bluuebunny/crossref_metadata_2025_split huggingface dataset:
Initiation And Elongation Of RNA Polymerase II Are Regulated By Multiple OGlcNAcylation Events
Authors: Lewis Brian A 
Published: November 2023

RNA pol II is thought to be regulated at two positions on a gene and this regulation determines whether a gene is transcribed. The first is at the promoter, where pol II and the general transcriptional machinery are recruited to form a pre‐initiation complex (PIC). Upon receiving the appropriate signal, pol II initiates transcription and elongates into the gene body. The current dogma states that a hypophosphorylated RNA pol II (pol IIA) is the initiation‐competent species. However, this pol IIA was shown to also contain glcnacylated pol II. Thus, although the O‐GlcNAc modification of the RNA polymerase II CTD was described twenty years ago it has never been clear what the initiation‐specific species of RNA pol II is, or whether O‐GlcNAcylation regulates PIC formation.The second position is located immediately downstream of the transcriptional start site (TSS). In the late 1980s, a transcriptionally engaged, paused pol II was found on several genes, roughly at +50 relative to the TSS. Recent genome‐wide approaches have shown that paused pol II exists on approximately 40% of promoters in the genome and thus understanding the regulation of pausing is a major issue in eukaryotic transcription. It is not known nor has it been suspected that O‐GlcNAcylation plays a role in regulating these events.We have made significant advances in showing functional requirements for O‐GlcNAcylation during both transcription initiation and elongation. Firstly, we have collected several lines of evidence that O‐GlcNAcylation events are required for transcription initiation and the recruitment of RNA pol II to promoters. Notably, OGT and OGA chemical inhibitors block transcription in vitro and inhibition of O‐GlcNAcylation prevents pol II entry into the promoter in vitro and in vivo. Secondly, we have found that chemical inhibition of the O‐GlcNAc aminidase (OGA) has pausing and elongation defects in vivo. In vitro elongation assays also showed a functional requirement for OGA activity in both crude nuclear extract and partially purified systems. Lastly, we identified a 600 kDa complex containing OGA and the pausing/elongation factor DSIF.These data indicate that O‐GlcNAcylation is essential for RNA pol II promoter recruitment and that RNA pol II goes through a cycling of O‐GlcNAcylation at the promoter, where before initiation pol II is in a glcnacylated state, and upon initiation, the O‐GlcNAc is removed. Our data also suggest a novel role of dynamic cycling of O‐GlcNAcylation in RNA pol II elongation control by showing a functional and physical coupling of OGA with the pol II elongation and the pausing machinery. In conclusion, our work has brought to light an entirely new level of regulation of both transcription initiation and elongation, mediated by O‐GlcNAcylation enzymes. These results are possibly manifestations of a direct link between the regulation of RNA pol II activity genome‐wide and the nutrient state of the cell.Support or Funding InformationCCR/NCI/NIH Intramural Research Program
Role of CD36 in ROS‐induced Calcium Influx in Human Lung Microvascular Endothelial Cells
Authors: Reyes Jose , Suresh Karthik , Servinsky Laura , Pearse David Bennett , Shimoda Larissa 
Published: November 2023

