bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enzyme Hydrophobic Sites and Allosteric Membrane Interactions Regulate Signaling and Mediators of Inflammation
Authors: Dennis Edward A 
Published: April 2020

We will discuss how membranes interact allosterically with enzymes to regulate cell signaling and metabolic pathways leading to inflammation
. We have recently used substrate lipidomics coupled with molecular dynamics to reveal enzyme specificity linked to hydrophobic binding sites for membrane phospholipid substrates
. We discovered unexpected headgroup and acyl chain specificity for each of the major human phospholipase A2 (PLA2) enzymes that explains the observed specificity at a new structural level. We discovered that a unique hydrophobic binding site — and not each enzyme’s catalytic residues or polar headgroup binding site — dominates each enzyme’s specificity. Each PLA2 shows unique specificity for its required fatty acid ranging from pro‐inflammatory arachidonic acid to membrane remodeling linolenic acid, antibacterial saturated fatty acids, and oxidized fatty acids in low‐density lipoproteins. Each PLA2 releases a specific fatty acid after the enzyme associates allosterically with membranes and extracts a single phospholipid substrate into its catalytic site. Each enzyme can be blocked by stereospecific inhibitors
specific for that PLA2. After decades of research advancements, we can now correlate PLA2 specificity and inhibition potency with molecular structure and physiological function using a novel lipidomics platform.From the cell signaling and inflammation perspective, the omics revolution began with genomics, proteomics and metabolomics, but lipidomics now dominates as the largest number of cellular metabolites are lipids, many playing critical roles in cell signaling, and over 40,000 distinct molecular species have been identified by the LIPID MAPS Consortium (www.lipidmaps.org). We have also employed lipidomics analysis to characterize the in vivo role of specific PLA2 enzymes in initiating the inflammatory response and cellular lipid signaling of Toll‐like (TLR4) and purinergic (P2X7) receptors in stimulated macrophages as models of bacterial infection and inflammation
. We can now explain in vitro enzyme specificity with in vivo specificity, inhibition and pro‐inflammatory and pro‐resolution lipid mediator formation pathways and inhibition
.Support or Funding InformationNIHGM20501‐45
Activation of the Exocyst Tethering Complex for SNARE Complex Regulation and Membrane Fusion
Authors: Munson Mary , Lepore Dante , Feyder Michael , Rossi Guendalina , Czuchra Alexander B , Kenner Lillian , Martínez-Núñez Leonora , Frost Adam , Brennwald Patrick 
Published: April 2020

A major challenge for a molecular understanding of membrane trafficking has been the elucidation of high resolution structures of large, multisubunit tethering complexes (MTCs) that spatially and temporally control intracellular membrane fusion. Exocyst is a hetero‐octameric protein complex, proposed to tether secretory vesicles at the plasma membrane, and to provide quality control of SNARE‐mediated membrane fusion. Breakthroughs in methodologies, including sample preparation, biochemical characterization, fluorescence and single‐particle cryo‐EM, are providing critical insights into the structure and function of the exocyst. We are investigating how the yeast exocyst interacts with SNARE proteins to control SNARE complex formation and membrane fusion. Intriguingly, fully assembled exocyst interacts weakly with the individual SNAREs, SNARE complexes, and the SNARE regulator Sec1, despite previously observed robust SNARE binding with recombinant proteins. Using an auxin‐inducible degradation system and mutant yeast strains, we purified exocyst subcomplexes and mutant complexes and showed that several have increased affinities for the different SNAREs. Negative stain EM was used to visualize the structure of an exocyst subcomplex, as well as the 3D structure of an activated mutant exocyst complex. Comparison of the negative stain images to the cryo‐EM structure of fully assembled exocyst revealed that several subunits become more dynamic and accessible for SNARE interactions. We propose that exocyst needs to become activated and undergo a conformational change, in order to efficiently interact with and regulate the SNAREs for membrane tethering and fusion.Support or Funding InformationWork in our laboratories is supported by US National Institutes of Health grants GM068803 (M.M.), GM054712 (P.B), F32GM123704 (D.L.) GM127673 (A.F.), DP2GM110772 (A.F.), A.F. is an American Asthma Foundation Scholar, an HHMI Faculty Scholar, and a Chan Zuckerberg Biohub Investigator. L.R.K. was supported by a graduate research fellowship from the NSF.
The RNA Exosome and Genetic Disease
Authors: Corbett Anita H. , Leung Sara W. , Sterrett Maria C. , de Amorim Julia , Enyenihi Liz C. , Morton Derrick J , van Hoof Ambro , Fasken Milo B. 
Published: April 2020

