bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maternal sleep disordered breathing during pregnancy as a cause of autism relevant neuronal and behavioral aberrations in the offspring.
Authors: Vanderplow Amanda M. , Kermath Bailey A. , Ewald Andrea C. , Gums Kimberly T. , Seablom Erin N. , Baker Tracy L. , Watters Jyoti J. , Cahill Michael E. 
Published: April 2020

Sleep disordered breathing (SDB) is characterized by recurring breathing cessations during sleep, causing intermittent hypoxia (oxygen deprivation) often several hundred times per night. SDB is present in ~15% of pregnant women as compared to ~2–4% of non‐pregnant women of childbearing age. Risk factors for gestational SDB development bear striking similarities to the collection of maternal risk factors for autism spectrum disorders (ASD), including advanced maternal age and maternal obesity. Maternal immune activation/inflammation is both a consequence of SDB, and an independent risk factor for, ASD. We posit that an overlooked, yet increasingly common inflammatory stimulus, maternal SDB, contributes to ASD etiology. We recently developed a rat model in which to study the consequences of maternal SDB on the offspring by exposing pregnant rats to nightly intermittent hypoxia from gestational days 10–21. Our findings indicate that offspring of mothers subjected to intermittent hypoxia during pregnancy exhibit deficits in several autism‐relevant behaviors, including a constellation of cognitive impairments, reduced interest in social interaction, and heightened anxiety. In most instances, the magnitude of behavioral disruption was more severe in male than female offspring. To begin to understand the mechanisms contributing to these behavioral aberrations, we examined the density of dendritic spines in the prefrontal cortex (PFC) and CA3 region of the hippocampus. Our data suggest that the offspring of mothers subjected to nightly intermittent hypoxia exhibited a striking increase in dendritic spine density in the PFC when compared to control offspring. However, the dendritic spine density in the CA3 region showed no significant differences between groups. Similar to our behavioral observations, dendritic spine aberrations were more prominent in male vs. female offspring. Additionally, these male offspring showed significant differences in spine morphology when compared to control offspring. Taken together, these findings indicate that maternal sleep disordered breathing not only induces autism‐relevant behavioral impairments, but also mimics the heightened cortical synaptic connectivity phenotypes recently uncovered in autism. Future studies are aimed at identifying a role for microglial inflammation and concomitant reduction in microglial‐mediated synaptic pruning in the behavioral and synaptic structural alterations that we have revealed in this model.Support or Funding InformationR25 Team‐ScienceUW2020 Pilot Award
Ectopically Expressed Human Olfactory Receptor OR51E1: a Search for Novel Molecular Probes
Authors: Pronin Alexey , Janczura Karolina , Volmar Claude-Henry , Brothers Shawn , Slepak Vladlen 
Published: April 2020

