bluuebunny/crossref_metadata_2025_split huggingface dataset:
Time‐dependent Development of Social Stress Caused by Repeated Exposures to Aggressors Simulating Features of Post‐traumatic Stress Disorder (PTSD)
Authors: Chakraborty Nabarun , Meyerhoff James , Gautam Aarti , Muhie Seid , Hammamieh Rasha , Jett Marti 
Published: June 2021

A utility criterion of a PTSD mouse model is recruiting a stressor with systematically variable intensity. An ideal stressor should maintain a ‘dose‐response relationship’ with the subjects’ behavioral shift. A typical PTSD model includes a brief exposure to foot or tail shock.We developed a model that involves repeated exposures (either 5‐day or 10‐day) to trained conspecific aggressors (Agg). A contextual reminder study evaluating a cluster of ethogram identified PTSD‐like (acute‐ and persistent‐) syndromes such as incubation, extinction and desensitization of fear responses. Pathophysiological consequences supported the model.This model reflects the combat‐like situation where life‐threatening events occur repeatedly and randomly. The direct relationship of PTSD‐risk with deployment frequency further justifies the model.Agg induced stress intensity can only be elevated by prolonging the exposure. The risk of habituation can thereby defeat the ‘dose‐response’ relationship. We addressed this concern by evaluating the time‐dependent behavioral shift of the subject mice (C57BL/6j). Subsets of subject mice were withdrawn from the Agg‐exposure (Agg‐E) schedule at regular intervals and their psycho‐patho‐physiological characters were evaluated. A regression model elucidated the temporal relationship of Agg‐E stress with the psychological alteration.
BNIP3 regulates mitophagy and apoptosis in delayed neuronal death in stroke
Authors: Shi Ruoyang , Kong Jiming 
Published: June 2021

Physiological level of autophagy promotes neuronal survival, while apoptosis contributes to delayed neuronal death in ischemic stroke. In this study, we show that the proapoptotic BCL‐2 family member BNIP3 is an upstream regulator for both processes. Specifically, deletion of BNIP3 gene is neuroprotective by affecting mitophagy and apoptosis pathways. We performed IHC, western blot, Co‐ip, ELISA, and cell transfection to analyze the BNIP3's regulation on mitophagy and apoptosis in cortical neurons and ischemic brains. Both BNIP3 wild‐type and knock‐out mice were used. In primary neurons exposed to OGD/reperfusion, BNIP3 was highly expressed, with the time course and expression levels of apoptosis‐related proteins (i.e. active caspase‐3, cytochrome C, and BAX) and autophagy‐related proteins (i.e. LC3, Beclin‐1, and LAMP‐2) positively regulated. Promoting or inhibiting autophagy pharmacologically didn't affect the expression patterns of BNIP3, indicating it is an upstream regulator. We also measured the brain damage of neonatal stroke in transgenic mice. TTC staining showed the infarct volume of ischemic brains was significantly reduced in BNIP3 KO mice compared to WT mice upon 3–7 days recovery. Silence of BNIP3 gene in cortical neurons of KO mice activated a robust autophagic response and decreased apoptosis, coordinately contributing to the neuroprotection in the KO animals after stroke.Grant Funding Source: Canadian Institute of Health Research, Canadian Stroke Network, Manitoba Health Research Council, Manitoba Institute of Child Health
Levels of nitric oxide synthase and cholecystokinin mRNA in the upper gastrointestinal tract of rats following experimental spinal cord injury
Authors: Swartz Emily , Holmes Gregory M. 
Published: June 2021

In addition to the loss of motor and sensory function, SCI causes immediate changes to gastrointestinal (GI) tract physiology and reflex function. Reflex control of the stomach is dominated by vago‐vagal circuits which remain anatomically intact following SCI. Our previous reports indicate that SCI a) reduces gastric motility, tone, and emptying; b) diminishes vagal afferent sensitivity to GI peptides such as cholecystokinin (CCK); and c) diminishes mesenteric blood flow.The mechanisms, interrelationships, and temporal patterning of post‐SCI gastroparesis remain poorly defined. We tested the hypothesis that acute SCI induces GI inflammation and a resulting reduction of CCK mRNA expression.We used qRT‐PCR to quantify the levels of CCK, iNOS, and nNOS in the GI tract, 1 week post‐SCI. Our data show a significant increase in CCK and iNOS in the duodenum; nNOS shows an upward non‐significant trend. Jejunal expression of CCK is reduced while both iNOS and nNOS levels show a non‐significant upregulation. Our data are similar to experimental ischemia‐reperfusion of the GI tract, in which the superior mesenteric artery (SMA) occlusion causes a dramatic inflammatory response within the GI tract. We conclude that our model of SCI represents a continuum of GI ischemia and that the increase in inflammatory mediators may play a role in decreasing the sensitivity of the gastric vagal afferents.Grant Funding Source: NINDS 49177
Ethanol down‐regulates microglia inflammatory responses augmented by endoplasmic reticulum stress
Authors: Goral Joanna , Meyer Alice 
Published: June 2021

