bluuebunny/crossref_metadata_2025_split huggingface dataset:
CCL2 Mediates Early Renal Leukocyte Infiltration During Salt‐Sensitive Hypertension
Authors: Alsheikh Ammar J. , Dasinger John Henry , Abais-Battad Justine M. , Fehrenbach Daniel J. , Yang Chun , Cowley Allen W. , Mattson David L. 
Published: April 2020

BackgroundHypertension is reported to be the largest independent risk factor for disease burden globally. Despite the prevalence and burden of hypertension on human health, the mechanisms leading to the development of hypertension in most individuals remain unknown. Both human and animal experimental evidence indicate that immune mechanisms may play a key role in the development of hypertension and associated end‐organ damage. Studies examining mechanisms of Dahl Salt‐Sensitive (SS) hypertension have implicated the infiltration of leukocytes into the kidneys as a key contributor to the hypertension and renal damage. However, the signaling pathways by which leukocytes traffic to the kidneys remain poorly understood.AimThe aim of the present set of studies was to use a unique unbiased discovery‐based approach to nominate signaling pathways which was followed by functional testing of the role of the identified pathways in renal leukocyte infiltration.Methods and ResultsWe analyzed a kidney RNA‐seq dataset from SS rats fed either a Low Salt (LS‐0.4% NaCl) or a High Salt (HS‐4.0% NaCl) diet. From this analysis, the chemokine (C‐C motif) ligand 2 (CCL2) and receptor CCR2 were nominated as a potential pathway modifying renal leukocyte infiltration. The functional role of the CCL2/CCR2 pathway was tested by daily administration of CCR2 antagonist (RS102895 at 5mg/kg/day in DMSO) or DMSO Vehicle for 3 days (n=13–15/group) or 21 days (n=9–11/group) by intraperitoneal injections to male SS rats during the HS challenge. A variety of parameters were measured throughout the study including blood pressure (measured continuously using radio‐telemetry), circulating leukocytes and renal leukocyte infiltration (by flow cytometry) and renal damage (assessed histologically and using urinary injury markers), body weight, food intake and electrolyte excretion. Results demonstrate that RS201895 treatment, compared to vehicle‐treated rats ameliorated renal damage (urinary albumin excretion; 43.4±5.1 vs 114.7±15.2 mg/day, P<001) and hypertension (144.3±2.2 vs 158.9±4.8 mmHg, P<0.001) after 21 days of HS diet. It was determined that renal leukocyte infiltration was blunted by day 3 of the HS diet (1.4±0.1 vs 1.9±0.2 ×106 CD45+ cells/kidney, P=0.034) in drug‐treated rats. A unique adaptation of an in vitro chemotaxis assay validated the ability of RS102895 to block leukocyte chemotaxis towards CCL2 at in vivo concentrations.Conclusion/SignificanceThe results demonstrate that increased CCL2 in SS kidney is important in early recruitment of leukocytes and blockade of this recruitment by administering RS102895, which subsequently ameliorated the renal damage and hypertension. These results provide a unique understanding to an important disease process that has potential therapeutic implications.Support or Funding InformationSupported by HL137748, HL116264.
Role of Zfra in mitigating epileptic seizure due to WWOX downregulation
Authors: Wen Kuang-Yu , Chang Nan-Shan 
Published: April 2020