Olfactory receptors comprise the largest family of G protein‐couples receptors (GPCRs) ‐ ~1400 functional genes in the mouse (Olfrs) and ~400 in the human (ORs). Most genes were assigned to it based solely on the sequence similarity. Recent studies demonstrated that many Olfrs are expressed outside of the nasal cavity and possibly involved in physiological processes such as muscle regeneration and controlling blood pressure. These ectopically expressed olfactory receptors might recognize cues other than odorants and be involved in processes independent of the sense of smell. Ligands were identified for fewer than 100 of ORs and Olfrs.Using transcriptomics analysis followed by RT‐PCR and in situ RNA hybridization we previously showed expression of several Olfrs and other olfactory signaling genes expressed in the mouse ocular tissues. We focused our attention on Olfr558 and demonstrated its expression in blood vessels of the eye. The human ortholog of Olfr558 (OR51E1 or PSGR2) was shown to be expressed in the prostate and dramatically up‐regulated in prostate cancer and is an established biomarker of malignancy. The human and mouse genes encoding this receptor are almost identical (94%), which is unique for GPCRs in general, and particularly for sensory chemoreceptors. This unusual evolutionary conservation points to the importance of its physiological function. While the function of this receptor or its native ligand is unknown, in vitro it responds to some short‐ and medium‐chain fatty acids (e.g., butyrate and valerate) by activating adenylyl cyclase. We found that growth of human prostate cancer cells that express OR51E1 is inhibited by valerate. The knockdown of OR51E1 gene with shRNA eliminated this effect of valerate.While butyrate and valerate can be used in such experiments, their low affinity and selectivity complicates interpretation of the data. Additionally, they are quickly metabolized in the body. Thus, there is a clear need for novel probes that can be used to dissect OR51E1 function. To address this need, we established a stable cell line overexpressing OR51E1 and demonstrated that in response to known OR51E1 agonists this cell line increases intracellular cAMP level. We then used this cell line for screening of a diverse chemical library of 9600 compounds, showing its suitability for high throughput screening. To demonstrate that our expression system can be used for expression of other ORs, we generated stable cell lines overexpressing two other receptors – OR51E2 and OR2AT4. We showed that both receptors are expressed as proteins of the expected molecular weight and localize on the cell surface. Since these ORs represents a large class of previously unexplored yet druggable targets, this project facilitates investigation of the entire family of ectopic olfactory receptors.
Measuring Effects of Treatment with a Novel Metalloprotease Inhibitor, Extracellular Matrix Protection Factor‐2, on Total Protein Production by Human Gingival Fibroblast Cultures
Authors: Maloney Trevor J. , Mattioli Patrisia , Laird Patrick , Musiol Joseph , Popper Hannah , Otto Ashley , Nguyen Stephanie , Graham Morgan , Green Kevan , Shamseddin Seyed , Borghaei Ruth , D’Angelo Marina 
Published: April 2020

Collagen, produced by fibroblasts, serves an integral role in the structural integrity of the extracellular matrix of the human gingiva, but during periodontal disease, matrix metalloproteases (MMPs) are upregulated and contribute significantly to the degradation of collagen. In this study, we test the effects of the novel MMP inhibitor, Extracellular Matrix Protection Factor‐2 (ECPF‐2) on total protein production by human gingival fibroblasts (HGVFs) reared in serum‐free media. Cells are enzymatically released from gingival tissue isolated during oral surgery and cultures produced from patient samples: normal, non‐inflamed (NTN); inflamed non‐smoker (IFN); inflamed previous smoker (IFP); inflamed current smoker (IFC). Subconfluent cultures were switched to 0.1% fetal bovine serum containing DMEM media overnight and then treated for 24 hours with complete serum‐free DMEM (Control); 5ug ECPF‐2; or 50ug ECPF‐2. Conditioned media was collected and concentrated and the cell layer was extracted with 0.5% CHAPS buffer. Total protein of the samples was quantified using the Pierce Modified Lowry Protein Assay. Regardless of treatment or patient pathology, all cultures tested produced approximately 1.3–1.5 ug/ml/culture of total protein. There was no statistically significant differences between patient samples or within treatment conditions per patient. These data suggest that our culture system allows for viable metabolism in serum‐free medium and treatment with the novel MMP inhibitor, ECPF‐2, does not affect the metabolic activity of HGVF cultures. Therefore, this culture system can be used to measure the therapeutic effects of ECPF‐2 on collagen metabolism associated with periodontal disease.Support or Funding InformationSupport from the PCOM Division of Research
Cardiomyocyte SNAP29 Insulates Desmosomes from Targeted Protein Degradation by Selective Autophagy to Restrict Arrhythmias
Authors: Wang Jie , Pellman Jason , Liang Yan , Lyon Robert , Mezzano Valeria , Zanella Fabian , Zhang Jing , Nigam Vishal , Gu Yusu , Dalton Nancy , Peterson Kirk L. , Sheikh Farah 
Published: April 2020

