bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic changes of intracellular Ca2+ content of rats brain after traumatic brain injury and its correlation with the concentration of calcitonin gene‐related peptide
Authors: Xue Wen , Duan Hubin , Qiao Xiaohong , Hao Chunyan , Zhang Xiaoyi , Du Yanrong , feng zhigang , Guo Zhiyong , Zhen Junping , Li Youliang 
Published: April 2020

ObjectiveTo investigate the dynamic changes of intracellular Ca2+ content of rats brain after traumatic brain injury (TBI) and its correlation with the concentration of calcitonin gene‐related peptide (CGRP)MethodsThe rats model with TBI was prepare by Feeney’s methods. 144 male Wistar rats were randomly divided into Sham operation group(Sham group, 36 rats) and traumatic brain injury group (TBI group, 108 rats ). Above two groups subdivided into six groups by 2h,6h,12h,24h, 48h and 72h after TBI( 6 rats in each group. The calcium content in the rats brain cells were detected by laser scanning confocal microscope, and the CGRP concentration in plasma of rats were got by enzyme‐linked immunosorbent assay.ResultsThere were significant differences of calcium fluorescence intensity in the rats brain cells between the Sham group and the TBI group (P<0.05). In the meantime there were also significant differences of the CGRP concentration in plasma of rats between the Sham group and the TBI group (P<0.05). After the injury, calcium fluorescence intensity in the rats brain cells began to rise within 2 hours, reached its peak in 24 hours, and decreased in 48 hours (P<0.05). In the TBI group, the CGRP concentration in plasma of rats increased at 2h, then decreased at 24h,48h, and increased slowly at 72h after TBI (P<0.05).ConclusionThere was a negative correlation between CGRP concentration in rats plasma and the intracellular calcium content of rats brain. Calcium overload played an important role in the brain secondary damage after TBI.Support or Funding Information• This study was supported by the National Natural Science Foundation Youth Fund (30600637), China Postdoctral Science Foundation Grant (2019T120195), China Postdoctral Science Foundation Grant (2014M561207), the Shanxi Province Basic Research Program ( 2010021034‐4, 201601D011119), the Shanxi Scholarship Council of China (2011‐096 and Key Projects No.4), Department of Human Resources and Social Security of Shanxi province fund grant, The science and technology research projects of health department of Shanxi Province(2016D011119), The Teaching Reform Program of Shanxi Higher Education, The Education Reform Project of Postgraduate Students in Shanxi Province. The Project of “131” Leading Talents of Shanxi Higher Education, The Education Reform Project of Shanxi Medical University, the fund grant of the eleventh college of shanxi medical university(Lvliang Hospital).
Charge Neutralization Tunes Dynamic Arrest of Initially Disordered Reflectin Proteins
Authors: Levenson Robert , Gordon Reid , Lee Tyler , Malady Brandon , Morse Daniel E. 
Published: April 2020

The intrinsically disordered reflectin proteins fill the reflective Bragg lamellae of iridescent cells in squid. In vivo, phosphorylation of the cationic reflectins leads to protein condensation and hierarchical assembly, driving osmotic dehydration of the lamellae and causing enhancement of intensity and tuning of the color of reflected light. In vitro, purified monomeric reflectin protein can be driven to cyclably and tunably assemble by pH‐neutralization or addition of salt, forming spheres of low polydispersity and reproducible size. Analysis of reflectin assembly by dynamic light scattering, x‐ray scattering, and transmission electron microscopy shows that the calibration between charge‐neutralization and assembly size is enabled by the rapid dynamic arrest of particle growth, as controlled by an electrostatic switch spatially distributed across the reflectin chain. Confocal microscopy of fluorescently labeled micron‐sized reflectin assemblies shows that they exhibit internal dynamics that rapidly slow following assembly, suggesting that assembly occurs through a transient liquid‐liquid phase separation that undergoes gelation to form stable protein‐dense condensates. Electron paramagnetic resonance (EPR) analysis shows the initially disordered reflectin monomers form ordered secondary structure that may be critical in the arrest of growth and stabilization of particles. These results provide new insights into the assembly of these unique intrinsically disordered proteins and the biophotonic systems they form, and suggest pathways for the creation of novel tunable biomaterials.Support or Funding InformationThis research was supported by the U.S. Department of Energy, U.S. Army Research Office, and Institute for Collaborative Biotechnologies.
Inhibition of Toll‐Like receptors alters the ability of tributyltin to increase production of interleukin 1 beta by human immune cells
Authors: Alcala Aliyah , Whalen Margaret 
Published: April 2020

