is phosphorylated in response to insulin in skeletal muscle. To explore the effect of phosphorylation of nNOSα on muscle cell differentiation, we generated C2C12 myoblast cell lines stably expressing IRES‐EGFP, IRES‐EGFP‐nNOSα, or IRES‐EGFP‐nNOSα S1412E (a phosphomimetic mutation). Differentiation was induced in all these stable cell lines by serum starvation, and was evaluated by morphological and immunoblot analyses. The phosphomimetic of nNOSα (S1412E) greatly stimulated the differentiation of myoblasts to myotubes in comparison to the vector only or the nNOSα‐expressing C2C12 myoblasts, as revealed by phase contrast microscopy and by the increased expression of myosin heavy chain (mYHC), a marker of differentiation, suggesting a critical regulation of muscle cell differentiation by nNOSα phosphorylation at this particular residue. Recombinant nNOSα S1412E showed no apparent increase in NO production, compared to nNOSα WT, suggesting that phosphorylation at this site in nNOSα promotes differentiation through alternate protein‐protein interactions, translocation or other signaling pathways. Future studies will involve distinguishing the distinct roles played by various nNOS splice variants and phosphorylation events in muscle cell differentiation.

Grant Funding Source: Supported by NIH #HL07446 and GM052419
Indomethacin increases organ damage and heat stroke mortality in mice (1104.13)
Authors: Leon Lisa , Dineen Shauna , Plamper Mark
Published: June 2021

The effect of non‐steroidal anti‐inflammatory drugs (NSAIDs) on the progression of organ damage during heat stroke (HS) recovery is unknown. We examined the effect of oral indomethacin (INDO; 5 mg/kg) on core temperature (Tc; ±0.1°, radiotelemetry), organ (kidney, liver) gene expression of IL‐6 and lipocalin2 (LCN2; an injury biomarker), and survival of male C57BL/6J mice (23.7±0.5g; 6‐9 mice/group) at Tc,Max (42.4°C), 3h and 24h of HS recovery. INDO had no effect on Tc during heat exposure or recovery. INDO caused ~50% increase in HS mortality, which was associated with increased gut hemorrhaging. In HS survivors, kidney IL‐6 (4‐ to 38‐fold) and LCN2 (6‐ to 9‐fold) expression was increased throughout recovery and INDO potentiated these responses (5‐ to 280‐fold and 20‐ to 56‐fold, respectively; P<0.05). Liver IL‐6 (2‐to 4‐fold) and LCN2 (14‐ to 16‐fold) expression was lower than the kidney, but these responses were also potentiated by INDO (IL‐6: 3‐17‐fold; LCN2: 20‐ to 120‐fold; P<0.05). Plasma LCN2 was increased by INDO in non‐heated control and HS mice at 24h of recovery indicating an effect of the drug alone on organ damage. NSAIDs are known to cause gut bleeding, but these studies suggest there is a heat‐NSAIDs interaction that exacerbates the toxic effects of this drug. Plasma LCN2 may be a sensitive biomarker of organ damage in our model. Author views not official US Army or DoD policy.

Grant Funding Source: DoD Medical Research and Materiel Command
Acute heat stress responses of mice with sickle cell trait (1104.18)
Authors: Chen Yifan , Islam Aminul
Published: June 2021

It has been a long standing controversy about whether individuals with sickle cell trait (SCT) are vulnerable to injury or even sudden death during physical activity in hot climates. Experimental studies assessing susceptibility of SCT to heat stress are lacking. Our objective was to examine whether transgenic SCT mice had an altered thermoregulatory response to heat exposure. All SCT mice (n =16) and majority of age‐ and gender‐matched normal mice (29 of 45) completed two separate heat tests (2 hr each at 39.5 degree C) without severe hyperthermia (42.4 degree C). SCT mice had a significantly lower body weight than normal mice (n = 19) with similar peak core temperature (Tc). There was no difference in weight loss between SCT and normal mice during heat exposure. No significant correlations between peak Tc and body weight were found among SCT or normal mice. Compared to their normal counterparts, both non‐heat exposed (n = 9) and heat exposed SCT mice had significantly higher levels of heat shock protein 72 (HSP72) in various tissues, slightly lower hematocrit values, but similar plasma lactate dehydrogenase concentrations. No differences in plasma inflammatory and redox makers were found between SCT and normal mice following heat exposure. We conclude that SCT mice reveal neither thermoregulatory dysfunction nor increased oxidative/inflammatory stress when exposed to moderate heat. The physiological significance of increased tissue HSP72 remains to be determined.