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a fatal and untreatable inherited cardiomyopathy associated with ventricular dysfunction and cardiac arrhythmias leading to sudden cardiac death in the young. Early detection is hindered as people present with complex disease patterns and harbors a “concealed phase”, where lethal arrhythmias are observed in the absence of overt structural changes to the heart. Mutations and loss of components of the desmosome, a cardiac mechanical cell‐cell junction, have been linked to the development of ARVC through human and mouse genetic studies. However, limited information exists on how desmosomal protein dysregulation/loss can trigger the complex features of ARVC, especially the asymptomatic “concealed phase”. Using a yeast two‐hybrid screen we identified Synaptoso‐malassociated protein 29 (SNAP29) as a novel desmosomal (desmoplakin, DSP)‐interacting protein. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy in non‐cardiomyocytes; however, its role at the desmosome and/or heart is undefined. We hypothesize that SNAP29 may play a critical role in maintaining desmosomal protein homeostasis and have direct relevance to ARVC disease features, as it is a disease associated with desmosomal protein dissolution. We show that SNAP29 co‐localizes with DSP in both the adult mouse and human heart. Cardiac‐specific SNAP29‐deficient mice (SNAP29‐cKO) displayed baseline and pacing‐ induced ventricular arrhythmias in an age‐dependent manner in the absence of cardiac structural and functional deficits. Furthermore, molecular analyses revealed that the arrhythmias found in SNAP29‐cKO hearts and cardiomyocytes were associated with a molecular loss of a subset of desmosomal proteins (DSP and plakophilin‐2) and the gap junction protein, connexin43. Furthermore, an accumulation of autophagic markers (p62 and LC3BII), as well as machinery, was observed at the cardiac cell‐cell junction in SNAP29 deficient cardiomyocytes. Acute blockade of autophagy using specific inhibitors (chloroquine and bafilomycin A1) was sufficient to rescue levels of desmosomal and gap junction proteins as well as arrhythmias in SNAP29 deficient cardiomyocytes. Our data suggest that SNAP29 insulates a subset of desmosomal proteins from selective autophagy‐mediated degradation to restrict cardiac arrhythmias. Thus, SNAP29 loss may directly contribute to the “concealed phase” of ARVC. Our results further identify autophagy defects as an underlying mechanism in desmosomal disease pathogenesis and highlight new potential therapeutic targets for ARVC.Support or Funding InformationNIH/NHLBI (HL09780)
Assessing Paracoccus denitrificans zinc metallochaperone AztD as a potential novel antibiotic drug target
Authors: Hendricks Grace , Meni Anusha , Yukl Erik 
Published: April 2020

Zinc is an important cofactor required for the catalytic activity and structural stability of enzymes. Additionally, zinc plays critical roles in host‐pathogen interactions during bacterial infection. As such, zinc homeostasis and its regulation are vital for every organism. Bacteria use ATP Binding Cassette (ABC) transporters to acquire zinc from their extracellular surroundings. In Paracoccus denitrificans, the ABC transporter consists of a cytoplasmic ATPase (AztA), a transmembrane permease (AztB), a periplasmic solute binding protein (AztC), and a metallochaperone (AztD). The P. denitrificans zinc import system is closely related to that of carbapenem resistant Enterobacteriaceae (CRE), a group of pathogenic organisms considered an urgent threat to human health by the Centers for Disease Control. These zinc import system proteins are necessary for bacterial virulence, have no known homologs in eukaryotes, and are conserved in human pathogens. Thus, this study aims to assess AztD as a potential novel antibiotic drug target. Current work involves using site‐directed mutagenesis to delete the N‐terminal domain of AztD in order to further understand its role in zinc binding and transfer to AztC. Future experiments include screening for pore blocking molecules that could act as zinc transfer inhibitors.Support or Funding InformationNational Institutes of Health 1R01GM122819‐01A1
Beta‐cell Prohormone Convertase 1/3 Plays a Larger Role in Glucose Homeostasis Than Beta‐cell Prohormone Convertase 2 in Mice
Authors: Taylor Austin James , Chen Yi-Chun , Verchere C. Bruce 
Published: April 2020