RationaleAcute respiratory distress syndrome (ARDS) is characterized by disruption of the endothelial barrier in lung microvascular endothelial cells (LMVEC). Reactive oxygen species (ROS) and increased intracellular calcium concentration ([Ca2+]i) have been implicated in the endothelial barrier dysfunction in ARDS, but the mechanisms linking these processes are not fully understood. We previously showed that exogenous ROS (H2O2) increases intracellular calcium in human LMVEC (HLMVEC) and that the calcium channel TRPV4 contributes to H2O2‐induced Ca2+ influx in HLMVEC. Additionally, our prior data suggest that phosphorylation of TRPV4 by the Src kinase Fyn is necessary for ROS‐induced Ca2+ influx. In endothelial cells, Fyn is known to be tethered to the cell membrane by CD36, a fatty acid scavenger receptor. In mouse LMVEC, we have shown that loss of CD36 significantly attenuates ROS‐induced Ca2+ influx, but the role of CD36 in ROS‐induced Ca2+ influx in HLMVEC is not known. We hypothesized that CD36 is present in HLMVEC, and that it acts as a scaffold for Fyn; thus, we hypothesized that direct inhibition of CD36 would not alter ROS‐induced Ca2+ influx in HLMVEC.MethodsWe measured [Ca2+]i by loading HLMVEC with the ratiometric calcium sensor Fura‐2AM and measuring the change in [Ca2+]i following exposure to H‐2O2 (250 μM) while in a temperature‐controlled flow chamber. Following excitation at 340 and 380 nM, emissions at 510 nM were recorded. Immunoflourescence was performed using fluorescently labeled AcLDL and DAPI nuclear stain in HLMVEC.ResultsCD36 was present in HLMVEC by both immunoblot and immunofluorescence analysis. To determine the effects of direct inhibition of CD36, we treated cells with the CD36 inhibitor succinimidyl oleate (SSO; 100 μM), which has previously been shown, in macrophages, to inhibit fatty acid uptake by CD36 but not the interaction between CD36 and Fyn. To ensure that adequate inhibition had occurred, we assessed uptake of Acetylated‐LDL in HLMVEC with and without SSO treatment. SSO significantly inhibited AcLDL uptake by HLMVEC. Untreated HLMVEC exhibit a large increase in [Ca2+]i following exposure to H2O‐2 that was not attenuated in HLMVEC treated with SSO. These data suggest that while CD36 is necessary for ROS‐induced Ca2+ influx, this mechanism, in HLMVEC, does not occur via direct binding and activation of CD36 by lipid moieties generated by increased intracellular ROS.
Hemoglobin Induced Lipid Peroxidation as an Initiating Factor of Pulmonary Hypertension
Authors: Irwin David , Loomis Zoe L , Redinius Katherine , Eigenberger Paul , Lisk Christina , Buehler Paul 
Published: November 2023

Pulmonary hypertension (PH) is a common etiology of both inherited and acquired forms of hemolytic diseases. It is suspected that cell free hemoglobin (CFH) may initiate vascular remodeling and increased pulmonary pressures, however, the mechanisms behind this pathology remain unclear. It is understood that CFH increases reactive oxygen species and lipid peroxidation through reactions which are exacerbated in pro‐oxidative microenvironments. Recently, oxidative stress has been implicated in NFkB activation through a Toll like receptor 9 (TLR9) mediated pathway; it has been proposed that TLR9 is activated by either nuclear or mitochondrial DNA that has been liberated by stress induced cellular trauma. In this regard, the purpose of this investigation was to test whether hemoglobin induced oxidative stress triggers TLR9 mediated inflammation. We hypothesized that Hb induced lipid peroxidation is linked to TLR9 activation, resulting in IL6 induction and a corresponding activation of the smooth muscle cell proliferation indicative of pulmonary hypertension.MethodsRat pulmonary artery endothelial cells and smooth muscle cells (RPAEC and RPASMC) or HEK TLR9 reporter cells were incubated for 6–24h with HbFe2+ and HbFe3+ in the presence or absence of glucose oxidase (GOX). Lipid peroxidation, TLR9 activity, IL6 expression, and SMC activation were determined using CLICK chemistry, NFkB inducible enzymatic assay, qRTPCR and MTT assay, respectively.ResultsHbFe2+ and HbFe3+ (with or without GOX) induced lipid peroxidation in RPAEC and reporter cell TLR9 activation; IL6 expression in RPAEC was increased when incubated with HbFe2+ and HbFe3+; RPASMC demonstrated a more proliferative state when co‐cultured with RPAEC treated with HbFe2+ and HbFe3+. Glucose oxidase augmented the Hb induced phenotypes in all experiments.ConclusionsHb‐induced lipid peroxidation is associated with TLR9 activation, IL6 induction, and SMC activation. As such, CFH may be an initiating factor and mediator of pulmonary hypertension associated with hemolytic diseases, thus providing clinicians with a potential therapeutic target.Support or Funding InformationNIH NLBI 1RO1HL125642‐01A1
The Role of PP5 in the FKBP51 Inhibition of Isoc Current
Authors: Hamilton Caleb L , Kadeba Pierre I , Abney Kevin A , Honkanen Richard , Scammell Jonathan G , Cioffi Donna L 
Published: November 2023