As a tumor suppressor, WWOX is a recently defined risk factor for Alzheimer’s disease (AD). WWOX limits AD progression due, in part, to its suppression of tau tangle formation by direct binding of tau and tau‐phosphorylating enzymes. Deficiency of WWOX leads to severe neural diseases, including epileptic encephalopathy, microcephaly, intractable seizures and developmental delay, and early death in human and animal newborns. Wwox knockout mice, which survive in less than 1 month, display spontaneous and audiogenic seizures and rapid formation of tau and amyloid aggregates in the brain in less than 3 weeks after birth. During AD progression in humans and mice, pS14‐WWOX is shown to accumulate in the brain hippocampus and cortex. Suppression of pS14‐WWOX by a short synthetic peptide Zfra (zinc finger‐like protein that regulates apoptosis) leads to clear up of tau tangles and amyloid plaques, and restoration of memory loss in triple transgenic mice for AD. Here, we determined that when Wwox wild type and heterozygous mice were pre‐injected with a Zfra4–10 peptide via tail veins, these mice resisted pentylenetetrazone (PTZ)‐induced seizure. Mechanistically, Zfra significantly suppressed pS14‐WWOX in the auditory cortex and hippocampal CA1, CA3, or DG areas. Zfra blocked PTZ‐induced activation of inflammatory microglia and astrocytes in mouse hippocampus. Also, Zfra significantly blocked the expression of RE1‐silencing transcription factor (REST) (>90%), which is a regulator of ion channels and neurotransmitter receptors associated with epilepsy. Together, our observations suggest that Zfra peptide is a potent agent in mitigating epileptic seizure caused by WWOX deficiency.Support or Funding InformationMinistry of Science and Technology, Taiwan, National Health Research Institute, Taiwan, and Department of Defense, USA.
The Deletion of AMPK β1 Worsens Glucose Tolerance and Liver Lipid Accumulation, but Doesn’t Impact the Metabolic Effects of Olanzapine in Male Mice
Authors: Shamshoum Hesham , Ashworth Kristen E. , Medak Kyle D. , Kemp Bruce E. , Wright David C. 
Published: April 2020

Second generation antipsychotic (SGA) medications, like olanzapine (OLZ), are an important component in the pharmacological management of many conditions including schizophrenia. These drugs, have been linked with a neuroendocrine signaling axis that, leads to weight gain, however, peripheral mechanisms and predictors are currently poorly understood. 5’AMP activated protein kinase (AMPK) is a ubiquitously expressed, energy‐sensing enzyme and significant associations between polymorphisms in AMPK subunit genes and OLZ‐induced weight gain have been demonstrated. Conversely, others have suggested that the central activation of AMPK mediates the deleterious metabolic effects of SGAs. The purpose of this study was to determine if reductions in AMPK activity, primarily in the liver, would impact the chronic effects of OLZ treatment. We hypothesized that the absence of AMPK β1, a subunit which is primarily found in the liver, at least in mice, would worsen the metabolic side effects of OLZ. To address this question, we housed male AMPK β1 knockout (KO) and wildtype (WT) mice at thermoneutrality (29°C) and fed them either a control diet (HFD, 45% kcal from fat) or a diet supplemented with OLZ (HFD+OLZ, 50mg/kg diet) for 10 weeks. OLZ treatment did not increase food intake or weight gain to a greater extent than the control diet in either genotype. There was a main effect of the OLZ containing diet to increase heat production, TEE and fat oxidation during the light and dark cycles. The OLZ‐fed mice also demonstrated suppressed RER during the dark cycle. Interestingly, the OLZ‐fed KO mice were more physically active during the light cycle than the OLZ‐fed WT mice. The AMPK β1 KO mice were more glucose intolerant than the WT control mice. On the other hand, the AMPK β1 KO male mice on both diets demonstrated lower circulating serum glycerol and NEFA levels but increased liver TAG accumulation. In conclusion, our findings provide evidence that the absence of AMPK β1 does not potentiate the effects of OLZ on weight gain, but worsens liver lipid accumulation and glucose tolerance.Support or Funding InformationThis study was supported by Canadian Institutes of Health Research
Concentration‐Time profiles of Glyburide in Rats: Predictions using a Continuous Absorption Model
Authors: Radice Casey , Korzekwa Ken , Nagar Swati 
Published: April 2020

