bluuebunny/crossref_metadata_2025 huggingface dataset:
Peripheral Coupling Sites Formed by STIM1 Govern the Contractility of Vascular Smooth Muscle Cells
Authors: Krishnan Vivek , Ali Sher , Gonzales Albert , Thakore Pratish , Griffin Caoimhin , Yamasaki Evan , Alvarado Michael G. , Johnson Martin T. , Trebak Mohamed , Earley Scott
Published: May 2022

Peripheral coupling between the sarcoplasmic reticulum (SR) and plasma membrane (PM) forms signaling complexes that regulate the membrane potential and contractility of vascular smooth muscle cells (VSMCs). However, the mechanisms responsible for these membrane interactions are poorly understood. In many cells, STIM1 (stromal‐interaction molecule 1), a single transmembrane‐domain protein that resides in the endoplasmic reticulum (ER), transiently moves to ER‐PM junctions in response to depletion of ER Ca2+ stores and initiates store‐operated Ca2+ entry (SOCE). Fully differentiated VSMCs express STIM1 but exhibit only marginal SOCE activity. We hypothesized that STIM1 is constitutively active in contractile VSMCs and maintains peripheral coupling. In support of this concept, we found that the number and size of SR‐PM interacting sites were decreased and SR‐dependent Ca2+ signaling processes were disrupted in freshly isolated cerebral artery SMCs from tamoxifen‐inducible, SMC‐specific STIM1‐knockout (Stim1‐smKO) mice. VSMCs from Stim1‐smKO mice also exhibited a reduction in nanoscale colocalization between Ca2+‐release sites on the SR and Ca2+‐activated ion channels on the PM, accompanied by diminished channel activity. Stim1‐smKO mice were hypotensive and resistance arteries isolated from them displayed blunted contractility. These data suggest that STIM1 – independent of SR Ca2+ store depletion – is critically important for stable peripheral coupling in contractile VSMCs.
FOXM1 Promotes Intestinal Vascular Endothelial Cell Proliferation and Protects Neonatal Mice against Necrotizing Enterocolitis
Authors: Yan Xiaocai , Managlia Elizabeth , Zhao You‐Yang , Tan Xiao‐Di , De Plaen Isabelle G.
Published: May 2022

Necrotizing enterocolitis (NEC) is a life‐threatening gastrointestinal illness affecting mostly premature infants characterized by intestinal inflammation and necrosis. While several factors such as abnormal intestinal colonization and an immature immune response have been shown to play a role in NEC, we previously found that defective intestinal microvascular development promotes NEC development. FOXM1 is a protein expressed in cells undergoing proliferation and a member of the Forkhead box family, a master regulator of cell cycle progression. FoxM1 has been shown to mediate endothelial cell proliferation under stress conditions. Here we hypothesize that promoting FOXM1 expression protects neonatal mice against intestinal injury in an experimental NEC model by promoting endothelial cell proliferation. To test our hypothesis, we first examined FOXM1 mRNA expression during NEC development in a neonatal mouse model. Second, we investigated the role of FOXM1 in NEC development using FOXM1 transgenic and knockout animals. Finally, we examined in vitro whether FOXM1 can protect endothelial cell proliferation under stress condition. We found that FOXM1 expression was significantly decreased in the intestine of pups exposed to the NEC model for 24 and 48 hours compared to dam‐fed littermate controls. Neonatal mice that lack of FOXM1 in endothelial cells showed higher mortality rate and more severe intestinal injury when exposed to the experimental NEC model. While their 3‐day survival remained unchanged, pups with endothelial‐cell specific overexpression of FOXM1 had decreased incidence of severe intestinal injury. Furthermore, when cultured in vitro, intestinal endothelial cells obtained from pups overexpressing FOXM1 showed increased proliferation, which was unaffected by VEGFR2 inhibition using VEGFR2 kinase inhibitor Ki‐8751. In conclusion, FOXM1 protects against neonatal NEC by promoting endothelial cell proliferation.
Liver energy homeostasis impacts hypothalamus gene expression and food intake regulation
Authors: Ponte Michael , Prom John , Newcomb Mallory , Morris E. Matthew
Published: May 2022

Several neuronal populations within the hypothalamus are known to modulate feeding behavior in mammals. Specifically, the arcuate nucleus contains appetite‐suppressing neurons pro‐opiomelanocortin (POMC), and appetite‐stimulating neurons neuropeptide Y (NPY)/agouti‐related peptide (AgRP) neurons. These neurons receive peripheral signals through blood and peripheral vagal afferents fibers. However, it is not well understood how peripheral signals, such as liver energy metabolism, may impact these neuronal populations to modulate feeding behavior. Herein, we used a hepatocyte‐specific PGC1a heterozygous (LPGC1a) mouse model, with associated reductions in mitochondrial fatty acid oxidation and respiratory capacity, to assess the role of liver energy homeostasis in the expression of arcuate nucleus genes involved in food intake regulation, and acute feeding behavior after peripheral delivery of nutrient or peripheral satiation hormones. To assess the impact of liver energy changes on arcuate nucleus gene expression, we used a fasting/refeeding paradigm (16‐hour fast, 2‐hour food withdrawal, and 16‐hour fast with a 4‐hour refeed). We observed reductions in liver PGC1a mRNA expression in LPGC1a compared to wildtype (WT) during all fasting and refed states. LPGC1a mice had significantly lower liver ATP and increased AMP during a fasted state. Suggesting that during a fasted state, the LPGC1a mice struggle to maintain liver energy homeostasis. Furthermore, LPGC1a mice had significantly increased AgRP mRNA expression in the hypothalamus during a fast. Interestingly, POMC expression was lower in LPGC1a mice at 2hrs and refed compared to WT and showed no change across any of the energy states. Importantly, acute food intake following 2 hr food withdrawal was not inhibited by intraperitoneal glucose or GLP‐1 in LPGC1a mice. Together these results suggest that a reduced liver energy state modulates activity of hypothalamic POMC and AgRP neurons, potentially contributing to alterations in feeding behavior of LPGC1a mice previously observed.
Effects of Dibutyltin Exposures on Translation Regulatory Factors eIF4E, eIF4B, and S6 in Human Immune Cells
Authors: Ruff Amanda , Whalen Margaret
Published: May 2022