The RNA exosome is an evolutionarily conserved, riboexonuclease complex that processes/degrades numerous classes of non‐coding RNA. The 10‐subunit core exosome forms an ring‐like structure composed of an upper ring of three S1/KH cap subunits (yeast Rrp4/40/Csl4‐human EXOSC2/3/1), a lower ring of six PH‐like subunits (yeast Rrp41/42/43/45/46/Mtr3‐human EXOSC4/7/8/9/5/6), and a 3′‐5′ riboexonuclease subunit, Rrp44/DIS3, at the base. The nuclear exosome contains an eleventh, cap‐associated riboexonuclease subunit, Rrp6/EXOSC10. RNAs are targeted directly to Rrp6 or threaded through the central channel of the exosome to Rrp44 for processing/degradation. Recently, mutations in four exosome subunit genes, EXOSC2/RRP4, EXOSC3/RRP40, EXOSC8/RRP43, and EXOSC9/RRP45 have been linked human diseases. Mutations in EXOSC2 cause a novel syndrome characterized by retinitis pigmentosa, hearing loss, and mild intellectual disability. In contrast, mutations in EXOSC3 and EXOSC8 cause pontocerebellar hypoplasia type 1b and 1c, respectively ‐ autosomal recessive diseases characterized by cerebellar hypoplasia and neuronal degeneration with early mortality ‐ and mutations in EXOSC9 cause cerebellar atrophy. We also recently collaborated to identify novel mutations in the EXOSC5 gene. To gain insight into the functional consequences of the mutations in these genes encoding structural subunits of the RNA exosome that have been identified in patients, we generated the corresponding mutations in the S. cerevisiae genes and examined their function in budding yeast. We find that rrp variants cause differential effects on cell growth and RNA exosome function, which could shed light on why impairments in different exosome subunits cause distinct and tissue specific phenotypes. We have complemented these functional studies with RNA‐Seq analysis to identify specific RNA targets that are differentially affected by the changes in RNA exosome subunits that model human disease as well as a high copy suppressor screen. To extend our functional studies of EXOSC2 and EXOSC3 mutations to mammalian cells, we have performed a proteomic analysis of variant and wildtype EXOSC2 and EXOSC3 proteins expressed in neuronal cells. These data support a model where differential interactions with RNA exosome cofactors could underlie disease‐specific phenotypes.Support or Funding InformationNIH grant 5 R01 GM130147‐02 to van Hoof and Corbett
Connections between mRNP Composition and mRNA Fate
Authors: Singh Guramrit , Yi Zhongxia 
Published: April 2020

Throughout their lifetime, messenger RNAs (mRNA) exist decorated with proteins as mRNA‐protein particles, or mRNPs. A key component of all spliced mRNPs is the exon junction complex (EJC), which assembles during pre‐mRNA splicing ~24 nucleotides (nt) upstream of exon‐exon junctions. The stable EJC core thus assembled serves as an interaction platform for peripheral proteins that direct mRNA export, localization, translation and nonsense‐mediated mRNA decay (NMD). Both mRNPs and EJCs and are widely presumed to be "dynamic" entities that change during an mRNAs lifetime. We recently discovered that EJCs, and hence spliced mRNPs, undergo an extensive compositional overhaul after their export to cytoplasm. This compositional switch has important implications for the EJC‐dependent NMD pathway, which can now be divided into at least two distinct phases. We find that two compositionally distinct EJCs differentially associate with the NMD factor UPF3B, which is also a peripheral EJC factor that is required for efficient NMD of only a small subset of mRNAs. We will discuss new insights gained from our work into the mechanism of EJC‐dependent NMD in the absence of UPF3B, and about the function of UPF3B in NMD. Overall, our work is shedding new light on how mRNP composition specifies distinct phases or branches of the NMD pathway, an essential post‐transcriptional mechanism that controls cellular adaptation, differentiation and development.Support or Funding InformationThis work is supported by NIH (R01 GM120209). ZY is supported by graduate fellowships from Pelotonia and OSU Center for RNA Biology.
Effects of chlorogenic acid on serum nuclear factor κB and motor function of rats with traumatic spinal cord injury
Authors: Qiao Xiaohong , Duan Hubin , Hao Chunyan , Xue Wen , Du Yanrong , Zhang Xiaoyi , Liu Wenyun , Wang Zhiqiang , Li Youlian , Guo Jianxun 
Published: April 2020