Toll‐like receptors (TLR) are integral for activating the innate immune system responsible for initial detection of infection through recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs). MAP Kinases are downstream signaling components that lead to the production of chemokines, type I interferons, and pro‐inflammatory cytokines, such as interleukin 1β (IL‐1β), in response to TLR activation. IL‐1β is a primary pro‐inflammatory cytokine necessary for an appropriate response to injury or infection. Dysregulation of IL‐1β levels leads to chronic inflammation, which is implicated in the development of diseases such as atherosclerosis, cancer, and autoimmunity. Tributyltin (TBT) is an environmental contaminant due to its use as a biocide in numerous products and in anti‐fouling paints. Previous studies have found that exposure to TBT, which is present in human blood, increases the secretion and intracellular concentrations (production) of the pro‐inflammatory cytokine IL‐1β in peripheral blood mononuclear cells (PMBCs) and this increase requires MAPK activation. The current study examines whether the upstream regulators of MAPK activation in immune cell production of IL‐1β, the TLR, are also required for TBT to induce increases in IL‐1β. TLRs were inhibited with compounds specific for selected TLRs and the ability of TBT (100, 50, and 25 nM) to cause increased production of IL‐1β after 24 h of exposure was examined using ELISA and western blot. Results indicate that when TLR are inhibited there is a decrease in TBT‐induced production IL‐β. This suggests that TBT utilizes the TLR to stimulate IL‐1β production.Support or Funding InformationThe National Science Foundation Tennessee Louis Stokes Alliance for Minority Participation (TLSAMP) Bridge to the Doctorate (BD) program
Elucidating the role of the axial cysteine residue in NHase catalysis and the enzyme maturation
Authors: Ogutu Irene , Lankathilaka Wasantha K. P. , Holz Richard C. , Bennett Brian A. 
Published: April 2020

Nitrile hydratases are metalloenzymes that contain trivalent Fe/or Co ions in their active site, coordinated in an N2S3 ligand environment. The sulfur atoms are post‐translationally modified and have three different oxidation states (αCys, Cys‐SOH and Cys‐SOO−) while backbone amines account for the nitrogen ligands and are deprotonated. Theoretical calculations assume that the αCys ligand is a thiolate, making it a strong π‐donor ligand and a good a nucleophile. A critical outstanding mechanistic question is whether the formation of a transient disulfide bond is essential for catalytic turnover. Such a transient disulfide bond was proposed based on QM/MM calculations between the αCys sulfur and the sulfur of the proposed sulfenic acid cyclic intermediate. However, more recent theoretical studies is self‐inconsistent in its conclusions regarding a transient disulfide formation. To examine the role of the αCys108 ligand in catalysis, it was mutated to A, M, S, and H in the Co‐type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). ICP‐MS demonstrated reduced metal ion content for each mutant: <5% (αC108A), ~44% (αC108M), ~50% (αC108S) and ~55% ( αC108H) and catalytic activity also decreased to ~0.3%, ~0.8%, ~1.6%, and ~6.7%, respectively, towards acrylonitrile. The X‐ray crystal structure of αC108A confirmed the mutation and the decreased active site metal content. Therefore, the axial thiolate is important to properly tune the electronic properties of the active site metal ion, but is not essential to catalysis. These data provide insight into the critical mechanistic question of whether the axial αCys ligand forms a transient disulfide bond during turnover.Support or Funding InformationThis work was supported by (CHE‐1808711, RCH; CHE‐1308672, BB; CHE‐1532168 BB & RCH), the Todd Wehr Foundation, Bruker Biospin, and the National Institutes of Health/NIBIB National Biomedical EPR Center (P41‐EB001980)
Investigating Novel Turnover Mechanisms of Huntingtin in Yeast
Authors: Warner Amanda , Buchan Ross 
Published: April 2020