RationaleEndoplasmic reticulum (ER) stress is involved in pathology of neuroinflammatory disorders, multiple sclerosis among them. Ethanol is known to affect immune function, including inflammation. In this study we examined the effect of ER‐stress inducer thapsigargin on inflammatory responses of BV2 mouse microglia. We also examined whether ethanol and inflammatory agents could induce ER stress in BV2 cells.MethodsBV2 microglia were stimulated with LPS (100 ng/ml) with or without ethanol (100 mM) for 24 h. The cells were treated with thapsigargin for 2–18 h. The inflammatory response was evaluated by measurement of nitric oxide (NO) production. XBP1 and CHOP, proteins induced by ER stress, were detected by western blot.ResultsThapsigargin did not induce, but significantly increased (x 2.5), LPS‐induced NO production by BV2 microglia. This effect was attenuated by ethanol (by 38%). XBP1 and CHOP were detected in BV2 cells exposed to thapsigargin, LPS, and ethanol.ConclusionsThe presence of XBP1 and CHOP shows that ER stress can be induced in BV2 cells by thapsigargin, LPS and ethanol. LPS‐induced inflammatory responses can be augmented by additional ER stress. However, ethanol – ER stress‐inducing agent itself – can reduce thapsigargin‐augmented inflammatory responses.
Modulation of Mcm2–7 activity by Cdt1
Authors: DaSilva Lance F , Kolaczyk Tomasz , Ma Xiaoli , Davey Megan J 
Published: June 2021

Genome duplication occurs once and only once in each cell cycle. During G1 phase, pre‐replicative complexes (pre‐RC), composed of ORC, Cdc6, Cdt1 and Mcm2–7, are assembled at replication origins. Mcm2–7 is the replicative helicase in eukaryotic cells; however, it does not unwind DNA in the pre‐RC. It is only in S‐phase, after activation of Mcm2–7, that DNA unwinding is detected. Cdt1 co‐purifies from yeast cells in a complex with Mcm2–7. Beyond being an escort for entry of Mcm2–7 into the nucleus and into the pre‐RC, the role of Cdt1 in DNA replication is not known. Using purified components from bacterial expression systems, we assembled Mcm2–7•Cdt1 complexes and compared its activity to that of Mcm2–7 alone. We show that Mcm2–7•Cdt1 has lower ATPase activity than Mcm2–7. In addition, DNA unwinding by Mcm2–7•Cdt1 is significantly lower than by Mcm2–7 alone. Furthermore and consistent with published reports, in vitro pre‐RC reconstitution experiments showed loading of Mcm2–7 to origins was dependent on ORC, Cdc6 and Cdt1. Together, the effects of Cdt1 on Mcm2–7 suggest mechanisms by which Cdt1 may carry out its essential role in the initiation of DNA replication.Research funded by CIHR
RNA Polymerase primed DNA synthesis in T7 Bacteriophage system
Authors: Nandakumar Divya , Patel Smita S 
Published: June 2021

DNA replication initiation is divided into three steps: Recognition of the replication origin by the initiator protein, melting of the duplex DNA, and synthesis of RNA primer. Primer synthesis is required as there are no known prokaryotic DNA polymerases that can carry out DNA synthesis de novo. In the T7 bacteriophage system and in mitochondrial DNA, the RNA polymerase catalyzes all three steps to initiate DNA replication. The primary replication origin in the T7 genome has two T7 RNA polymerase promoters separated by 52 bp sequence and followed by an AT rich region. There is almost nothing known about the mechanism of RNA‐primed DNA synthesis, including the length of the RNA primer synthesized by T7 RNA polymerase, the mechanism of switching from transcription to DNA synthesis, and why these events occur only at specific promoters in the T7 genome. Whether specific interactions exist between the two polymerases is also not known. Our results suggest that on short DNA substrates, one of the two promoters at the replication origin is sufficient for initiation of DNA synthesis. The RNA polymerase synthesizes primers of 8–12 nucleotide length that are utilized by the DNA polymerase. In the T7 bacteriophage system, this is the critical length at which the transition from transcription initiation to elongation occurs. Our results also show that an actively transcribing RNA polymerase is necessary for the primers to be utilized by the DNA polymerase.Source of research support: NIH
Transcriptional regulation of trocarin D, a prothrombin activator from Tropidechis carinatus
Authors: Han Xia , Kwong Shiyang , Ge Ruowen , Kolatkar Prasanna , Kini R. Manjunatha 
Published: June 2021