Insulin is synthesized in pancreatic beta cells from its precursor proinsulin, which is cleaved by the prohormone convertases 1/3 (PCSK1) and 2 (PCSK2) to generate mature insulin. In patients with type 1 or type 2 diabetes, proinsulin processing to mature insulin is impaired and elevated circulating proinsulin:insulin ratios are found in both conditions. It is unknown whether the beta‐cell prohormone processing impairments observed in diabetes contribute to or are secondary consequences of beta‐cell failure. Given the key role of PCSK1 and PCSK2 in insulin synthesis and beta‐cell function, we hypothesized that beta‐cell PCSK1 or PCSK2 deficiency would result in beta‐cell dysfunction and glucose intolerance.Beta‐cell specific knockout mouse models of Pcsk1 (Pcsk1betaKO) and Pcsk2 (Pcsk2betaKO) were generated using cre‐lox recombination and monitored for changes in glycemia on chow and high‐fat (HF) diets. Neither Pcsk1betaKO or Pcsk2betaKO chow‐fed animals displayed hyperglycemia or impaired glucose tolerance in the first 14 weeks of life. By 26 weeks of age Pcsk1betaKO, but not Pcsk2betaKO, chow‐fed male mice displayed fasting hyperglycemia and impaired glucose tolerance. After 19 weeks of HF diet, 3 of 5 Pcsk1betaKO male mice developed diabetes (fasting glycemia > 16mM), while no hyperglycemia was observed in Pcsk2betaKO mice up to 30 weeks of diet duration. Pcsk1betaKO mice displayed elevated islet and plasma (pro)insulin levels, decreased islet mature insulin levels, translucent insulin granules, and trended toward increased beta‐cell area in the pancreas. Despite altered insulin biosynthesis and immature granules, Pcsk1betaKO islets displayed similar insulin secretion kinetics to Pcsk1 wild‐type islets ex vivo.Our data suggest that Pcsk1 plays a larger role in proinsulin processing and beta‐cell function in mice than Pcsk2. However, despite severe impairments in insulin biosynthesis, Pcsk1‐deficient beta cells compensate remarkably well at early ages and hyperglycemia is only observed in states of increased beta‐cell secretory demand (mild insulin resistance associated with aging or HF diet in mice). Thus, impaired Pcsk1‐mediated prohormone processing, but not Pcsk2‐mediated processing, in beta cells increases diabetes susceptibility in mice.Support or Funding InformationFunding was provided by the Canadian Institutes of Health Research and Canucks for Kids Fund Childhood Diabetes Laboratories
Effects of nicotine + morphine on reward‐related behavior and nicotinic acetylcholine receptor regulation in mouse midbrain.
Authors: Avelar Alicia Jo , Akers Austin T. , Cooper Skylar Y. , Kasireddy Vishnupriya , Henderson Brandon J. 
Published: April 2020

In the United States about 480,000 deaths/year are caused by cigarette smoking and about 50,000 due to opioid abuse. Co‐use of more than one drug is common, therefore, it is vital to study the effects of drug combinations to understand how co‐use alters the brain. Especially given that in America, > 90% of opioid dependents are heavy smokers. The objective for this study is to determine how combined nicotine + morphine alters reward‐related behavior and nicotinic acetylcholine receptor (nAChR) expression in the midbrain. Our hypothesis is that co‐use of nicotine + morphine leads to enhanced GABA neuron disinhibition of VTA dopamine neurons. Female and male (3–5 months old) C57BL/6J background strain mice with or without α4‐mCherryα6‐GFP nAChRs were used in conditioned place (CPP) preference assays to examine reward‐related behavior with nicotine + morphine. We used confocal microscopy to analyze the density of α4‐containing (α4*), α6*, and α4α6* nAChRs in ventral tegmental area (VTA), substantia nigra pars compacta (SNc), and substantia nigra pars reticulata (SNr). In our CPP assays, a Two‐way ANOVA (factors: treatment and sex) showed a significant effect of sex (F (1, 42) = 5.75; p = 0.02) on reward‐related behavior, suggesting a sex‐dependent dose effect with morphine. In our microscopy assays, nicotine + morphine treatment downregulated α4* nAChRs on SNr GABA cells. Our CPP data supports that sex is an important variable in the effects of drugs on reward‐related behavior. Our receptor regulation data suggests that downregulation of α4* nAChRs on SNr GABA cells is important to the combined effects of nicotine + morphine.Support or Funding InformationSupport: Funding was provided through start‐up funds through the Marshall University Research Corporation.
Interaction between Ras and the two Ras‐Binding Domains of Raf kinase
Authors: Cookis Trinity , Volmar Alicia , Merritt Kathleen , Mattos Carla 
Published: April 2020