Dibutyltin (DBT) is an organotin that contaminates the environment due to its uses as a stabilizer in polyvinylchloride, PVC plastics and as a deworming agent in certain poultry products. DBT has been found in drinking water and other beverages due to leaching from PVC plastics used during the distribution of drinking water and storage of beverages such as beers and wines. DBT has been found in human blood at levels as high as 0.3μM. Inflammatory cytokines are key mediators in the response to injury or infection. However, if their levels are increased in the absence of a necessary immune response, chronic inflammation is possible. Chronic inflammation is associated with various disorders including, rheumatoid arthritis, Crohn’s disease, atherosclerosis, and cancer. DBT can increase the production of pro‐inflammatory cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL‐1β), and interleukin 6 (IL‐6) in human immune cells. DBT appears to utilize the ERK 1/2 and/or p38 MAPK pathways to stimulate pro‐inflammatory cytokine production in immune cells. MAPK pathways are capable of regulating translation including processes leading to the phosphorylation (activation) of eukaryotic initiation factor 4E (eIF4E), eIF4B, and the S6 ribosomal subunit. The current study examines the levels and phosphorylation state of eIF4E, eIF4B and S6 after 6‐hour and 24‐hour exposures to DBT in peripheral blood mononuclear cells (PBMCs). DBT (at all concentrations) caused a robust increase in phosphorylation (activation) of eIF4B (S406) within 6‐hour exposure across all donors. Additionally, elevated levels of phosphorylated and total S6 were observed at 0.1 μM in all donors in 6‐hour exposures. At 24 hours of exposure, increases in P‐S6, S6 and P‐eIF4B (S406) begin to taper off with increases observed sporadically amongst donors. These results suggest that DBT may be in part elevating the production of key pro‐inflammatory cytokines in immune cells by its ability to activate translation.
Exercise and Hypercapnia Differentially Modify Ratios of Extracranial and Intracranial Pulsatility
Authors: DuBose Noah , Ainslie Philip N. , Sherman Sara R. , Baynard Tracy , Hoiland Ryan , Smith Kurt J. , Green Daniel J.
Published: May 2022

INTRODUCTION

Damping of pulsatile flow between extracranial and intracranial cerebral arteries is an essential allostatic mechanism protecting cerebral microvessels from recalcitrant hemodynamics. The ratio of pulsatility between proximal and distal cerebral arteries may provide a measure of cerebrovascular hemodynamic damping. This might prove useful as an evaluation of cerebrovascular regulation in response to pulsatile perturbations.

PURPOSE

To characterize cerebral pulsatile damping between extracranial and intracranial environments in response to perturbations eliciting matched shear stress, such as exercise and hypercapnia.

METHODS

Participants (n=10) completed two 30‐min experimental conditions aimed at matching cerebral artery shear stress, each separated by 48 hrs: (1) mild hypercapnia (CO2; FICO2:0.045) and (2) submaximal cycling (EX; 60%HRreserve). Cerebral pulsatility index (PI: (systolic velocity‐diastolic velocity)/mean velocity)) was assessed at baseline, during, and following each condition in the internal carotid artery (ICA) and middle cerebral artery (MCA) using Doppler ultrasound. Heart rate (HR) and blood pressure (BP) were assessed continuously using ECG and photoplethysmography, respectively. Cerebral pulsatile damping was calculated: (ICA PI / MCA PI) to investigate ratios of cerebral pulsatile hemodynamics between extracranial and intracranial arteries.

RESULTS

Cerebral pulsatile damping was greater during CO2 (1.66 ± 0.31) than EX (1.22 ± 0.35) (time*condition effect, p=0.002). The change in cerebral pulsatile damping was related to the change in heart rate (r = ‐0.70, p = 0.04), but not BP between baseline and the experimental conditions.

CONCLUSIONS

As evidenced by the response in cerebral pulsatile damping, exercise and hypercapnia result in different ratios of extracranial to intracranial pulsatility despite inducing similar vasodilation when shear stress was matched. This might in part be explained by differences in HR between conditions. Further research is important to elucidate the mechanisms behind the deviance in hemodynamic responses.
Is HFpEF Caused by Coronary Microvascular Disease?
Authors: Sompalle Saiaravind , Domingo Catherine N. , Kim Parker , Kegecik Karlina , Pucci Thomas , Shockling Lindsay , Yin Liya , Dong Feng , Chilian William , Ohanyan Vahgan
Published: May 2022