ObjectiveTo investigate the effects of chlorogenic acid on serum nuclear factor κB (NF‐κB) and motor function of rats with traumatic spinal cord injury.MethodsForty‐five male Sprague‐Dawley rats were randomly divided into sham operation group( group S), injury group ( group I) and chlorogenic acid treatment after injury group( group C),15 rats in each group, weight 180 20g. Rats model of spinal cord injury was established in accordance with the model of Allen ‘s hit. The rats in the group C were given chlorogenic acid (50mg/kg) by lavage in 1 hours after injury. The rats in the group S and group I were given the same dosage normal saline to fill the stomach. The rats in all groups were given the same dose daily for 7 days. On the seventh day, Basso‐Beattie‐Bresnahan hindlimb motor score was used to measure the effect of chlorogenic acid on the recovery of motor function after spinal cord injury. The rat was sacrificed and the tissue of spinal cord was taken out on the same day. The gene and protein expression of NF‐κB in the spinal cord tissue was detected by reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemical assay.ResultsThe rats of group I and group C had severe lower limb neurological dysfunction than that of group S(p<0.05), and the BBB motor score of group C was significantly higher than that of group I (p<0.05). The gene and protein expression of NF‐κB in the spinal cord tissue of group C were significantly lower than that of group I (p<0.05), but were higher than that of group S (p<0.05).ConclusionChlorogenic acid can reduce gene and protein expression of NF‐κB in spinal cord tissue, reduce inflammation after spinal cord injury, and improve motor function of rats with spinal cord injury.Support or Funding InformationThis study was supported by the National Natural Science Foundation Youth Fund (30600637), the science and technology research projects of Lvliang, China Postdoctral Science Foundation Grant (2014M561207 and 2019T120195), 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, 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 Education ReformProject of Shanxi Medical University.
Differential roles of GluN2B in two types of chemical‐induced LTP‐mediated phosphorylation regulation of GluA1 at serine 845 in hippocampal slices
Authors: Zhang Bin , Lu Wen , Fang Weiqing , Ma Wu , Xue Fusheng 
Published: April 2020

Synaptic plasticity, such as long‐term potentiation (LTP) and long‐term depression (LTD), underlies the cellular mechanism of learning and memory. Chemical‐induced LTP, which facilitates biochemical analysis of molecular changes in brain slices or neuronal cultures, was accepted as an in vitro model for exploring synaptic plasticity. Chemical‐induced LTP, either by forskolin and rolipram or by glycine, is thought to be dependent on NMDA receptor. However, subunit specific dependence and regulation of NMDA receptor in chemical‐induced LTP remain poorly understood so far. In the present study, we found that phosphorylation of tyrosine 1472 at GluN2B was modulated by forskolin and rolipram‐induced LTP but not glycine‐induced LTP in hippocampal slices. Furthermore, elevated phosphorylation level of serine 845 at GluA1 by forskolin and rolipram‐induced LTP rather than glycine‐induced LTP was dependent on the activation of GluN2B, which is supported by the results from GluN2B antagonists, small interfering peptide and CRISPR‐Cas9‐mediated knock out of GluN2B. Taken together, we reveal a significant role of GluN2B in forskolin and rolipram‐induced LTP, uncovering NMDA receptor subunit specific dependence in a specified chemical‐induced LTP.Support or Funding InformationThis work was supported by National Natural Science Foundation of China Grant 31700882 (to Z.B.) and 31600863 (to W. L.), the start‐up fund of Hainan Medical University (to W. L.), the Tri‐Five Cultivating Project from Hangzhou Normal University 2017XJSGWXM209 (to Z.B.).
Key Dynamic Residues in the Catalytic Activity of Human Cyclophilins Identified with a Dynamical Evolution Analysis
Authors: Ahmed Furyal , Yao Xin-Qiu , Hamelberg Donald 
Published: April 2020