The interaction between Ras and Raf kinase at the membrane promotes cell proliferation through the mitogen activated protein kinase (MAPK) pathway. Ras mutations drive 20% of all human cancers and despite great efforts, there are currently no drugs targeting Ras. Raf interacts with Ras via its two N‐terminal Ras‐binding domains: the Ras‐binding domain (RBD) and the cysteine‐rich domain. Binding of both the Raf‐RBD and CRD are required for Ras‐mediated activation of Raf kinase, however, the mechanism that results in the activation of the C‐terminal Raf kinase domain remains unknown. Here we present the 2.8 Å crystal structure of Ras in complex with a Raf construct containing both the RBD and CRD, revealing the interface for Raf‐CRD binding. In combination with molecular dynamic simulations, we identify allosteric effects induced by Raf‐CRD binding that stabilize the Ras active site to promote a Ras conformation poised for intrinsic hydrolysis.Support or Funding InformationNSF, MCB‐1517295. Northeastern Office of Undergraduate Research and Fellowships
Structure‐Activity Relationships of a Negative Allosteric Modulator of the D3 Dopamine Receptor and Investigation of its Binding Site
Authors: Moritz Amy E. , Inbody Laura R. , Gandhi Disha M. , Akano Emmanuel , Fearce Chelesa , Free R. Benjamin , Barnaeva Elena S. , Hu Xin , Southall Noel T. , Ferrer Marc , Keck Thomas M. , Frankowski Kevin J. , Sibley David R. 
Published: April 2020

Dopamine receptors (DARs) are G protein‐coupled receptors (GPCRs) that regulate diverse physiological functions including movement, cognition, mood, and reward‐related behaviors, and are involved in the treatment or etiology of many neuropsychiatric disorders including schizophrenia, substance use disorder (SUD), and Parkinson’s disease. DARs are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological properties. Antagonists of D2‐like DARs are currently used as drug therapies for many psychiatric disorders. While the D2R is highly expressed in the dorsal striatum, which regulates movement, the D3R has a more limited distribution to limbic regions associated with the control of mood and emotion. D3R‐selective antagonists may therefore be useful as schizophrenia or SUD therapeutics as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R‐preferring antagonists. Discovery of subtype selective compounds for the D3R and D2R has been challenging due to high sequence homology within their orthosteric binding sites. However, compounds that modulate receptor activity through interactions with a less conserved allosteric site of the receptor have the potential to be highly selective. To find highly selective allosteric antagonists of the D3R, our lab utilized a high‐throughput screen of the NIH Molecular Libraries Program 400,000+ small molecule library using a D3R‐mediated β‐arrestin recruitment assay. We found one compound, MLS6357, that was selective for the D3R versus the D2R in several functional outputs including β‐arrestin recruitment and G‐protein activation. Further, radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross‐reactivity with other GPCRs. Additionally, Schild‐type functional assays found that MLS6357 is a purely non‐competitive negative allosteric modulator of the D3R. We synthesized 46 analogs of MLS6357 using iterative medicinal chemistry and tested them for activity which revealed structure activity‐relationships and enabled further refinement of the scaffold. These efforts produced modulators that are 5‐ to 9‐fold more potent than the original hit compound. To identify the allosteric site on the D3R, we utilized D3R/D2R chimeras which revealed receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R‐related pathophysiology.Support or Funding InformationNINDS Intramural Research Program