Heart failure is responsible for one of eight deaths in the United States. Despite advances in the treatment of heart failure, e.g., beta1‐adrenergic antagonists, angiotensin converting enzyme inhibitors, these therapies are successful only in one type of HF—heart failure with reduced ejection fraction (HFrEF). Another type of heart failure termed HFpEF (heart failure with preserved ejection fraction) presents a far more difficult condition, because there have been no successful clinical trials demonstrating an effective therapy. Moreover, epidemiological data show the prevalence of HFpEF is increasing while that of HFrEF is decreasing. In some studies of patients with non‐obstructive coronary disease, categorized by poor coronary reserve without obstructive coronary artery lesions, HFpEF is observed. Because such patients have coronary microvascular disease, we proposed that a murine model of coronary microvascular disease may also present with HFpEF. In mice null for Kv1.5 channels (KO), a model of inadequate coronary metabolic dilation, we have observed poor exercise tolerance, shortened lifespan, and ventricular fibrosis. We hypothesized that inadequate metabolic dilation in the KO mice leads to chronic coronary insufficiency, i.e., inadequate myocardial blood flow, which would lead to cardiac apoptosis resulting in diffuse fibrosis, stiffening, and culminating in HFpEF. Although many laboratories use standard echocardiographic measurements of diastolic function as verification of HFpEF, such measurements may be spurious in small animals, such as mice, because several indices of diastolic function are heart rate dependent. To overcome this hindrance, we directly evaluated left ventricular diastolic stiffness by measuring LV pressure‐volume (PV) loops, using the slope of the end‐diastolic pressure and volume points (in anesthetized mice using a PV solid‐state transducer). LV PV loops were measured in anesthetized, wild‐type control (WT) and the KO mice during changes in preload induced by slow volume infusion. The series of PV loops were superimposed, and a computer best fit the diastolic PV points to a line, where the slope of the PV line reflects LV diastolic elastance (stiffness). The average slope of the LV end‐diastolic PV lines were roughly doubled in the KO vs WT (P<0.05), indicating increased stiffness. Moreover, LV end‐diastolic pressures were nearly tripled in the KO vs WT, 7.3±1.6 vs 2.8±1.7 mmHg, respectively. (P<0.05). In conclusion, a model of inadequate coronary dilation in mice, which we believe parallels patients with coronary microvascular disease, leads to increased stiffening of the left ventricle and HFpEF.
Membrane Targeting of Constitutively Active Gαq Regulates Inhibition by YM‐254980
Authors: Dwyer Morgan , Randolph Clinita , Aumiller Jenna , Inoue Asuka , Wedegaertner Philip
Published: May 2022

Uveal melanoma, the most common cancer of the eye in adults, is driven by an activating glutamine to leucine or proline mutation at residue 209 of the α subunit of G proteins Gq or G11 (Gαq/11QL or Gαq/11QP). This mutation is found in more than 90% of cases and up to 50% of patients experience untreatable metastases. YM‐254980 is a promising inhibitor of constitutively active (CA) Gαq/11, though its mechanism of action is not well understood. Evidence supports that although Gαq/11 has 2 sites of palmitoylation that allow for association with the plasma membrane (PM), these proteins are still found in subcellular locations. We have shown that more strongly targeting GαqQL to membranes by adding an N‐terminal myristoylation site renders it insensitive to YM, suggesting a requirement of Gαq/11 translocating off the membrane to be inhibited. To further understand how PM‐restricted GαqQL loses sensitivity to YM inhibition of signaling, we have generated additional membrane targeting mutants of GαqQL. Although palmitoylation at cysteines 9 and 10 of GαqQL is essential for signaling, preventing palmitoylation by mutating both cysteines 9 and 10 to serines in the context of myristoylated GαqQL does not abolish signaling, as assayed by TEAD‐ and SRE‐dependent luciferase reporter assays in HEK293 q/11 knockout cells. Moreover, signaling by this myristoylated, palmitoylation‐deficient GαqQL mutant remains insensitive to YM, indicating that introduction of a single site for myristoylation, in the absence of any palmitoylation, is sufficient to render GαqQL resistant to YM. To further define the mechanism of YM insensitivity of membrane targeting mutants of GαqQL, we are currently examining other CA Gα subunits, specifically Gα16 (3 sites of palmitoylation) and Gα13 (2 sites of palmitoylation), that have been mutated to introduce a YM binding site. We have generated myristoylated mutants of these Gα subunits, and are testing if they, like myristoylated GαqQL, are resistant to inhibition by YM. These experiments will highlight the requirements of membrane association for signaling by GαqQL and reveal insights into the mechanism of YM inhibition.
The Interaction of the AAT Protein with Alpha‐1 Antitrypsin Deficiency and Other Respiratory Disorders
Authors: Pore Aarushi , Paul Meirah , Faseru Kunmi , Awe Taiwo
Published: May 2022

Alpha‐1 antitrypsin (AAT) deficiency is a codominant genetic disease. Ordinarily, the liver makes large amounts of the protein AAT and releases it into the blood in its normal M form. It was hypothesized that if there is a deficiency of the AAT protein in the human body, which is caused by the misfolding of the Z variant of the protein in the liver, then AAT deficiency disorder occurs, which contributes to a multitude of lung and liver diseases. This Z variant results from an encoded misfolded variant where the glutamic acid in position 342 of the protein is replaced by a lysine. Structurally, AAT is a proteinase inhibitor and binds to human leukocyte elastase, which prevents damage to the lower respiratory tract. AAT deficiency is not a disease itself, but is considered a predisposition for the development of a number of diseases, mainly pulmonary emphysema and other chronic respiratory disorders. Through literature review as well as conversing with an expert on AAT protein and deficiency, it was found that the insufficient release of AAT into the blood causes low blood levels and increased susceptibility to respiratory diseases, as the function of the AAT protein is to provide protection to the lungs against such diseases. In its ideal functionality, conditions such as emphysema, cirrhosis, and COVID‐19 are protected against, but with AAT deficiency, the individual is more susceptible to these conditions. Although AAT deficiency is inherited genetically, environmental factors can contribute to the early onset of the disorder. Some examples are the utilization of lung irritants, such as smoking or vaping, as well as irritants in the forms of particles found in coal mining and steel millwork. Avoiding these environmental factors as much as possible can allow for an individual to mitigate symptoms of AAT deficiency for a prolonged period of time. Currently, there is no permanent cure to this disease other than frequent infusions, but there are various treatments under research. Studies have been focused on the geographical reach of AAT, as well as newly discovered and efficient treatment options for AAT deficiency. Researchers have been additionally synthesizing information pertaining to the AAT protein being heavily correlated with COVID‐19, which can aid in the research of similar respiratory diseases.
Adaptation of thermophilic enzymes from an ancestral reductive TCA cycle for carbon fixation in plants
Authors: Rose Robert B. , Buhrman Greg , Baer Hayden
Published: May 2022