Trocarin D from the Tropidechis carinatus venom (TrTD) is a prothrombin activator sharing similar structure and function with blood coagulation factor Xa (FXa). It is probaly evolved from its plasma counterpart (TrFX) by gene duplication. The gene sequences of TrTD and TrFX are highly similar, except for the promoter and intron 1 regions. The insertion in the promoter region of TrTD (VERSE—VEnom Recruitment/Switch Element) accounts for elevated but not tissue specific expression of TrTD. The insertions/deletions intron 1 were examined for their “silencing” role in non‐venom gland cells. Using a series of deletion luciferase reporter assays, the strongest silencing cis‐elements were located in a 193 bp fragment within the insertion 2 segment (1975 bp). This segment has multiple silencing cis‐elements that turn off gene expression synergistically. Several silencer binding transcription factors (TFs) were identified using DNA affinity chromatography and mass spectrometry. The findings here highlight the importance of intron 1 for TrTD venom gland‐specific expression, and will contribute to understanding the transcriptional silencing mechanisms of TrTD in non venom gland tissues.Source of research support: Academic Research Grants from NUS
Rab4 GTPase regulates cell surface expression of hERG channels through Nedd4–2
Authors: Cui Zhi , Zhang Shetuan 
Published: June 2021

The human ether‐a‐go‐go‐related gene (hERG) encodes the pore‐forming subunits of the cardiac rapidly activating delayed rectifier K+ channel (IKr) which is important for cardiac repolarization. Dysfunction of IKr causes long QT syndrome (LQTS), a cardiac electrical disorder which can lead to cardiac arrhythmias and sudden death. Currently, trafficking mechanisms of the hERG channel are poorly understood. We investigated the effects of Rab4 GTPase on the function and expression of hERG channels using Western blot, patch clamp and immunofluorescence staining. Our data revealed that Rab4 selectively decreased the hERG expression on the plasma membrane. Mechanistically, Rab4 did not directly target the hERG protein. Instead, it increased the expression of ubiquitin ligase Nedd4–2, which mediates hERG ubiquitination and degradation. Mutations that disrupt the Nedd4–2 targeting site in hERG completely abolished the effect of Rab4. Our data further revealed that Rab4 inhibited Nedd4–2 self ubiquitination, leading to a decreased degradation rate of Nedd4–2. We conclude that Rab4 regulates the cell surface expression of hERG channels through Nedd4–2.Supported by the Canadian Institutes of Health Research – CIHR.
Activation of muscarinic receptor increases cardiac potassium hERG channel through phosphorylation of E3 ubiquitin ligase Nedd4–2
Authors: Wang Tingzhong , Zhang Shetuan 
Published: June 2021

The human ether‐a‐go‐go‐related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (IKr) which is critical for repolarization of cardiac action potential. Loss of function of hERG channels due to gene mutations or drug blockade causes long QT syndrome. Presently, little is known about the restoration or enhancement of hERG channel function. In the present study, we found that muscarinic receptor agonist, carbachol, specifically increased hERG expression and IhERG, but had no effect on IKs, IEAG and IKv1.5 channels in HEK expression systems. Carbachol treatment decreased hERG‐ubiquitin interaction and hERG degradation. It is known that hERG channels are degraded by the Nedd4–2, we found that disrupting the binding sites of Nedd4–2 by the Y1078A point mutation or C‐terminal truncation Δ1073 mutation in hERG completely eliminated the effects of carbachol on hERG channels. Our data further indicate that carbachol treatment inhibited Nedd4–2 activity by enhancing its phosphorylation level. Another muscarinic receptor agonist, oxotremorine, increased hERG function, and the muscarinic receptor antagonist 4‐DAMP abolished the effects of both carbachol and oxotremorine on hERG channels. Enhancement of hERG function by muscarinic receptor activation represents a novel pathway in hERG regulation, and this finding may have potential for the management of long QT syndrome patients.Supported by: Canadian Institutes of Health Research (CIHR).
Cross‐Presentation of peptides Chaperoned by Heat Shock Protein gp96
Authors: Phillips Shantell Sherie , Binder Robert J 
Published: June 2021