BackgroundIn acute respiratory distress syndrome, inflammatory signals increase calcium influx into endothelial cells through store‐operated calcium entry (SOCE). SOCE plays a major role in the promotion of inter‐endothelial cell gap formation. SOCE is comprised of two major currents: the calcium release activated current (Icrac) and the store‐operated calcium current (Isoc). Our laboratory has shown that FK506 binding protein 51 (FKBP51) inhibits Isoc current in pulmonary artery endothelial cells (PAECs). However, the mechanism(s) by which FKBP51 inhibits Isoc is unknown. FKBP51 has been shown to mediate its effects in neuronal cells through dephosphorylation events of the microtubule associated protein tau. We propose that a similar cellular event may be responsible for the action of FKBP51 on Isoc. FKBP51 has no endogenous phosphatase activity and is likely dependent on other cellular phosphatases for downstream dephosphorylation of tau. In particular, the role of the protein phosphatase five (PP5) might be implicated because, like FKBP51, it is a co‐chaperone of heat shock protein 90 (HSP90) that has been shown to decreases tau phosphorylation in vivo. We have shown that HSP90 and FKBP51 both co‐precipitate with the Isoc heterocomplex in PAECs. We hypothesize that the FKBP51‐dependent inhibition in Isoc requires PP5.MethodsWe utilize immunocytochemistry and a membrane‐cytoskeletal preparation to determine the localization of PP5 in PAECs. Whole‐cell patch clamp electrophysiology was used to measure Isoc in HEK293 cells and PAECs. Dexamethasone treatment was used to increase FKBP51 expression. CRISPR/Cas9 was used to generate a PP5 knockout HEK293 cell line.ResultsWe show that a subset of PP5 is located at the cell‐cell borders of PAECs and in both the cell membrane and spectrin‐actin cytoskeleton fractions associated with the Isoc heterocomplex. Additionally, we show that dexamethasone treatment leads to decreased Isoc in HEK293 cells. However, in the CRISPR/Cas9 knockout PP5 HEK293 cells, the dexamethasone‐mediated decrease in Isoc was prevented.ConclusionTogether, these findings suggest a role for PP5 in the FKBP51‐mediated inhibition of Isoc.Support or Funding InformationSupported by NIH 5R00HL089361.
Hypocapnia aggravates hypoxia‐induced inhibition of alveolar Na‐transport
Authors: Mairbaurl Heimo , Sahinoz Melis , Baloglu Emel 
Published: November 2023

Hypercapnia that is often associated with ARDS inhibits alveolar fluid reabsorption by PKC‐dependent mechanisms. Also Hypocapnia seems to reduce lung fluid clearance. In both cases inhibition was associated with internalization of Na/K‐ATPase. Hypocapnia is a consequence of hyperventilation in hypoxia. Hypoxia also inhibits alveolar fluid clearance by decreasing Na/K‐ATPase‐ and Na‐channel dependent transepithelial Na transport. It is not known, however, how altered CO2 affects hypoxia‐induced alveolar reabsorption. To answer this question we exposed primary rat alveolar epithelial cells to acute and 24h of hypoxia with 5% and 3% CO2, and measured trans‐monolayer ion transport as short circuit currents (ISC) in Ussing chambers. We show here that inhibition of alveolar reabsorption by hypoxia and hypocapnia was additive. This effect was small after 30 min but well pronounced after 24h of treatment. Ion transport at both CO2‐levels was not affected by acetazolamide, an inhibitor of carbonic anhydrase. In contrast to earlier reports we found no hypocapnia‐induced inhibition of Na/K‐ATPase, i.e. ISC after apical permeabilization with amphotericin A, although there was a decrease in whole‐cell α1‐Na/K‐ATPase protein measured by Western blot. However, we found a decrease in amiloride‐sensitive ISC indicating inhibition of epithelial Na‐channels (ENaC), which was associated with a decrease in apical αENaC protein in hypoxia, which was enhanced by hypocapnia. Our results indicate that hypocapnia aggravates hypoxia‐induced inhibition of alveolar reabsorption which may enhance the risk of formation alveolar edema in hypoxia.Support or Funding InformationTLRC‐H, DZL
Alterations in the Expression of Profilin1/pVASP‐S157 and Cofilin1/pVASP‐S239 in Perinatal Inflammation/Neonatal Hyperoxia‐Induced Lung Injury
Authors: Ali Mehboob , Heyob Kathryn M , Rogers Lynette K 
Published: November 2023