The reductive TCA cycle (rTCA) is one of 5 carbon‐fixing cycles in prokaryotes other than the Calvin‐Benson cycle ubiquitous in plants. Adaptation of these cycles to biofuel crops is one strategy for reducing the greenhouse gas CO2 and simultaneously increasing crop yields. A minimal 5‐enzyme synthetic rTCA cycle was designed at NCSU utilizing 2 unique enzymes to carboxylate and reduce 2‐oxoglutarate to isocitrate. The enzymes are derived from an evolutionarily primitive configuration of the rTCA cycle that persists in the order Aquificales in hot springs and deep ocean thermal vents. Two strategies are being pursued to engineer mesophilic forms of these enzymes: 1) reducing the temperature of activity of the Aquificales enzymes, and 2) modifying the substrate specificity of homologous mesophilic enzymes. The first enzyme, 2‐oxoglutarate carboxylase (OGC), is a member of the biotin‐dependent carboxylase family homologous to pyruvate carboxylase. Biotin‐dependent carboxylases are multi‐subunit complexes. So far, we have identified the temperature‐dependent steps of the biotin carboxylase domain of OGC and predicted mutations to reduce the temperature of activity based on the crystal structure. Phylogenetic analysis suggests that OGC and pyruvate carboxylase diverged prior to Aquificales evolution. An ancestral promiscuous enzyme has been proposed to carboxylate both pyruvate and 2‐oxoglutarate. Ancestral enzymes are being reconstructed from phylogenetic analyses to switch the substrate specificity of mesophilic pyruvate carboxylase to carboxylate 2‐oxoglutarate. The second enzyme, oxalosuccinate reductase (OSR), is homologous with isocitrate dehydrogenase. In fact, isocitrate dehydrogenase has replaced OGC/OSR in most organisms utilizing the rTCA cycle. Isocitrate dehydrogenase favors the oxidative decarboxylase of isocitrate in the TCA cycle, while OGC/OSR favors the reductive cycle. OSR is an ancestral, non‐decarboxylating isocitrate dehydrogenase. We propose to engineer a mesophilic OSR by mutating isocitrate dehydrogenase to inhibit decarboxylation, based on phylogenic and structural studies of OSR. Ultimately engineering a mesophilic OGC/OSR will be utilized to increase carbon capture in seed oil crops like Camelina sativa that are carbon limited. Oil from Camelina seeds can be converted to jet fuels.
Early Life Stress and Heart Function in the Pristane‐Induced Model of Systemic Lupus Erythematosus (SLE) in Mice
Authors: Cruz‐Ojeda Felix O. , Kellum Cailin E. , He Lihao , Xie Min , Pollock Jennifer S.
Published: May 2022

Early life stress (ELS) is defined as childhood adversity such as maltreatment (e.g., verbal and physical abuse), sexual abuse, and household dysfunction occurring prior to age 18. ELS is associated with increased risk of chronic disease and poorer health outcomes earlier in adulthood compared to those without exposure to ELS. SLE is an autoimmune disease most prevalent in women with high mortality from cardiovascular disease and influenced by environmental factors including psychosocial stress such as ELS. SLE affects multiple organs including the heart. Pericarditis and heart failure are known pathologies affecting SLE patients at a higher rate than patients without SLE, however the influence of ELS on heart disease development is unknown. We hypothesized that a mouse model of ELS, maternal separation with early weaning, MSEW, have increased risk of heart disease particularly in response to the development of SLE in adulthood. MSEW entails separating pups from the dam 4h/day (postnatal, PD 2 to 5) and 8h/day (PD 6 to 16) followed by weaning at PD17. Normally reared (NR) litters remain with the dam and are weaned at PD21. The pristane‐induced model of SLE uses a single intraperitoneal (i.p.) injection to induce SLE‐like symptoms in female mice. Previous data have shown that Anti‐Smith autoantibody is not elevated in the SLE animals at 5 months after the i.p. injection compared to the NR and MSEW controls (P=0.0514). However, there is an increase in the presence of Anti‐Smith autoantibody in the SLE animals at 6 months after the i.p. injection (P=0.0007). Therefore, our objective was to determine parameters of systolic and diastolic heart function in NR control mice (NR+CON), NR+SLE, MSEW, and MSEW+SLE at time points early in the development of SLE at 4 months post‐injection with a VEVO 3100 high resolution echocardiography. Systolic function assessed by Ejection Fraction (P=0.3535), Fractional Shortening (P=0.3301), Left ventricular Mass (P=0.9216), Left Ventricular Diastolic Volume (P=0.4767), and Left Ventricular Systolic Volume (P=0.9568) showed no significant differences. Similarly, diastolic function assessed by E/e’ ratio (P=0.9159), Myocardial Performance Index (P=0.2867), MV E/A ratio (P=0.9231), and e’/a’ ratio (P=0.1104) showed no significant differences. These results suggest that the pristane‐induced model of SLE or ELS did not induce systolic or diastolic dysfunction early in the development of SLE. Future work will focus on a longitudinal strategy for the identification of heart dysfunction in mice with SLE.
Alcohol Dysregulates CD4 T Cell Mitochondrial Homeostasis
Authors: McTernan Patrick M. , Siggins Robert W. , Simon Liz , Molina Patricia E.
Published: May 2022