Most drugs are administered orally due to cost‐efficacy and increased patient compliance. However, the absorption of these drugs is subjected to complex biological processes and depends on the drug’s physicochemical properties. Prior to human trials, preclinical species such as rodents are utilized to evaluate efficacy and safety in vivo. Because animal studies are expensive and time‐consuming, it is beneficial to predict a drug’s pharmacokinetic profiles prior to in vivo studies. Pharmacokinetic modeling and simulation is useful for such predictions.The small intestine and colon of male Sprague Dawley rats were divided into 10 unequal segments to measure the change in luminal pH. Luminal contents were collected and directly measured under fed (n=4) and 12‐hour fasted (n=4) conditions. Glyburide is an oral anti‐diabetic medication that stimulates insulin secretion by blocking ATP‐dependent potassium channels in pancreatic beta cells. In vivo pharmacokinetic studies were performed to determine the effect of food. Fasted (n=4) and fed (n=4) male Sprague Dawley rats were dosed 5mg/kg of glyburide by oral gavage. Rats (n=2) were dosed intravenously to collect pharmacokinetic parameters. Blood samples were collected at 0.1, 0.25, 0.5, 1, 2, 4, 6, 10, and 24 hours post dose. Data were analyzed with non‐compartmental analysis. A continuous rodent absorption model was developed to predict the concentration‐time profiles of compounds based on rodent anatomical data collected in‐house and from the literature. Several physiological parameters were incorporated in the model, including small intestinal transit time, luminal pH, and transporter expression along the intestines.Rats fasted for 12 hours had a higher pH along the intestine that increased slowly, compared to fed rats. While the pH of the colon was higher in fasted rats, the cecum was not altered by fasted conditions. Fasted rats had an average glyburide AUC(0–24) of 2.7 μg*h/ml, while fed rats had an AUC(0–24) of 0.97 μg*h/ml. The mean apparent clearance (CL/F) was 1.8 L/min/kg for fasted rats and 5.1 L/min/kg for fed rats. The mean half‐life increased slightly from 10.6 hours in fasted rats to 11.6 hours in fed rats. Current predictions using the rodent continuous absorption model do not accurately describe the exposure profile of glyburide. To refine the model, further anatomical data collection and incorporation of fasted versus fed conditions are underway.Support or Funding InformationThis work was supported by NIH grants R01GM114369 and R01GM104178 to Swati Nagar and Ken Korzekwa.
TRPML channel activation partially rescues Ca2+ spark activity in sheep fetal pulmonary arterial myocytes following intrauterine long‐term hypoxia
Authors: Ang Dylan , Romero Monica M. , Puglisi Jose L. , Zhang Lubo , Wilson Sean M. 
Published: April 2020

Ca2+ sparks result from the activation of ryanodine receptors located on the sarcoplasmic reticulum of pulmonary arterial smooth muscle cells. These Ca2+ sparks activate potassium channels on the plasma membrane and are important to arterial vasodilation and increases in vascular blood flow. Previous studies showed that long‐term high altitude hypoxia restricted membrane depolarization elicited Ca2+ spark activity in fetal lamb pulmonary arterial myocytes, but had little effect on pulmonary myocytes in adult sheep. Moreover, the studies also indicated that there was preservation of the sarcoplasmic reticulum Ca2+ stores in pulmonary arterial myocytes of both fetuses and adults as determined through direct activation of ryanodine receptors with caffeine. Given that the sarcoplasmic reticulum Ca2+ stores are intact and have functional ryanodine receptors we hypothesize that aberrations to the canonical membrane depolarization Ca2+ spark triggering pathway in fetal pulmonary arterial myocytes due to intrauterine hypoxia can be resolved through activation of alternate pathways. Recent studies illustrated that Ca2+ sparks were triggered in various vascular myocytes by releasing Ca2+ from lysosomes through activation of TRPML (transient receptor potential cation channel, mucolipin subfamily) channels. To evaluate the potential that TRPML activation may elicit Ca2+ sparks in fetal and adult pulmonary arterial myocytes we studied isolated pulmonary arteries from near term fetal and adult sheep from low altitude (700 m) or those that had been exposed to high altitude (3801 m) for 110+ days. Ca2+ sparks were recorded using laser scanning confocal microscopy while 10 μM MLSA1 was used to activate TRPML channels. MLSA1 enhanced the activity of Ca2+ sparks in myocytes of adult pulmonary arteries to a greater extent than those from fetuses, suggesting developmental changes in the communication of lysosomes with the sarcoplasmic reticulum. Of importance, TRPML activation by MLSA1 partially recovered the aberrant Ca2+ spark activity in fetal pulmonary arterial myocytes caused by hypoxia. The findings suggest that ryanodine receptor activation through pathways alternate to membrane depolarization may hold promise for improving pulmonary vascular function following intrauterine hypoxic exposure.Support or Funding InformationImaging was performed in the LLUSM Advanced Imaging and Microscopy Core with support of NSF Grant MRI‐DBI 0923559 (SMW) and the Loma Linda University School of Medicine. Additional support provided through NIH grant HD083132 (LZ).
Molecular Determinants of Signaling and Posttranslational Modifications of GPR64
Authors: Gad Abanoub A. , Azimzadeh Pedram , Balenga Nariman 
Published: April 2020