Cell proliferation and migration have been shown to be regulated by actin dynamics/cytoskeletal remodeling. Profilin1, cofilin1 and VASP (phosphorylated at serine‐157 (pVASP‐S157) or serine‐239 (pVASP‐S239)) are the major regulators of actin remodeling within the cell. Infants that develop Bronchopulmonary Dysplasia (BPD) exhibit decreased alveolarization resulting from altered cellular proliferation and migration during septal formation. Using our mouse model of BPD, we tested the hypothesis that changes in expression and cellular localization of profilin1, cofilin1 and phosphorylated VASP play a key role in decreases in secondary septal formation and thus alveolarization.Human lung autopsy tissues from infants that died with BPD or controls were assessed for total F‐actin content and expression of profilin1, colfilin1, and phosphorylated VASP by immunofluorescence. Lung tissues were obtained from our murine model of maternal LPS exposure followed by 7 days of 85% hyperoxia. Murine tissues were harvested at 7 days of life and lung homogenates were assessed for profilin1, colfilin1, and VASP phosphorylation by western blot.A significant decrease in F‐actin content, pVASP‐157, pVASP‐S239, and profilin1 and an increase in cofilin1 were observed in BPD tissues compared to controls. Western blots from lung tissue homogenates indicated increases in cofilin1, profilin1, and pVASP‐S239 but a decrease pVASP‐S157 at 7 days.In conclusion, we propose that these alterations in expression and phosphorylation of ABP may be contributing to the decrease in cell migration and septal formation in immature lungs injured by hyperoxia exposure. The differences between the findings in humans and mice are likely to be due to the early age of the mice, prior to onset of severe symptoms. Further investigations are needed to define the specific cell types affected and the role of actin binding proteins in lung development.Support or Funding InformationThis work was supported by the National Institutes of Health, NCCIH/ODS (LKR R01AT006880).
An Acute Sustained Hypoxic Stress is Sufficient to Cause Respiratory Muscle Weakness in the Mouse
Authors: O'Leary Andrew J. , O'Halloran Ken D. 
Published: November 2023

Respiratory muscle weakness is a strong predictor of poor outcome in patients. Acute hypoxia (AH) is a feature of respiratory conditions such as acute respiratory distress syndrome (ARDS) and ventilator‐associated lung injury (VALI). However, the effects of AH on the respiratory muscles are largely unknown despite the clinical relevance. We assessed the effects of AH exposure on ventilation, respiratory muscle contractile function and gene expression.Adult male C57BL6/J mice (n=8 per group) were exposed to 1, 4 or 8 hours of hypoxia (FiO2 = 0.10) or normoxia (FiO2 = 0.21). Ventilation was assessed using whole‐body plethysmography during gas exposure. Sternohyoid, diaphragm and EDL muscles were excised post‐mortem. Respiratory muscle contractile performance was analysed ex‐vivo. Muscle gene expression was examined using qRT‐PCR.Minute ventilation was increased (p<0.01, two‐way ANOVA & Bonferroni post hoc test) after 10mins of AH compared with normoxia, returning to levels equivalent to normoxia by 20mins and remaining similar to normoxia thereafter. AH decreased tetanic force production in the sternohyoid (12.80 ± 1.15 vs. 9.72 ± 1.06 N/cm2, p=0.0683, unpaired t‐test) and in the diaphragm (29.53 ± 3.15 vs. 20.88 ± 1.98 N/cm2, p=0.0334). AH differentially altered PGC1α, NFκB1 and selenoprotein N1 mRNA levels in the diaphragm while decreased NRF1, NFκB1, junctophilin 1 & 2, ryanodine receptor 1, calsequestrin 1, dihydropyridine receptor, and selenoprotein N1 mRNA levels were observed in the sternohyoid (p<0.05, one‐way ANOVA & Tukey's post hoc test). No significant changes were observed in the mRNA expression of PPARα, PGC1β, or NRF2 in either muscle in response to AH exposure. UCP‐3 gene expression was increased (p<0.05, one‐way ANOVA & Tukey's post hoc test) in AH diaphragm, sternohyoid and EDL (limb) muscle.Diaphragm weakness is reported in mechanically ventilated patients, which is primarily attributed to inactivity of the muscle per se, although this is controversial. A potential role of hypoxia in the development and/or exacerbation of this weakness is unclear. Here we show that AH is sufficient to cause respiratory muscle weakness with resultant decreased mechanical power outputs. Functional changes may relate to altered mitochondrial function, atrophy, and/or SR Ca2+ release. Following the acute hypoxic ventilatory response at 10mins, ventilation in AH remains at normoxic levels thereafter (most likely due to a decreased metabolic O2 demand during AH exposure), suggesting that the altered gene expression and muscle weakness observed are not due to enhanced respiratory muscle activity but relate to hypoxic stress per se. These findings highlight a potentially critical role for hypoxia in the respiratory muscle dysfunction observed in acute respiratory‐related disorders and mechanically ventilated patients.Support or Funding InformationSupported by the Department of Physiology, UCC and the UCC Strategic Research Fund.
Developmental Window Exists in the Hypoxic Ventilatory Response of Juvenile and Adult Male Rats Following Carotid Sinus Nerve Transection
Authors: Coffee Gregory A. , Getsy Paulina M. , Lewis Stephen J. 
Published: November 2023