Cross‐presentation is the ability of Dendritic Cells (DCs) to uptake and process extracellular antigens and present them on their major histocompatibility class I molecules (MHC). Heat shock protein (HSP)‐chaperoned peptides are cross‐presented by DCs. It has been demonstrated that CD91 acts as a cell surface receptor for heat shock protein gp96, binding gp96 directly and allowing the HSP‐peptide to be up taken by the cell into the endosome. Previous studies have shown that HSP90 is critical for the translocation of peptides from the endosome to the cytosol for proteasomal degradation. These peptides are then translocated to the endoplasmic reticulum where they are loaded onto the MHCs for cell surface display. The fate of gp96 after endocytosis is unknown and is the focus of this study.In the current study, we investigate the fate of gp96 after internalization. We used selective cell fractionation of DCs to identify the location of gp96. Through this procedure, we were able to initially separate the intracellular membranes and plasma membranes of the DCs and showed that gp96 was primarily associated with intracellular membranes. With further fractionations, we were able to isolate the cytosol, mitochondria, and nucleus and showed that at 30mins gp96 was primarily located in the cytosol and not the nucleus or mitochondria. These study results show the route of trafficking gp96 associated with cross‐presentation.
Quantitative internalization kinetics of the green fluorescence protein‐tagged guanylyl (guanylate) cyclase/natriuretic peptide receptor‐A in Human Embryonic Kidney‐293 cells
Authors: MANI INDRA , Garg Renu , Nguyen Vickey A , Pandey Kailash N 
Published: June 2021

Cardiac hormone atrial natriuretic peptide (ANP) modulates blood pressure and blood volume by activation of the guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and subsequent generation of intracellular second messenger cGMP. Advances in the development of proteins conjugated with the enhanced green fluorescence protein (eGFP) provide fluorescence based analysis of protein trafficking in individual cells. The aim of this study was to quantify internalization kinetics of NPRA in human embryonic kidney‐293 (HEK‐293) cells through confocal microscopy. Cells were stably transfected with cDNAs encoding NPRA‐eGFP. HEK‐293 cells were treated with natural ligand ANP at different time course (0, 5, 10, 15, and 30 minutes) and internalization of receptor was analyzed through confocal immunofluorescence microscopy. In control cells without ANP treatment, all receptors were localized on plasma membrane. Internalization of receptor from the plasma membrane was analyzed as 58.4% at 5 min, 69.0% at 10 min, 70.8% at 15 min, and 75.6% at 30 min with compare to controls. Similarly 51.4% receptor population was localized in cytoplasm at 5min, 62.5% at 10 min, 65.5% at 15 min and 69.9% at 30 min. In conclusion these data demonstrate that after ligand binding, NPRA is rapidly internalized and trafficked from the cell surface to cell interior.This work was supported by the NIH grant (HL57531).
A Semisynthetic Approach to Understanding PTEN Regulation
Authors: Bolduc David , Gabelli Sandra , Raben Dan , Devreotes Peter , Amzel Mario , Cole Phil 
Published: June 2021

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressing, lipid phosphatase that negatively regulates the PI3K/AKT signaling pathway by dephosphorylating the lipid second messenger PIP3. PTEN function is frequently lost in cancer and its normal functions are regulated by a variety of mechanisms within cells. One such mode of regulation acts through the phosphorylation of a cluster of serine and threonine residues on PTEN's C‐terminal tail. Phosphorylation at this cluster has been proposed to induce a conformational change in the PTEN protein which is essential for its tumor suppressive functions. To better understand how phosphorylation at this regulatory cluster affects PTEN's activities we have generated semisynthetic PTEN in its phosphorylated form by expressed protein ligation. An interfacial kinetic analysis reveals that phosphorylation causes an increase in the membrane binding constant Ks, while having no change in the interfacial Km. These results are corroborated using a vesicle binding assay. Using Small Angle X‐ray Scattering and other biochemical techniques we have shown that phosphorylation of PTEN's tail induces an intramolecular conformational change that stabilizes the phosphatase domain of the protein. Using protein semisynthesis we have developed a valuable tool for future studies involving PTEN phosphorylation.Source of Funding:NIH Grant: R01CA074305
Structure‐function studies of the Golgi Dsc E3 ligase complex required for SREBP activation in yeast
Authors: Espenshade Peter J , Lloyd S. Julie‐Ann , Tong Zongtian , Raychaudhuri Sumana 
Published: June 2021