Grant Funding Source: Supported by The Office of Naval Research Grant N0001411MP2002 and USU Intramural Grant R091EH
Electric fans mitigate cardiovascular and thermal strain during extreme heat events (1104.5)
Authors: Ravanelli Nicholas , Jay Ollie
Published: June 2021

Extreme heat events (EHEs) increase mortality rates due to heat related illness and are known to exacerbate underlying health conditions. Current international health guidelines warn against the fan use when ambient temperature exceeds 35°C; however no evidence exists supporting or refuting their use during EHEs. Our aim was to evaluate the critical environmental limits for cardiovascular and thermal strain with (F) and without (NF) a fan (v=4.0 m•s‐1) at an ambient temperature (Ta) of 36°C and 42°C in 4 separate trials. Six males (23.8 ± 3.5 y) sat in a climatic chamber regulated at either 36°C or 42°C while ambient water vapor pressure (Pa) was raised by 0.3 kPa every 7.5 min from 1.6 to 5.6 kPa. Heart rate (HR), esophageal temperature (Tes), and whole body sweat loss (WBSL) were measured. The critical Pa (Pa_crit) at which an upward inflection of HR was observed was significantly higher with F at both 36°C (F: 5.1±0.2 kPa; NF: 3.9±0.4 kPa, P<0.001) and 42°C (F: 4.4±0.4 kPa; NF: 3.2±0.4 kPa; P=0.01). Likewise, the Pa_crit for an upward inflection of Tes was significantly higher with F at 42°C (F: 4.8±0.1 kPa; NF: 4.1±0.4 kPa; P=0.01). However, WBSL was significantly greater with F at 42°C (F: 877±23 g; NF: 540±125 g, P=0.005) and 36°C (F: 391±23 g; NF: 353±32 g, P=0.02). Thus, contrary to current guidelines, fans mitigate cardiovascular and thermal strain at Ta values as high as 42°C, but necessitate a greater fluid intake.

Grant Funding Source: Supported by NSERC
Effects of hindlimb unloading and radiation on vasodilator responses in skeletal muscle arteries (1106.18)
Authors: Ghosh Payal , Stabley John , Kilar Cody , Behnke Bradley , Alwood Joshua , Shirazi Yasaman , Globus Ruth , Delp Michael
Published: June 2021

Long‐term spaceflight leads to extensive changes to the microvasculature as a result of weightlessness, which may be compounded by the effects of radiation exposure. The purpose of this study was to assess the individual and combined effects of hindlimb unloading (HU) and radiation (Rad) on vasodilator responses in the skeletal muscle microcirculation.

Adult male C57BL/6 mice were randomized to one of four groups: control, HU (tail suspension for 10‐11 days), Rad (100cGy of 150MeV protons and 20cGy of 800MeV 56Fe), and HU‐Rad. Gastrocnemius feed arteries were isolated, cannulated and pressurized for in vitro study. Endothelium‐dependent (ACh, 10‐9‐10‐4 M) and ‐independent (Dea‐NONOate, 10‐9‐10‐4 M) vasodilator responses were evaluated. NOS and COX inhibitors (L‐NAME and Indomethacin) were used to determine which endothelium‐dependent signaling pathways were altered.

In all treatment groups endothelium‐dependent and ‐independent vasodilator responses were significantly impaired compared to controls (max ACh dilation: 91Â±2%). ACh‐induced vasodilation was less in the HU‐Rad (68Â±3%) group than in HU (79Â±1%) or Rad (80Â±2%) alone. Independent and simultaneous treatment with L‐NAME and indomethacin reduced vasodilation and eliminated group differences. Dea‐NONOate‐induced vasodilation was reduced to a greater degree in the Rad and HU‐Rad groups.