The carotid bodies (CB) are peripheral chemoreceptors that respond to hypoxia by stimulating breathing and increasing blood pressure in order to restore oxygen (O2) levels. The carotid body activates chemosensory afferent terminals of the carotid sinus nerve (CSN), which stimulate the brainstem to increase ventilation. Data from our lab suggests a critical developmental window exists in which the hypoxic ventilatory response (HVR) switches from neonatal to adult. This hypoxic ventilatory response (HVR) seen in male adult rats is blunted after bilateral carotid sinus nerve transection (CSNX). Our objective is to determine the HVR of juvenile male Sprague Dawley rats before and after bilateral CSNX. To test this we performed CSNX at postnatal (P) age P21, and exposed the rats to hypoxia (10% O2) using the BUXCO unstrained, awake, whole‐body plethysmography system four days post surgery. Our results suggest the HVR seen in control juvenile rats is blunted compared to adult rats (n= 12 juvenile and n = 9 adult). Also, after CSNX the juvenile rats exhibit a more diminished HVR compared to adult rats (n = 3 juvenile and n = 9 adult). This data suggests that in control male rats, a critical developmental window may exist in which the carotid body becomes fully sensitized to low blood O2, thus allowing the greater HVR seen in the adult. In addition, our data shows that a secondary means of responding to hypoxia after CSNX may not have functionally developed in the juvenile rats.Support or Funding InformationNIH PO1‐HL1018710
To Sigh Or Not To Sigh: Role Of Glia In Sigh Generation
Authors: Dashevskiy Tatiana , Bloom Jacob , Ramirez Jan‐Marino 
Published: November 2023

The respiratory network generates two distinct rhythmic activities: normal respiration (eupnea), which is relatively fast and sighs that occur at a very slow, but regular frequency. Understanding how the slow periodicity of sighs is generated has been challenging. Here, we propose that glia cells play a critical role in setting up the periodicity of sighs. In mouse transverse brain slice preparations containing the respiratory network, specifically the pre‐Bötzinger complex (PreBötC) we recorded Ca2+ signals over a time interval of 2 to 5 min to capture sighs, and subsequently applied aCSF solution containing 0KCl to differentiate glia from neurons. Neurons drastically decrease their Ca2+ signal, while glia increase intracellular Ca2+ in response to 0KCl. Among the glia cells we observed Ca2+ signals that were synchronized with the periodic sighs. In fact, the phase shift analysis between sighs and Ca2+ signal from non‐neuronal cells showed that on average Ca2+ spikes preceded sigh activity. Furthermore, glia toxins inhibit sighs while eupneic activity was preserved. Based on our pharmacological manipulations in vitro and in vivo we propose the hypothesis that release of glial ATP through activation of P2Y receptors activates glial and neuronal Gq/11 protein. Gq/11 in turn increases glial and neuronal intracellular Ca2+, which in turn leads to the release of glial ATP, thus locking the cycle. Our experimental data are consistent with our computational approach that models the neuronal‐glial interaction. The network model predicts that the presence of IP3‐like channels is necessary to produce “sigh” behavior and to mimic transitions between activity states caused by neuromodulators. We analyze the model dynamics and describe dose‐response curve to neuromodulator such as norepinephrine. Our theoretical data are not only similar to our in vivo and in vitro experimental data, but the model also predicts that depending on the modulatory state of the respiratory center, the network can exhibit bi‐stable behavior, meaning that sighs can be triggered between “on” and “off” states by short perturbation. We detected two different mechanisms that support sigh bi‐stability. The first mechanism is based on the presence of a saddle equilibrium and the range of Ca2+ parameters where the observed bistability is limited by sub‐critical Andronov‐Hopf bifurcation and homoclinic bifurcation for periodic cycle. The second mechanism is based on the presence of a saddle periodic orbit and the parameter range is defined by sub‐critical Andronov‐Hopf bifurcation and saddle‐node for periodic orbits bifurcation. This theoretical prediction is consistent with our experiments in in vivo anesthetized mice: Freely breathing anesthetized mice that did not spontaneously produce sighs were stimulated by repeated, brief pulses of hypoxia, which elicited sustained periodic sighs.Support or Funding InformationNIH: F32 HL121939‐01