CD4 T cell differentiation to pro‐inflammatory and immunosuppressive subsets requires distinct metabolic pathways. Pro‐inflammatory CD4 subsets rely on glycolysis, while immunosuppressive (Treg cells) subsets, require functional mitochondria for their differentiation and function. Previous studies have shown that binge alcohol (ethanol, EtOH) administration increased Tbet‐expressing (Th1) and decreased FOXP3‐expressing (Treg) CD4 T cells in the colons of mice. We tested the hypothesis that EtOH dysregulates normal CD4 T cell differentiation, after stimulation, by impairing mitochondrial homeostasis. Human naïve CD4 T cells were isolated from buffy coats from blood bank donors (N = 6) using MACS sorting. Cells were stimulated using anti‐CD3‐coated dishes in the presence of anti‐CD28 and IL‐12, and exposed to EtOH (0 and 50 mM) for 3 days. Mitochondrial content was measured with Mitotracker Deep Red. Gene expression indicative of: autophagosome formation (ATG5, ATG7, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1), mitophagy (PINK1, PRKN),mitochondrial fusion (MFN1, MFN2, OPA1), mitochondrial fission (MFFand FIS1), and mitochondrial biogenesis (PPARC1A, PPARC1B, TFAM) was determined by RT2 profiler arrays. EtOH‐treated CD4 T cells had increased mitochondrial content (p = 0.0008) with Tregs accounting for the greatest increase in mitochondria (p = 0.04). There was a main effect of stimulation (p < 0.05) to increase ATG5, ATG13, MAP1LC3B, BECN1, BNIP3L, ULK1, MFF, PPARC1B, and TFAM, and a main effect of EtOH (p < 0.05) to increase PINK1 and decrease ATG7. There was a main effect of both EtOH and stimulation (p < 0.05) to increase MFN2, and OPA1.Taken together, these results indicate that EtOH increases mitochondrial content in Treg cells and dysregulates mitochondrial gene expression important for mitochondrial repair and mitophagy. These EtOH‐mediated alterations in gene expression could result in an inability of CD4 T cells to maintain mitochondrial homeostasis and remove damaged mitochondria that is required for normal differentiation and function of anti‐inflammatory Treg cells.
Processing of DNA Clamp Loader Subunit DnaX Is Important in the Absence of Caulobacter Cell Division Inhibitors
Authors: Tashjian Tommy F. , Chien Peter
Published: May 2022

The DNA clamp loader is critical to the processivity of DNA replication and to the coordination of synthesis on the leading and lagging strands. In bacteria, the major subunit of the clamp loader, DnaX, has two forms: the essential full‐length DnaX tau and a shorter DnaX gamma. These forms are conserved across bacterial species and three distinct mechanisms have been found to create them such as ribosomal frameshift or transcriptional slippage. In Caulobacter crescentus, the full‐length DnaX tau is partially proteolyzed by ClpXP into two shorter DnaX gamma forms. Despite being discovered over 30 years ago, the purpose of maintaining multiple DnaX forms is unknown.

To examine the purpose of encoding two forms, we created a nonproteolyzable allele of DnaX (tau‐only) which had no observable growth or morphological defects but showed a mild sensitivity to DNA damage. Adding an additional plasmid encoded copy of dnaX(tau‐only) to the dnaX(tau‐only) strain causes growth defects, filamentation, and increased RecA induction. These defects are not observed when adding a plasmid copy of dnaX(tau‐only) to the wild type strain, suggesting that an excess of DnaX tau is toxic, and likely causes replication stress.

To better define the pathways that are affected in the absence of DnaX gamma, we performed transposon sequencing analysis to identifying genes that cause synthetic interactions with the dnaX(tau‐only) allele. We found that the insertions in two cell division inhibitors, sidA and didA, were underrepresented in the dnaX(tau‐only) strain compared to wild type. Consistent with this finding, we observed that adding a plasmid copy of dnaX‐tau‐only in a ΔsidA or ΔdidA strain causes an extreme growth defect and filamentation. Interestingly, although sidA is expressed as part of the RecA/LexA‐dependent DNA damage response in Caulobacter, we do not see a similar filamentation when we add an exogenous copy of the dnaX‐tau‐only allele to a ΔrecA strain. This indicates that the uninduced level of sidA expressing is sufficient to suppress the growth and filamentation phenotypes. The observation that cell division inhibitors are critical in the absence of proper DnaX processing suggests that the coordination of DNA replication and cell division is not intact in the dnaX‐tau‐only allele.
A protein mutated in Parkinson's disease prevents damage of metabolites and proteins caused by a glycolytic metabolite
Authors: Heremans Isaac P. , Caligiore Francesco , Gerin Isabelle , Bury Marina , Lutz Marilena , Graff Julie , Stroobant Vincent , Vertommen Didier , Teleman Aurelio , Van Schaftingen Emile , Bommer Guido T.
Published: May 2022

The accumulation of cellular damage is suspected to contribute to aging and neurodegenerative diseases. Oxidative stress and environmental factors likely play a role, but other damaging agents likely remain to be discovered

Here we report that an enzyme mutated in hereditary Parkinson’s disease achieves the seemingly impossible task to prevent damage caused by a glycolytic metabolite without affecting the metabolite itself. Inactivation of this enzyme in model systems ranging from flies to human cells leads to the accumulation of a wide range of metabolites and proteins damaged by a newly discovered covalent modification.