These findings suggest that the combination of insults experienced during spaceflight lead to significant impairment of vasodilatory function, mediated through endothelium‐dependent reductions in NOS signaling and vascular smooth muscle dysfunction.

Grant Funding Source: Supported by NSBRI, NCC 9‐58‐44
Transcriptome‐wide RNA sequencing analysis of rat skeletal muscle feed arteries: impact of exercise training in obesity (1106.23)
Authors: Padilla Jaume , Jenkins Nathan , Thorne Pamela , Martin Jeffrey , Rector R , Davis J , Laughlin M
Published: June 2021

We employed next generation RNA sequencing (RNA‐Seq) technology to determine the extent to which exercise alters global gene expression in skeletal muscle feed arteries and aortic endothelial cells (AEC) of obese OLETF rats. Transcriptional profiles of the soleus and gasctrocnemius muscle feed arteries (SFA and GFA, respectively) and AEC‐enriched samples from rats that underwent an endurance exercise training program(EndEx; n=12), interval sprint training program (IST; n=12), or remained sedentary (Sed; n=12) were examined. In response to EndEx, there were 39 upregulated and 20 downregulated genes in SFA and 1 upregulated and 1 downregulated gene in GFA (FDR < 10%). In response to IST, there were 305 upregulated and 324 downregulated genes in SFA and 101 upregulated and 66 downregulated genes in GFA, with an overlap of 32 genes between arteries. Furthermore, in AEC, there were 183 up‐regulated and 141 downregulated genes with EndEx and 71 upregulated and 69 downregulated genes with IST, with an overlap of 35 between exercise programs. Only expression of two genes (i.e., Tubb2b and Slc9a3r2) was altered (i.e., increased) by exercise in all three arteries. The finding that both EndEx and IST produced greater transcriptional changes in the SFA compared to the GFA is intriguing when considering the fact that treadmill exercise is associated with greater relative increases in blood flow to the gastrocnemius muscle compared to the soleus muscle.

Grant Funding Source: NIH RO1HL036088
Total sleep deprivation and pain perception during cold noxious stimuli in humans: role of sex (1106.8)
Authors: Larson Robert , Yang Huan , Durocher John , Carter Jason
Published: June 2021

Several studies indicate a higher incidence of chronic pain in women compared to men, and that women report higher perceived pain (PP) responses to acute experimentally induced noxious stimuli. To date, studies examining the relations between sleep deprivation and pain have focused on pain threshold methodologies, not sustained noxious stimuli, and potential sex differences have not been explored. Therefore, we hypothesized that 24‐hour total sleep deprivation (TSD) would augment PP during the cold pressor test (CPT), and that women would report higher PP than men. Mean arterial pressure (MAP), heart rate (HR), and PP responses to CPT were recorded in 27 subjects (14 men, 13 women; age 22±1 yrs) after a normal night of sleep (NS) and TSD (randomized, crossover design). PP (Δ6.9±0.5 vs. Δ8.3±0.5 a.u., p<0.01) and HR (Δ9±1 vs. Δ12±1 beats/min, p<0.04) responses to CPT were augmented by TSD. MAP responses to CPT were similar in both conditions (Δ12±1 vs. Δ13±1 mmHg, p=0.57). Contrary to our hypothesis, there were no sex differences in the PP response or cardiovascular reactivity to CPT. Interestingly, changes in MAP were significantly correlated to changes in PP in women, but not men, during both NS and TSD trials (r 蠅 0.67, p 蠄 0.02). In conclusion, TSD augments PP and HR responses to CPT; however, these responses were similar between sexes. Moreover, PP and MAP responses to CPT demonstrated an association in women, but not men.
Anillin regulates cell‐cell junction integrity by organizing junctional accumulation of RhoA‐GTP, F‐actin, and Myosin‐2 (1109.17)
Authors: Miller Ann , Reyes Ciara , Jin Meiyan , Arnold Torey , Breznau Elaina , Espino Rhogelyn , Goryachev Andrew
Published: June 2021