Some of the adhesion G protein‐coupled receptors (aGPCRs) show constitutive activity in the absence of their large N‐terminal fragment (NTF). We previously demonstrated that GPR64/ADGRG2, an orphan aGPCR, regulates the secretion of parathyroid hormone by interacting with the calcium‐sensing receptor and elevating the cAMP levels in parathyroid adenoma cells. In HEK293 cells, we described the role of beta‐arrestins, dynamin, and GPCR kinases (GRKs) in the signaling of NTF‐truncated GPR64. Whether structural domains and motifs other than the NTF also regulate signaling, trafficking, and posttranslational modifications of GPR64 remains unknown. In the current study, we mutated a number of extracellular motifs that are conserved among zebrafish, mouse, and human GPR64. We measured cAMP production (time‐resolved FRET), surface expression (ELISA and biotinylation assays), and ubiquitination (co‐immunoprecipitation) of WT and mutant GPR64 in HEK293 cells. We discovered that several residues in the extracellular domains alter the surface expression, ubiquitination, and degradation of GPR64 at basal and tethered agonist‐stimulated conditions. The molecular mechanisms by which these residues regulate trafficking, signaling, and ubiquitination of GPR64 are currently under investigation.Support or Funding InformationThis work was supported by NIH grant 1R01GM13061701A1 (to N.B.). Student support was provided by an NIH‐NIGMS Initiative for Maximizing Student Development Grant (R25‐GM55036) (to A.A.G.).
Structure‐Based Insights into Control of Heme Biosynthesis
Authors: Brown Breann L. , Kardon Julia R. , Baker Tania A. 
Published: April 2020

Protein assembly drives many aspects of cellular and mitochondrial physiology. We are interested in uncovering and characterizing the protein interactions that support proper heme biosynthesis. Heme is a critical biomolecule that carries out several functions in nearly all life forms, including humans where its most widely‐known role is mediating oxygen transport in blood. It is imperative that heme production is tightly controlled as alterations in cellular heme levels can have drastic consequences for human health. The first and rate‐limiting enzyme controlling heme biosynthesis is aminolevulinic acid synthase (ALAS). ALAS is conserved in α‐proteobacteria and non‐plant eukaryotes, there are two isoforms in vertebrates, with the mitochondrial protein ALAS2 being responsible for heme synthesis during erythropoiesis. There are two separate diseases that may result from one of over 50 mutations in the ALAS2 gene. Mutations decreasing optimal activity lead to the disease X‐linked sideroblastic anemia (XLSA), whereas other mutations causing hyperactivity underlie X‐linked protoporphyria (XLPP). Importantly, several disease‐causing mutations are located in a eukaryote‐specific C‐terminal extension, a region absent from bacterial ALAS enzymes. Certain mutations also result in an inability of ALAS2 to assemble with other proteins, including proteins involved in separate metabolic pathways. However, we lack an understanding of how these mutations lead to a change in ALAS2 structure, and therefore, function. We seek to understand how ALAS interacts with accessory proteins as well as organism‐specific differences in assembly that may alter regulation of heme production. Therefore, we use X‐ray crystallography combined with in vitro activity assays of various eukaryotic ALAS enzymes to parse apart the role of this key regulatory region in eukaryote ALAS function. Our work is beginning to reveal key structure‐function relationships between the orientation and molecular contacts mediated by the C‐terminus and overall ALAS enzyme function, thus controlling heme biosynthesis.Support or Funding InformationJane Coffin Childs Memorial Fund for Medical Research Award and a Burroughs Wellcome Postdoctoral Enrichment Program Fellowship Award 1015092 (B.L.B); NIH Ruth L. Kirschstein National Research Service Award (F32DK095726) (J.R.K.); National Institutes of Health Grant (R01 DK115558) (T.A.B.), and the Howard Hughes Medical Institute. X‐ray data collected at NE‐CAT (P41 GM103403).
CaMKII is Necessary for Proliferation and Migration of Pulmonary Arterial Smooth Muscle Cells
Authors: Huetsch John , Jiang Haiyang , Shimoda Larissa 
Published: April 2020