Thus, this enzyme represents a highly conserved strategy to prevent damage in cells that perform glycolysis. Thereby, it interrupts a fundamental link between carbohydrate metabolism and a type of cellular damage that might contribute to the development of Parkinson’s disease.
Distinct Effects of Mesterolone on Oxidative and Glycolytic Fibers of Avian Skeletal Muscle
Authors: Allouh Mohammed Z. , Ouda Zakaria H. , Ouda Sara H.
Published: May 2022

Aim

Oxidative and glycolytic muscle fibers differ in their responses to physiological stimuli and pathological insults. We examined whether these fibers respond differentially to exogenous anabolic androgenic steroids (AASs) by comparing morphological and histological changes between the oxidative anterior latissimus dorsi (ALD) and glycolytic pectoralis major (PM) fibers in adult avian muscles.

Methods

The AAS mesterolone was selected for this study. Mesterolone is a synthetic steroid that has no estrogenic side effects, low hepatotoxicity, and confers oral activity that allows its oral route of administration. Adult female White Leghorn chickens (Gallus gallus) were randomly divided into five groups: a vehicle control and four mesterolone treatment groups (4, 8, 12, and 16 mg/kg). Mesterolone was administered orally every three days for 4 weeks. Immunocytochemical techniques and morphometric analyses were employed to measure the changes in muscle weight, fiber size, satellite cell (SC) composition, and number of myonuclei.

Results

Mesterolone increased both body and muscle weights and induced hypertrophy in glycolytic PM fibers but not in oxidative ALD fibers. Mesterolone induced SC proliferation in both muscles; however, the myonuclear accretion was noticeable only in the PM muscle. In both muscles, the collective changes maintained a constant myonuclear domain size and the changes were dose independent.

Conclusion

Mesterolone induced distinct dose‐independent effects in avian oxidative and glycolytic skeletal muscle fibers; these findings might be clinically valuable in the treatment of age‐related sarcopenia.
Identifying Predictive Markers of CNS Oxygen Toxicity and Ketone Ester Effects on Latency to Seizure and Antioxidant Capacity
Authors: Stavitzski Nicole M. , Landon Carol S. , D'Agostino Dominic P. , Dean Jay B.
Published: May 2022

Hyperbaric oxygen (HBO2) is used in hyperbaric oxygen therapy and in undersea and aerospace medicine. What limits the use of HBO2 is the risk of seizures, i.e. central nervous system oxygen toxicity (CNS‐OT). In undersea medicine, the threat of CNS‐OT is what limits the time that the warfighter remains at depth while breathing HBO2. In addition, when breathing HBO2, various non‐convulsive signs and symptoms often precede onset of seizures, including bradycardia followed by tachycardia, hypothermia, and hyperventilation. We hypothesized that the early cardiorespiratory responses to HBO2 during the safe latent period could be used as predictive physiological markers (or physio‐markers) prior to the onset of CNS‐OT. Furthermore, using a ketone ester (KE) supplement, R,S‐1,3‐butanediol diacetoacetate ester, we posited that the KE would increase the antioxidant capacity and the latency time to seizure (LSz) when exposed to HBO2. Accordingly, 18 male, Sprague Dawley (SD) rats were implanted with DSI 4‐ET radio telemetry to record EEG, ECG, and respiratory EMG for offline analyses of physio‐markers. At least 1 week following surgery, each rat was placed in a hyperbaric chamber and “dived” to 5 ATA O2 (1 ATG/min) after oral gavage of either water (control group) or KE (treatment group). An additional 9 SD rats were also dived to 5 ATA O2, but not implanted with telemetry, to collect blood samples to measure antioxidant capacity with the RedoxSys® device before and after HBO2 exposure. HBO2 caused a 3‐phase hyperoxic ventilatory response: decreased respiration, transient hyperventilation, and a secondary later hyperventilation prior to seizures. Results showed that LSz increased in the KE group by 307% (controls: 6.42 ± 0.44 (n=15); KE: 22.16 ± 5.10 (n=18)). Additionally, minute ventilation and RR‐Interval (1/heart rate) were significantly different beginning ~15 minutes prior to onset of seizures. Receiver operating characteristic (ROC) analyses indicated that both hyperoxic hyperpnea and bradycardia were reliable physio‐markers for seizure prediction; that is, ROC for minute ventilation in both control and KE group, and for RR‐Interval in controls, was 1 (excellent predictor), and in the KE group was 0.84 (good predictor). However, core body temperature did not decrease significantly during HBO2 exposure and thus was not a physio‐marker for CNS‐OT. In addition, KE increased antioxidant capacity of blood from animals that were exposed to HBO2 with (p=0.0399) or without (p=0.0018) the development of CNS‐OT. In conclusion, hyperoxic hyperpnea and bradycardia can be used as predictive physio‐markers in conjunction with ketone therapy to reliably predict and delay CNS‐OT in rodents, and KE delays seizures in part by increasing overall antioxidant capacity during exposure to HBO2. Together this may enable safer and longer exposures for applications in undersea/aerospace medicine as well as hyperbaric oxygen therapies.
The Acyltransferase Gpc1 is Both a Target and an Effector of the Unfolded Protein Response (UPR)
Authors: Hrach Victoria L. , Nelson Laura , King Will , Conklin Shane , Patton‐Vogt Jana
Published: May 2022