Anillin is a scaffolding protein that organizes and stabilizes actomyosin contractile rings and was previously thought to function primarily in cytokinesis. Using Xenopus laevis embryos as a model system to examine Anillin’s role in the intact vertebrate epithelium, we find that a population of Anillin surprisingly localizes to epithelial cell‐cell junctions throughout the cell cycle, whereas it was previously thought to be nuclear during interphase. Further, we show that Anillin plays a critical role in regulating cell‐cell junction integrity. Both tight junctions and adherens junctions are disrupted when Anillin is knocked down, leading to abnormal intercellular spaces and increased permeability to fluorescent dextran. Further, fluorescence recover after photobleaching (FRAP) studies show that Anillin perturbation affects the dynamics of the tight junction protein ZO‐1. Anillin interacts with RhoA, F‐actin, and Myosin‐2, all of which regulate cell‐cell junction structure and function. When Anillin is knocked down, active RhoA (RhoA‐GTP), F‐actin, and Myosin‐2 are misregulated at junctions. Indeed, while overall accumulation of RhoA‐GTP is reduced when Anillin is depleted, increased dynamic “flares” of RhoA‐GTP are observed at cell‐cell junctions. Junctional accumulation of F‐actin and Myosin‐2 are also reduced when Anillin is depleted. We propose that Anillin is required for proper RhoA‐GTP distribution at cell‐cell junctions in order to maintain a robust apical actomyosin belt, which is required for cell‐cell junction integrity. These results reveal a novel role for Anillin in regulating epithelial cell‐cell junctions.

Grant Funding Source: Supported by R00GM089765, start‐up funds, NSF Predoc Fellowships & T32GM007315 to C.R. and E.B.
The kinase WNK1 is a powerful inhibitor of the K+:Cl‐ cotransporters (1109.6)
Authors: Melo Zesergio , Murillo Adrián , Chávez‐Canales María , Vázquez Norma , Hadchouel Juliette , Gamba Gerardo , Mercado Adriana
Published: June 2021

The K+:Cl‐ cotransporters (KCC1‐4) belong to the SLC12 family of electroneutral cation‐chloride cotransporters that translocate ions outside the cells to regulate cell volume and intracellular chloride concentration. We previously shown that WNK3 and WNK4 inhibit the activity of KCCs and the elimination of kinase activity turned them into a powerful activators of all KCCs. WNK1 is a ubiquitously expressed kinase that produces two distinct hereditary diseases: pseudohypoaldosteronism type II and hereditary sensory neuropathy 2. Its effect on the regulation of KCCs activity is yet unknown. In the present study, we show using Xenopus laevis oocytes as expression system, that activation of all four KCCs by cell swelling was prevented by coinjection with the various WNK1 isoforms cRNA. The negative effect of WNK1 on the KCCs is kinase dependent, as it was not present when the kinase dead WNK1‐D368A was used. Elimination of the SPAK binding site (WNK1‐F316A) or the HQ motif of WNK1 (required for WNK to WNK interaction; WNK1‐HQ) prevented the inhibitory effect of WNK1 on KCCs, suggesting that interaction between WNK1 monomers and with SPAK is required. In contrast to the kinase dead WNK3, the kinase dead WNK1‐D368A had no effect on the KCCs activity in isotonic conditions. Altogether, our data support that under hypotonic conditions KCCs are regulated by WNK1 and this modulation requires SPAK binding sites in the kinase.

Grant Funding Source: SEP‐CONACyT 165815 GG, 132503 AM
Enteric glial nitric oxide potentiates P2X7‐mediated neuron death (1110.12)
Authors: Brown Isola , Gulbransen Brian
Published: June 2021