Huntington’s disease is a devastating neurodegenerative disease which leads to symptoms of involuntary movements, memory loss, and ultimately death. The disease is characterized by a CAG repeat expansion mutation in the huntingtin (HTT) protein which leads to its aggregation inside the cytoplasm of striatum neurons. It is unknown whether these aggregates are directly toxic or if they reflect a cellular protection mechanism. Recently, endocytosis activity has been shown to reduce the toxicity, protein levels and propensity to aggregate of TDP‐43, a protein that forms potentially toxic aggregates in amyotrophic lateral sclerosis (ALS) patient motor‐neurons. Conversely, high expression of aggregation‐prone TDP‐43 can impair endocytosis function. Thus, there is a possibility that HTT aggregates, similar to TDP‐43 in ALS, will also inhibit endocytosis and be cleared by the endocytosis pathway. Using a Huntington’s disease yeast model, wild type and mutant HTT localize to the cytoplasm as well as the vacuole, an acidic organelle where proteins are trafficked by endocytosis for degradation. “WT” HTT with 25 CAG repeats is expressed diffusely throughout the cytoplasm while “pathological” HTT with 103 CAG repeats forms cytoplasmic aggregates, in addition to exhibiting diffuse cytoplasmic localization. Pathological HTT exhibits slight growth defects in vps34Δ and vps15Δ strains; these PI3K subunit genes are key to normal endocytic (and autophagic) function. Protein half‐life assays indicate that WT HTT has a slower turnover rate compared to the pathological HTT. Proteasome inhibition also slows WT HTT turnover but does not impact pathological HTT turnover rate. A genetic screen using the tet‐off regulatable promoter library will be conducted to determine for the first time what essential genes genetically interact with mutant HTT. Further microscopy and turnover assays will be conducted to determine if enhancement or suppression of endocytosis leads to clearance or accumulation of HTT in the vacuole respectively, and whether endocytosis activity represents a viable therapeutic target for Huntington’s disease.Support or Funding InformationThis project was funded by the University of Arizona Undergraduate Biology Research Program with funds from the Arnold and Mabel Beckman Foundation.
Identifying protein expression changes in acute myeloid leukemia
Authors: Caplan Mika , Hynes-Smith Willow , Swenson Samantha , Wittorf Karli , Gilbreath Tyler , Vahle Heather , Buckley Shannon 
Published: April 2020

Acute myeloid leukemia (AML) is a devastating cancer affecting the hematopoietic system. It is characterized by infiltration of abnormal proliferating immature myeloid cells originating in the bone marrow. Although this disease represents only 35% of diagnosed leukemias, it accounts for nearly 50% of leukemia‐related deaths, making it the leading cause of leukemia‐related mortality. Disease initiation often occurs through common recurrent genetic aberrations resulting in the formation of oncogenic fusion proteins. Two common mutations include mixed lineage leukemia (MLL) rearrangements and an inversion of chromosome 16, known as Inv(16). Although many of these AML initiating mutations have been identified, the downstream effects leading to disease progression are still largely unknown. Previous research has relied on RNA sequencing and microarray techniques to study the downstream effects, providing data at the transcriptional level. While these studies have proven efficacious, they fail to capture the changes that occur at the proteomic level. To interrogate the effect of protein expression alterations in AML, we performed a quantitative mass spectrometry (MS) analysis using mouse models to compare three tumor types (Inv(16), MLL‐AF9, and MLL‐ENL) to untransformed cells from the tumor‐initiating population. In parallel, we performed RNA sequencing for the same populations. With these combined results, we identified 61 proteins whose expression was upregulated in AML tumors, but strikingly, were unaltered at the transcriptional level. These proteins are shown to be associated with mitochondrial function as well as RNA processing. In addition, analysis of patient expression data sets in adult and pediatric AML reveal that a number of the proteins differentially expressed have no significant RNA expression alterations. These studies identify a set of proteins that have not previously been associated with leukemia, and may ultimately serve as potential targets for therapeutic manipulation to hinder AML progression and help contribute to our understanding of this disease.Support or Funding InformationNational Institute for General Medical Science (NIGMS) INBRE ‐ P20 GM103427, NIH P20GM121316
Cytosolic Ca2+ modulates Golgi structure through PKC‐mediated GRASP55 phosphorylation
Authors: Ireland Stephen Curtiss , Ramnarayanan Saiprasad , Fu Mingzhou , Zhang Xiaoyan , Emebo Dabel , Wang Yanzhuang 
Published: April 2020