The unfolded protein response (UPR) is sensitive to both proteotoxic stress and membrane bilayer stress. These stresses are sensed by the ER transmembrane protein Ire1. When activated, Ire1 uses its endonuclease activity to splice HAC1mRNA, producing a mature transcription factor that binds to UPR elements (UPREs) in the promoters of target genes. Hac1 targets include not only genes involved in protein folding, secretion, and degradation, but also a subset of lipid metabolic genes. One aspect of lipid metabolism is the deacylation of phosphatidylcholine (PC) by phospholipases to produce glycerophosphocholine (GPC). In Saccharomyces cerevisiae, GPC can be reacylated in a novel two‐step process catalyzed first by GPC acyltransferase Gpc1, followed by acylation of the lyso‐PC molecule by Ale1. This metabolic cycle has been termed the PC deacylation/reacylation pathway (PC‐DRP). In prior studies, loss of Gpc1 was shown to result in an increase in di‐unsaturated PC species at the expense of mono‐unsaturated PC species, indicating a role for PC‐DRP in PC acyl chain remodeling. Here, we probe the role of Gpc1 as both a target and an effector of the UPR. Exposure to the UPR‐inducing compounds tunicamycin, DTT, and canavanine results in an increase in GPC1 message that is dependent upon the UPR transcriptional activator Hac1. The importance of this increased expression to cellular function is illustrated by the finding that cells lacking Gpc1 exhibit increased sensitivity to those compounds. In a converse set of experiments, we show that that loss of GPC1 results in upregulation of the UPR as measured by expression of the ER chaperone KAR2. Consistent with these findings, we show that Gpc1 primarily co‐localizes with the endoplasmic reticulum.
A Common Alpha Globin Gene Deletion is Associated with Decreased Arterial Alpha Globin Gene Expression and Altered Vasoreactivity among Black Americans
Authors: Brooks Steven D. , Zeng Xianke , Jackson Mary , Tesfuzigta Naomi , Laurence Kyeisha , Nino de Rivera Jessica , Jackson Jarrett , Emeh Robert , Smith Rachel , Kamenyeva Olena , Ganesan Sundar , Kabat Juraj , Cruz Phillip , Joyce Stacy , Davis Jeremy , Ruhl A. Parker , Ackerman Hans
Published: May 2022

Alpha globin binds to endothelial nitric oxide (NO) synthase (eNOS) and limits the diffusion of NO from endothelial cells into vascular smooth muscle. Previously, we demonstrated that pharmacologic disruption of alpha globin‐eNOS binding enhanced NOS‐dependent vasodilation in human arteries. Here, we explore the hypothesis that individuals with alpha globin gene deletions will have reduced alpha globin gene expression in resistance arteries and altered arterial vasoreactivity.

To test this hypothesis, we screened healthy Black Americans (clinical trial # 02692872) for a common 3.7 kb gene deletion in the alpha globin gene locus (HBA) that reduces the number of functional gene copies. We excluded people with risk factors for cardiovascular disease and created a nested case control study that included 15 people with four copies, 8 people with three copies, and 2 people with two copies of HBA. Each individual underwent an incisional subcutaneous adipose biopsy (clinical trial # 03937817), from which we isolated resistance arteries less than 200 μm in diameter. Resistance arteries were cannulated and perfused free of blood before undergoing multiphoton imaging, gene expression analysis, or pressure myography.

Multiphoton imaging of adipose resistance arteries revealed alpha globin to co‐localize with eNOS near the myoendothelial junction between endothelial and smooth muscle cells. Arteries from individuals with three functional copies of HBA had lower expression of HBA compared to arteries from individuals with four functional copies of HBA(1715 copies/ng cDNA vs 1176 copies/ng cDNA, p = 0.03). The concentration of phenylephrine required to constrict the human adipose arteries to 50% of baseline was 8 times greater for arteries from subjects with three copies of HBA compared to arteries from subjects with four copies of HBA (log10[EC50]: ‐4.78 (95% CI: ‐4.29 to ‐5.22) vs ‐5.68 ± (95%CI: ‐5.33 to ‐5.98) p = 0.003). Arteries from individuals with three copies of HBA also had reduced maximal constriction to phenylephrine compared to subjects with four copies of HBA(p < 0.01).

These results offer compelling new genetic evidence from humans supporting the role of alpha globin as a regulator of NO signaling in resistance arteries. We demonstrate that HBA deletions reduce arterial expression of HBA and change arterial vasoreactivity. This clinical investigation defines a novel vascular phenotype for individuals with alpha thalassemia and provides insight into a vascular mechanism by which alpha globin gene deletions protect against severe malaria, chronic kidney disease, and vascular complications of sickle cell disease.
Activation of Cold‐Sensitive Afferents Inhibits Aberrant Irritant‐evoked Cardiopulmonary Reflexes in the Spontaneously Hypertensive (SH) Rat
Authors: Hooper Justin S. , Taylor‐Clark Thomas
Published: May 2022

Inhalation of irritants activates airway vagal afferent nerves, evoking cardiopulmonary reflexes. Evidence suggests that cardiopulmonary reflexes evoked by irritant/pollution inhalation are remodeled from bradycardia in normal subjects to tachycardia and ventricular arrhythmia in patients with pre‐existing cardiovascular disease. We have shown similar remodeling in the spontaneously hypertensive (SH) rat: selective activation of airway vagal afferent nerves via inhalation of the TRPA1‐specific agonist allyl isothiocyanate (AITC) evoked parasympathetic‐mediated reflex bradycardia in normotensive wistar‐kyoto rats but evoked sympathetically‐mediated tachycardia and premature ventricular contractions (PVCs) in spontaneously hypertensive (SH) rats.