Enteric neuron death causes gut dysfunction in inflammatory bowel disease (IBD). Activation of neuronal P2X7 receptors (Gulbransen et. al. 2012) and oxidative stress (Abdo et. al. 2010) promote enteric neuron death. Yet, the source of oxidative stressors and how oxidative stress influences P2X7 activation in inflammation remain unclear. One potential source of oxidative stress is the enteric glial enzyme, inducible nitric oxide synthase (iNOS), which is upregulated in colitis (Green et. al, 2004). We hypothesize that NO produced by glial iNOS potentiates P2X7‐mediated neuron death. We tested our hypothesis using the 2,4‐dinitrobenzene sulfonic acid in vivo model of colitis and an in situ model of P2X7‐driven neuron death. We used the ratio of oxidized/reduced glutathione as an indicator of oxidative stress. We modulated iNOS activity and NO with the drugs 1400W (iNOS inhibitor) and PAPA NONOate (NO donor) and evaluated neuron survival and iNOS expression using immunohistochemistry. We find that glial oxidative stress is increased in inflamed animals (p<0.0001) and that glial NO production increases during colitis (p<0.05) with no change in glial iNOS expression. P2X7‐mediated neuron death in situ is protected against with 1400W (p< 0.05) and potentiated by PAPANONOATE. In conclusion, our results suggest that enteric glia contribute to P2X7‐mediated neuron death in IBD through NO‐dependent mechanisms.
The velocity of pellet propulsion in guinea pig distal colon is differentially modified by short‐chain fatty acids (1110.8)
Authors: Hurst Norm , Kendig Derek , Murthy Karnam , Grider John
Published: June 2021

INTRODUCTION: Short‐chain fatty acids (SCFAs) are produced by commensurate bacteria in the colon. The type and concentration of SCFAs depend on dietary intake of carbohydrate and fiber. While most fermentation occurs in proximal colon, unabsorbed SCFAs entering the distal colon are likely to modify propulsion initiated by fecal pellet‐induced distension. AIM: To determine the effect of SCFAs on pellet propulsion in distal colon. METHODS: The distal colon was removed from guinea pig and placed in an organ bath containing Kreb’s buffer. Following equilibration, video tracking software was used to measure the velocity of propulsion of a clay pellet placed in the orad end of a segment. Pellets propulsion was repeated during intraluminal perfusion of Kreb’s buffer containing individual SCFAs at 30 mM caudad to the pellet at 0.1 ml/min. RESULTS: The mean velocity of pellet propulsion in the absence of SCFAs was 1.5 ± 0.2 mm/sec. Luminal perfusion with 30 mM sodium butyrate increased the velocity of pellet propulsion by 30 ± 4% (p<0.05) whereas luminal perfusion with 30 mM sodium propionate decreased velocity of pellet propulsion by 75 ± 8%. Conclusion: Propionate had opposing effects to butyrate on the velocity of propulsion of artificial clay fecal pellets in the guinea pig distal colon. These studies suggest that the movement of feces in the colon depends not only on distension but also on the chemical (i.e. dietary) composition of the feces and the microbiota.

Grant Funding Source: Supported by DK34153
High fat diet alters homocysteine metabolism and hydrogen sulfide biosynthesis in mice (1116.3)
Authors: Sarna Lindsei , Hwang Sun‐Young , Siow Yaw , O Karmin
Published: June 2021

High fat diets (HFD) are obesogenic and predispose to metabolic syndromes. Non‐alcoholic fatty liver disease (NAFLD), a hepatic manifestation of the metabolic syndrome, is characterized by hepatic steatosis with progression to steatohepatitis in advanced stages. Homocysteine (Hcy) levels are reportedly altered in patients with NAFLD, although the mechanisms responsible remain undefined. Since the liver is one of the principal sites regulating systemic Hcy levels, the objective of the present study was to investigate the effect of a HFD on hepatic Hcy metabolism. Mice were fed a control diet (10% kcal fat) or a HFD (60% kcal fat) for 5 weeks. Compared to control mice, mice fed a HFD gained more weight and developed fatty liver. Serum Hcy levels were significantly lower and corresponded with increased mRNA and protein expression of transsulfuration enzymes cystathionine‐β‐synthase and cystathionine‐γ‐lyase in the liver. Enhanced expression of transsulfuration enzymes also increased hydrogen sulfide biosynthesis in the liver of HFD fed mice. Preliminary results associate these responses to the regulation of hepatic oxidative stress. The significance of our findings as they related to obesity and NAFLD are under investigation
Mesenchymal stem cells isolated from the bone marrow of mice with gastritis induce epithelial‐mesenchymal transition of gastric organoids (1119.5)
Authors: Turner Jacob , Feng Rui , Donnelly Jessica , Schumacher Michael , Zavros Yana
Published: June 2021