Fungal sterol regulatory element‐binding protein (SREBP) transcription factors are oxygen‐responsive transcription factors required for adaptation to hypoxia. Our recent studies identified five genes (dsc1–5, defective for SREBP cleavage) that code for subunits of the Golgi Dsc E3 ligase complex that is required for fission yeast SREBP activation. Dsc proteins have domains that are predicted to function in the ubiquitin proteasome pathway: Dsc1 is a ubiquitin E3 ligase with a RING domain, Dsc2 has a predicted ubiquitin‐associated (UBA) domain and Dsc5 contains a ubiquitin regulatory X (UBX) domain that binds Cdc48.To begin to define the mechanism for Sre1 cleavage in fission yeast, we performed structure‐function studies to elucidate the organization of this multi‐subunit complex. Using co‐immunoprecipitation analysis and Blue native PAGE, we found that Dsc1–5 form a stable, detergent solubilized complex with a discrete architecture with distinct subcomplexes. Parallel studies in budding yeast, which lacks SREBP homologs, demonstrate that the Dsc E3 ligase complex is structurally conserved and localizes to the Golgi. Future studies will focus on the identification of additional Dsc E3 ligase substrates and the function of this complex in Golgi protein degradation.Studies were supported by the National Institutes of Health (HL‐077588).
Apoptosis Associated with Osteoarthritis is Attenuated in Mice Lacking the Receptor for Advanced Glycation End Products (RAGE)
Authors: Wilhelm Spencer K , Larkin D Justin , Doxey Alexander S. , Kartchner Jeffrey Z. , Vanderuff James , Anderson Karl , Draper Christian S. , Chavez Elizabeth , Reynolds Paul R. , Kooyman David L. 
Published: June 2021

ObjectiveIt has been previously shown that chondrocyte apoptosis is associated with the progression of osteoarthritis. The objective of this project was to examine the effects of S100, RAGE, Tgf‐β1, HtrA1 and NF κβ on apoptosis in the progression of osteoarthritis (OA).MethodsSamples of wild type (WT) and RAGE knockout (KO) were assayed for HtrA1, S100, RAGE, TGF‐β1, NF‐kB, TUNEL and evaluated for cartilage degradation four weeks after a knee destabilization procedure.ResultsWe observed positive staining for all biomarkers except TGF‐β1examined in both WT and RAGE KO mice with high Mankin score OA. All biomarkers assayed were present, except HtrA1, in samples with low Mankin score OA. We also observed varying degrees of TUNEL assay staining in both WT and RAGE KO mice. Remarkably, despite similar IHC staining profiles, RAGE KO mice exhibited significantly less cartilage degradation and chondrocyte apoptosis, assayed by TUNEL, compared to WT mice.ConclusionWe conclude that RAGE mediated upregulation of TGF β1 leads to an upregulation of HtrA1 and apoptosis associated with osteoarthritis. This suggests that an inhibition of inflammatory processes involving RAGE may attenuate chondrocyte apoptosis, and slow the progression of OA.
Inositol biosynthesis is fine‐tuned by inositol pyrophosphates in Saccharomyces cerevisiae
Authors: Ye Cunqi , Greenberg Miriam L 
Published: June 2021

In yeast, inositol biosynthesis is coordinated with the synthesis of phospholipids by controlling the expression of enzymes in the biosynthetic pathways. Inositol pyrophosphates, most recently characterized in the inositide family, have diverse roles in phosphate signaling and other important cellular processes. However, little is known about the function of inositol pyrophosphates in the biosynthesis of inositol and phospholipids. The objective of this study is to understand the mechanistic links between inositol metabolism and inositol pyrophosphates. Our results indicated that inositol pyrophosphate deficient mutants kcs1Δ and vip1Δ exhibit decreased inositol biosynthesis due to decreased derepression of INO1, which encodes the rate‐limiting enzyme for inositol biosynthesis. Further studies revealed that the inositol pyrophosphate kinase activity and the basic leucine zipper (bZIP) domains in KCS1 are both required for optimal INO1 expression. Interestingly, the levels of Kcs1p correlate with INO1 expression. We conclude that conversion of IP5 to inositol pyrophosphates modulates INO1 transcription.This work is funded by grant R01 DK081367 from NIH and a travel grant from Wayne State University.