It has been well documented that the endoplasmic reticulum (ER) responds to cellular stresses through the unfolded protein response (UPR), but it is unknown how the Golgi apparatus responds to similar stresses. In this study, we treated HeLa cells with ER stress inducers, thapsigargin (TG), tunicamycin (Tu) and Dithiothreitol (DTT), and found that only TG treatment resulted in Golgi fragmentation. TG induced Golgi fragmentation at a low dose and short time when UPR was undetectable, demonstrating that Golgi fragmentation occurs independently of ER stress. Further experiments demonstrated that Golgi fragmentation was through elevated intracellular Ca2+ and protein kinase Cα (PKCα) activity, which phosphorylates the Golgi stacking protein GRASP55. Significantly, activation of PKCα with other activating or inflammatory agents, including Phorbol 12‐myristate 13‐acetate (PMA) and histamine, modulates the Golgi structure in a similar fashion. Hence, our study revealed a novel mechanism through which increased cytosolic Ca2+ modulates Golgi structure and function.Support or Funding InformationThis work was supported by the National Institutes of Health (Grants GM112786, GM105920, and GM130331), MCubed and the Fastforward Protein Folding Disease Initiative of the University of Michigan to Y. Wang. S. Ireland is a graduate student who has been partially supported by the Mary Sue and Kenneth Coleman Endowed Fellowship Fund from the University of Michigan.
Inhibition of Low density Lipoprotein Internalization and Transcytosis by HDL; an alternative role for “good” cholesterol
Authors: Fung Karen Yin Yue , Lee Warren , Fairn Greg 
Published: April 2020

Atherosclerosis results from the build‐up of low‐density lipoprotein (LDL) cholesterol and immune cells in the arteries leading to their occlusion. High‐density lipoprotein (HDL) is believed to reverse this process by removing the arterial cholesterol from the body. Both LDL and HDL reach beneath the artery by crossing the endothelium through Scavenger receptor B1 (SR‐B1) mediated transcytosis. ApoAI, the protein exclusively found on HDL, mediates the interaction between HDL and SR‐B1. One natural ApoAI variant called Milano (ApoAI‐Mil) is of interest because injections of lipidated ApoAI‐Mil led to enhanced plaque regression. We hypothesize that the natural ability of ApoAI‐Mil to dimerize leads to better interaction with SR‐B1. This could result in better inhibition of LDL transcytosis since both HDL and LDL bind to SR‐B1 for transcytosis and this could also improve HDL transcytosis so that more HDL will reach the target tissue to extract cholesterol. We took an in vitro microscopy approach to quantify internalization of fluorescently labeled LDL in coronary endothelial cells. The ApoAI variants were also lipidated to form fluorescent HDL‐like particles (DiI‐DMPC‐WT or DiI‐DMPC‐Mil) to measure its association with SR‐B1 overexpressing cells. We observed that recombinant ApoAI‐WT can inhibit LDL internalization in coronary endothelial cells. There was also about an 8x‐fold increase of DiI‐DMPC‐WT fluorescence signal in HeLas overexpressing SR‐B1 compared to GFP alone. Furthermore, there was an even higher fold increase (~11x) of DiI‐DMPC‐Mil fluorescence signal compared to GFP alone. Similarly, excess recombinant ApoAI‐Mil led to greater inhibition of LDL internalization compared to ApoAI‐WT. There was no difference in the amount of DiI‐DMPC‐WT or ‐Mil associated with HeLa cells overexpressing Alk1, a LDL‐specific transcytosis receptor. Together this suggests that compared to ApoAI‐WT, ApoAI‐Mil more readily associates with only SR‐B1 which may lead to better inhibition of LDL internalization and to more HDL available to remove arterial cholesterol.Support or Funding InformationEarly Research Award & CIHR
Protective effects of Gladiolus psittascinus bulb Aqueous extract on Nicotine induced Hepatotoxicity in Experimental Rats
Authors: Oyetayo Folake Lucy , Odeniyi Ifeoluwa Adebayo , Olaseigbe Olajumoke Esther , Jegede Funmilayo Olusola 
Published: April 2020