Background: Bone marrow‐derived MSCs exhibit characteristics that are reminiscent of a cancer‐associated fibroblast (CAF) phenotype in response to chronic gastritis, (Donnelly et al., 2013, Dig. Dis. and Sci.). Although it is well documented that MSCs migrate to the site of gastric inflammation, the precise role of these cells in cancer development is largely unknown. Hypothesis: MSCs, transformed to a CAF phenotype, induce epithelial‐mesenchymal transition (EMT) of the gastric epithelium. Methods: MSCs were isolated from the bone marrow of either uninflamed C57BL/6 mice (B6 MSCs) or gastrin‐deficient mice exhibiting chronic gastritis (GKO MSCs). B6 MSCs or GKO MSCs were then co‐cultured in transwells using mouse‐derived fundic gland gastric organoids. Gene expression changes were measured by qRT‐PCR in both MSCs and organoids. Results: Compared to B6 MSCs, GKO MSCs exhibited increased expression of IL‐6, TGFβ, VEGF, alpha smooth muscle actin (αSMA), gremlin1 and components of the Hedgehog signaling pathway. Organoids co‐cultured with B6 MSCs appeared as epithelial spheroids with strong E cadherin expression. In co‐culture with GKO MSCs, organoids exhibited a budding phenotype with decreased expression of E cadherin and increased expression of αSMA, Zeb1/2, TWIST1/2, N‐cadherin and VEGF. Conclusion: MSCs isolated from mice with chronic gastritis induce EMT of the gastric epithelium.

Grant Funding Source: 119072‐RSG‐10‐167‐01‐MPC (Y. Zavros)
DNA methylation differences in CD16+ cells between normal weight and obese women of childbearing age (1120.11)
Authors: Hohos Natalie , Park Hea Jin , Shade Deanna , Hausman Dorothy , Meagher Richard , Smith Alicia , Della‐Fera Mary Anne , Bailey Lynn , Baile Clifton
Published: June 2021

It is well established that DNA cytosine methylation epialleles (CG vs 5MeCG) alter the expression of obesity related genes. The current study examined differences in 5MeCG in CD16+ cells isolated from blood using antibody‐bound magnetic beads in normal weight (BMI 18.5‐24.9 kg/m2; n = 12) and obese (BMI > 30 kg/m2; n = 6) women (18‐35y). 5MeCG was measured across > 485,000 CG sites using the HumanMethylation450 BeadChip (Illumina©). 16,969 CG sites were differentially methylated in obese as compared to normal weight women (p < 0.05). 65.4% of these sites had decreased methylation. Six of these CG’s remained associated (p < 10‐7) after adjustment for multiple testing (False Discovery Rate < 0.05), including sites in CRYGB, CDH5, and VASH1. Our results have potential implications in obesity and CDH5 has been shown previously to have differential methylation in peripheral blood leukocytes in obese (Wang et al. doi: 10.1186/1741‐7015‐8‐87). Continued analysis will further examine these methylation differences.

Grant Funding Source: Supported by a grant from the UGA Obesity Initiative and the GRA Eminent Scholar Endowment (CAB)
Bbs1 gene deletion from the leptin receptor‐containing neurons causes obesity (1126.10)
Authors: Guo Deng Fu , Sheffield Val , Rahmouni Kamal
Published: June 2021