ObjectiveCa2+/calmodulin‐dependent protein kinase II (CaMKII) is a calcium‐ and oxidation‐activated serine/threonine kinase, with four known isoforms. CaMKII has been shown to be necessary for remodeling involving smooth muscle cell proliferation and migration in response to injury in the systemic vasculature. However, the role of CaMKII in pulmonary arterial smooth muscle cell (PASMC) function remains unknown.MethodsPASMCs were isolated from male Wistar rats and expanded in culture. In cell lysates, mRNA expression of CaMKII isoforms was measured by real‐time quantitative reverse transcription PCR; protein expression was assessed via immunoblot. PASMC proliferation was measured via BrdU incorporation. PASMC migration was assessed by the Transwell assay. PASMCs were treated with 10 μM KN‐93 in vitro to inhibit CaMKII or with either vehicle or 10 μM KN‐92 (an inactive analog of KN‐93) for control experiments. To deplete CaMKIIδ protein, PASMCs were treated with 100nM siRNA targeted to CaMKIIδ while PASMCs treated with scrambled siRNA were used for control experiments.ResultsThe CaMKIIδ and CaMKIIγ isoforms showed mRNA and protein expression in rat PASMCs. Both proliferation and migration of rat PASMCs were inhibited by pharmacologic inhibition of CaMKII using KN‐93, but were not inhibited by the inactive analog KN‐92. siRNA‐mediated knockdown of CaMKIIδ resulted in reduction of CaMKIIδ protein expression but did not have an effect on either PASMC proliferation or migration.ConclusionsCaMKII activity is necessary for PASMC proliferation and migration. As the CaMKIIδ isoform is not required for these functions, future work will examine the role of the CaMKIIγ isoform.Support or Funding InformationFunding SourcesNIH K08HL133475 & Pulmonary Hypertension Association K08 Supplement
The Effects of In Vitro Seizure‐like Activity on Structural Plasticity of Dendritic Spines
Authors: Nieder Cassidy , Ramon Alfonsina , Getzfread Mollie , Lipmann-Bell Jocelyn 
Published: April 2020

Most excitatory synapses in the mammalian brain connect to dendritic spines. These membranous protrusions function as chemical and electrical micro‐compartments, transducing individual signals of excitatory neurotransmitters from axonal boutons. Structural plasticity, the ability of spines to change their shape and thus their function in response to synaptic activity, underlies learning and memory. This shape change requires regulation of actin mesh‐works, which are regulated by actin binding proteins (ABPs) within spines. Decreased concentrations of functional ABPs induce abnormalities in the density, morphology, and dynamic nature of dendritic spines. Early‐life seizure models demonstrate altered spine morphology, and a decrease in ABPs hinders synaptic signal amplitudes. Further, neonatal seizures are linked to cognitive deficits later in life, including autistic‐like behaviors, and patients with autism spectrum disorder express depleted concentrations of endogenous ABPs and exhibit dendritic spine dysgenesis. Based upon these findings, we hypothesized that seizure‐like activity causes deficits in the structural plasticity of dendritic spines via depletion of functional ABPs. To investigate the physiological consequences of uncontrolled, synaptic excitability on intracellular actin regulation at the molecular level, we induced in vitro seizure‐like activity in primary, rat hippocampal neurons using a zero magnesium model. At multiple time points after this event, we assessed localization of ABPs using immunocytochemistry, actin turnover employing fluorescent recovery after photo bleaching, and spine and actin dynamics in response to stimulation utilizing live‐cell imaging in GFP‐actin transfected neurons. Preliminary results show a decrease in localization of ABPs to dendritic spines and an increase in spine motility after in vitro seizure‐like activity in cultured hippocampal neurons. Taken together, these data indicate that seizures may alter ABPs in a model that also shows changes in spine dynamics and structural plasticity. Ongoing work will examine this link further to determine if ABPs could be a possible therapeutic target to decrease cognitive deficits that can result from early‐life seizures and lead to intellectual disabilities.Support or Funding InformationCCDA: Center for Chronic Disorders of Aging