Aqueous extract of the bulb of Gladiolus psittascinus was evaluated for protective activities in nicotine induced hepatotoxic rats. Biomarker and antioxidative enzyme activities were analysed using standard analytical procedure. Rats were divided into three groups: Control, Nicotine induced hepatotoxic, nicotine induced but treated Gladiolus psittascinus group. Induction of hepatotoxicity in rats led to significant (P<0.05) reduction of antioxidant enzyme activities. Superoxide dismutase activity (μmol/L) decreased from 18.58± 3.50 in control rats to 9.65± 1.00 in the nicotine induced. This was ameliorated on administration of the bulb extract at 100mg/kg body weight to 17.55± 3.23 in the aqueous Gladiolus psittascinus bulb treated group. Catalase activities (μmol/L) increased significantly from 3.65± 1.43 (Nicotine induced group) to 10.65±1.65 in the bulb extract treated group. Furthermore, plasma aminotransferases activities (U/L) in hepatotoxic rats were significantly reduced to control levels in the bulb extract treated group with Aspartate aminotransferase (AST) showing almost a fourfold reduction (16.51±0.70 (induced rats) to 4.21±0.33(bulb extract treated)) while Alanine aminotransferase (ALT) showed a twofold reduction (5.11±0.70 (induced rats) to 2.50±0.34(bulb extract treated)). Hence, the foregoing shows the potentials of Gladilous psittascinus bulb aqueous extract in enhacing the production of antioxidant enzymes required to scavenge reactive oxygen species. This holds promise for its use as diet therapy in the management and control of hepatotoxicity and oxidative stress related disorders.Support or Funding InformationTertiary Education Trust Fund, Nigeria.
Investigating the Role of the Med Protein in Biofilm Formation
Authors: Lara-Garcia Juan , Wacker Sarah 
Published: April 2020

Bacillus subtilis is a soil‐dwelling bacterium that serves as a good model organism for the study of different adaptive responses to various environmental conditions. These adaptive responses, including spore formation, cannibalism, and biofilm formation, are controlled by the master regulator Spo0A whose phosphorylation is under the control of five kinases. Initial interest in the protein Med came from its role in the transcription of operons related to cannibalism, and studies show the effect of Med is dependent on one of the five kinases, KinD. These same studies suggest Med may play a role in biofilm formation in a KinD‐dependent manner. The goal of our study of the Med protein is to determine whether it plays a direct role in biofilm formation by directly detecting environmental signals or indirectly through interactions with KinD. To determine whether Med is involved in detecting environmental signals, Δmed and ΔkinD bacteria were compared to wildtype B. subtilis using luciferase reporter assays that quantify the effect of different small molecules on the transcription of biofilm genes. Luciferase assays were compared to biofilm assays under the same conditions to visualize the effect on the biofilm phenotypes. We find that Δmed mutants had a defect in biofilm formation and reduced transcription of biofilm genes compared with wildtype bacteria. However, Δmed mutants were the same as wildtype bacteria in terms of their pattern of stimulation by small molecules. These results indicate that Med is not directly sensing environmental signals and instead is likely to be involved in the biofilm formation pathway indirectly through KinD, which has been shown to bind small molecules through its extracellular CACHE domain. We therefore shifted our focus to investigating interactions between Med and KinD and how Med may affect KinD’s ability to sense small molecules. Med and KinD were purified by tagging them with GST and His tags respectively. Additionally, in the case of KinD we purified constructs of the extracellular and intracellular domains to more precisely determine where the Med protein may be interacting with KinD. Presence of the proteins was determined by SDS‐PAGE and interactions between them was determined by Western blot analysis.Support or Funding InformationResearch was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Number R15GM135861.