Bardet‐Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder with several features including obesity. Systemic knock‐out mice lacking expression of Bbs2, Bbs4 and Bbs6, or Bbs1M390R knock‐in, recapitulated many BBS phenotypes including obesity. However, the role and contribution of Bbs genes in different tissues to obesity remains unclear. To address this, we generated a new conditional knockout mouse where exon 3 of the Bbs1 gene is floxed. Cre‐mediated recombination causes a frame shift resulting in a premature stop. We assessed whether deletion of the Bbs1 gene in the leptin receptor neurons (LRb) affects body weight. Importantly, Bbs1flox/LRbCre mice display an obesity phenotype as indicated by the increased body weight and fat mass measured by MRI. We found that the obesity phenotype in Bbs1flox/LRbCre mice is due to both an increase in food intake and reduction in energy expenditure as indicated by the decreased O2 consumption and heat production. These results indicate that hyperphagia and low metabolic rate explain the development of obesity in Bbs1flox/LRbCre mice. Finally, in HEK 293 cells, silencing Bbs1 or Bbs2 genes significantly decreased LRb, but not LRa or transferrin receptor, trafficking to the plasma membrane highlighting the important role of Bbs gene in the regulation of LRb signaling. These findings demonstrate that Bbs genes are critical for energy homeostasis.

Grant Funding Source: NIH and AHA
Hydrogen sulfide decreases a slow rectifying potassium conductance in nucleus tractus solitarius neurons (1129.4)
Authors: Malik Rishi , Ferguson Alastair
Published: June 2021

Hydrogen Sulfide (H2S) is an endogenously produced gasotransmitter which has been shown to play a role within the central nervous system. Prior studies have shown that H2S has presynaptic effects on neurons of the nucleus tractus solitarius (NTS) through modulation of glutamate transmission. We have also recently demonstrated postsynaptic effects of H2S on the membrane potential of NTS neurons. The current study was undertaken to investigate the ion channels in NTS neurons modulated by H2S. Using whole cell voltage‐clamp recordings we found that H2S causes a decrease in current evoked in the depolarizing range of a slow voltage ramp indicative of modulation of the voltage activated potassium conductance IK. This conductance was found to decrease by a mean of 20.2 ± 2.5% (n=11) at the end of the ramp (10 mV) following application of 10 mM NaHS, a H2S donor, with all 11 neurons influenced in a similar manner. All cells demonstrated a partial to full recovery of this conductance after wash off of NaHS. The present work suggests that H2S controls the excitability of NTS neurons, at least in part through the modulation of the delayed rectifier potassium conductance, IK.

Grant Funding Source: This work was supported by the Canadian Institutes for Health Research.
The effects of altered extracellular pH on dDAVP‐induced phosphorylation (serine 256) and intracellular trafficking of AQP2 (1137.1)
Authors: Choi Hyo‐Jung , Kwon Tae‐Hwan
Published: June 2021

The effects of altered extracellular pH (pHe) on dDAVP‐induced phosphorylation (S256, p‐AQP2) and trafficking of AQP2 were examined in the rat kidney inner medullary collecting duct (IMCD) cells. Freshly prepared IMCD suspension was exposed to buffer with pH 6.4, 7.4, or 8.4 for 1 h. dDAVP (10‐10 M, 3 min)‐induced AQP2 phosphorylation was more prominent when tubule suspension was exposed to pH 7.4 or 8.4, compared to pH 6.4. When primary cultured IMCD cells were exposed to pHe 6.4, 7.4 or 8.4 for 1 h, intracellular pH was changed to 6.1, 7.2 and 8.1, respectively. IMCD cells cultured in transwell chamber were exposed to transepithelial pH gradient for 1 h (pH 6.4, 7.4 or 8.4 at the apical side vs. pH 7.4 at the basolateral side). Laser scanning confocal microscopy and cell surface biotinylation assay revealed that exposure to lower pH (pH 6.4, 1 h) significantly reduced dDAVP (10‐9M, 15 min, basolateral)‐induced AQP2 trafficking and surface expression of p‐AQP2. Importantly FRET analysis revealed that dDAVP (10‐10 M)‐induced increase of PKA activity was attenuated when cells were exposed to pHe 6.4, whereas forskolin (10‐7 M)‐induced PKA activation was not affected. Taken together, decreased pHe is likely to exert an inhibitory effect on dDAVP‐induced AQP2 phosphorylation and intracellular trafficking in IMCD cells, presumably via an inhibition of G‐protein‐cAMP‐PKA action.