Activation of TRPA1‐expressing airway vagal afferents also evokes cough in some conscious animal models. Cough reflexes have been shown to be inhibited by nasal stimulation with L‐menthol via activation of TRPM8 ion channels expressed on cold‐sensitive trigeminal afferents which regulate brainstem respiratory networks. Here, we investigated the cardiopulmonary reflexes evoked by inhalation of L‐menthol (150mg/ml) alone and in combination with AITC (4.7mg/ml) in SH rats. L‐menthol inhalation alone had no effect on respiration/heart rate, but, when inhaled during AITC inhalation, L‐menthol selectively inhibited the AITC‐evoked tachyarrhythmia and tachypnea. Importantly, the inactive isomer D‐menthol (150mg/ml) failed to affect AITC‐evoked sympathoexcitatory reflexes. In another set of studies, the selective TRPM8 agonist WS‐12 (300mM) also inhibited the AITC‐evoked tachyarrhythmia. Therefore, we conclude that aberrant cardiopulmonary reflexes evoked by irritant inhalation are reduced by activation of menthol‐sensitive pathways within the airways. This data supports the therapeutic potential of menthol treatment against irritant‐evoked cardiopulmonary reflexes.
Deletion of LDLRAP1 Induces Atherosclerotic Plaque Formation, Insulin Resistance, and Dysregulated Insulin Response in Adipose Tissue
Authors: Leigh Tani , Kawai Tatsuo , Kelemen Sheri , Okune Rachael , Preston Kyle , St. Paul Amanda , Corbett Cali , Yu Jun , Scalia Rosario , Autieri Michael
Published: May 2022

Hypercholesterolemia, vascular inflammation, obesity, and insulin resistance often overlap and exacerbate each other. Mutations in low density lipoprotein receptor adaptor protein‐1 (LDLRAP1) lead to LDL receptor malfunction and are associated with the Autosomal Recessive Hypercholesterolemia (ARH) disorder in humans; however, direct causality on atherogenesis in a defined pre‐clinical model has not been reported. The objective of this study was to test the hypothesis that deletion of LDLRAP1 will lead to hypercholesteremia and atherosclerosis. LDLRAP1‐/‐ mice fed a high‐fat western diet (HFD) had significantly increased plasma cholesterol and triglyceride concentrations (p<0.001), accompanied with significantly increased plaque burden compared with wild‐type controls. Unexpectedly, LDLRAP1‐/‐ mice gained significantly more weight compared with controls (p<0.001), and even on a chow diet, LDLRAP1‐/‐ mice are insulin resistant, and calorimetric studies suggests an altered metabolic profile. We determined that LDLRAP1 is highly expressed in white adipose tissue (VAT), and LDLRAP1‐/‐ adipocytes are significantly larger (p<0.01), and have reduced glucose uptake and AKT phosphorylation, but increased CD36 expression. VAT from LDLRAP1‐/‐ mice is hypoxic and has gene expression signatures of dysregulated lipid storage and energy homeostasis. These data are the first to indicate that lack of LDLRAP1 directly leads to atherosclerosis in mice and also plays an unanticipated metabolic regulatory role in adipose tissue. LDLRAP1 may link atherosclerosis and hypercholesterolemia with common co‐morbidities of obesity and insulin resistance.
The Effects of Activation and Blockade of Delta Opioid Receptors on the Acquisition of Responding for an Opioid‐Associated Stimulus
Authors: Robertson Stephen H. , Rice Kenner C. , Jutkiewicz Emily M.
Published: May 2022

Drug‐associated stimuli take on conditioned reinforcing properties that promote drug‐seeking and relapse. Previously, we have demonstrated that daily systemic injections of SNC80 (delta opioid receptor agonist) resulted in dose‐dependent increases in responding for the opioid‐associated stimulus. In the current experiment, we assessed the extent to which activating and/or blocking delta opioid receptors via a systemic injection of SNC80 or naltrindole, respectively, modified the conditioned reinforcing properties of an opioid‐associated stimulus. We hypothesized that a pre‐session injection of SNC80 would increase the conditioned reinforcing properties of the remifentanil‐associated stimulus; however, an injection of naltrindole would have no effect, and an injection of naltrindole would block the ability of SNC80 to enhance the conditioned reinforcing properties of the stimulus. First, we exposed rats to 20 i.v. infusions of remifentanil (3.2 ug/kg/infusion) and 20 stimulus presentations that were delivered response‐independently each day for 5 days. For the experimental group (Paired Conditioning), the remifentanil infusions and stimulus presentations were delivered concurrently. The control group (Random control) received the same number of infusions and stimulus presentations, but they were not explicitly paired. For the next 7 sessions (acquisition), we tested the extent to which the stimulus functioned as a conditioned reinforcer by allowing rats to freely respond via a nosepoke for presentations of the remifentanil‐associated stimulus only. On the fourth acquisition session, rats received one of the following combinations of injections: (a) vehicle + vehicle, (b) vehicle + naltrindole (3.2 mg/kg), (c) vehicle + SNC80 (3.2 mg/kg), or (d) SNC80 (3.2 mg/kg) + naltrindole (3.2 mg/kg). We found that rats in the Paired Pavlovian Conditioning group responded for the remifentanil‐associated stimulus significantly more than controls (p= 0.003, hp2= 0.15). Responding for remifentanil‐paired stimuli depended on treatment condition (p < 0.001, hp2= 0.35). Post‐hoc tests revealed that SNC80 administration increased responding for the remifentanil‐associated stimulus relative to all other treatment conditions (p’s < 0.001). These findings demonstrated that a remifentanil‐associated stimulus takes on conditioned reinforcing properties and that the conditioned reinforcing properties of the stimulus was augmented by delta opioid receptor activation and these effects were blocked via naltrindole. These findings support previous work that delta opioid receptors play a role in mediating conditioned reinforcing properties of drug‐associated stimuli.