Cyclophilins belong to peptidyl‐prolyl isomerases (PPIases), a group of enzymes catalyzing the interconversion of the peptide bond preceding a proline between the cis and trans isomer. Of the 17 unique human cyclophilins, five closely related homologues (CypA‐E) were the focus of this project. These five isoforms function in a variety of environments, and participate in diverse subcellular processes, yet all have highly conserved sequences, very similar structures, and the same core catalytic function. As dynamics are known to couple protein function, looking into the conserved dynamics can provide insights into the catalysis of peptidyl‐prolyl isomerization by cyclophilins. Functional dynamics were compared using multiple statistical analyses, including Cartesian and dihedral principal component analysis (PCA), root mean square fluctuation (RMSF), and angular variance analysis. A set of key dynamical residues, defined by the most dynamically conserved positions, were identified in the gatekeeper 2 region. The highly conserved glycine (GLY79) in this region was predicted to underlie the local flexibility. To test it, additional simulations were performed for CypE and CypA, where GLY79 was mutated to alanine. Mutation of GLY79 to ALA79 leads to decreased flexibility of CypE and CypA during substrate binding (free → cis) but increased flexibility during catalysis (cis → ts). Dynamical changes occur in the mutated region, as well as in a distal loop downstream of the mutation site in sequence. Allosteric effects on catalysis by the mutation were also examined. Both mutated CypE and CypA show shifted binding free energies of substrate under distinct substrate peptide cis‐trans conformations. The results suggest a reversal in the direction of catalysis in the mutated CypE and enhanced catalytic activity in the mutated CypA.Support or Funding InformationNSF‐REU program award no. 1757933, NSF grant (MCB‐1517617)
Isolated extensor digitorum longus muscles from old mdx dystrophic mice show little force recovery 120 minutes after eccentric damage
Authors: Kiriaev Leonit , Kueh Sindy , Morley John W. , North Kathryn N. , Houweling Peter J. , Head Stewart I. 
Published: April 2020

Duchenne muscular dystrophy is characterized by progressive wasting and cycles of regeneration in skeletal muscle. Our laboratory work suggests, branched fibres, could be responsible for the terminal phase of muscle damage in old (58–112 weeks) dystrophic mice. A recent study in 12 week old dystrophic mice reported that the majority of force loss produced by a series of eccentric contractions (EC) in extensor digitorum longus (EDL) muscles recovers (65%) within 120 minutes, concluding this is incompatible with the assumption that EC force loss is due to mechanical damage.The aim of this project was to assess the recovery post EC damage in adult (~16 weeks) and old (~88 weeks) dystrophic muscles. Male mdx mice and littermate controls were euthanized, EDL muscles dissected from the hind limbs and maintained in Krebs solution at room temperature. The muscles were maximally stimulated, and a series of 6× EC performed to assess force loss. Muscles were left to rest for up to 120 minutes whilst measuring recovery. Single muscle fibres were isolated enzymatically to assess the degree of fibre branching.Our findings replicated force recovery in young mdx mice with simple fibre branches. However, minimal recovery (~23%) in muscles of 88 week old mdx mice at 120 minutes post EC was observed. This data supports our “tipping point” hypothesis and shows a distinct pathophysiology in aged mdx mice with EC force loss due to acute fibre rupture at branch nodes that occurs in “old” dystrophic EDL muscles with >70% complex branched fibres. These findings have important implications for pre‐clinical drug studies that use protection from EC damage in young mdx mice as a marker for drug efficacy.A better understanding of the pathology of muscle damage in the mdx mice will improve our ability to test and interpret pre‐clinical drug studies using this model.
Extracellular Hsp70 is a mortality predictor in septic shock and correlates with APACHE II, SOFA and inflammatory cytokines
Authors: Carbajal Patricia Bautista , Molina Pablo Duarte , Contla Martínez Indira I. , García León Miguel L. , Ángel Ambrocio Antonio H. , López Neyla Baltazar , Chew Rosa María Wong 
Published: April 2020

BackgroundSeptic shock mortality ranges from 30 to 60%. Intracellular Hsp70 is a protein involved in the folding of mature proteins, its extracellular detection has been described in rheumatoid arthritis and several infectious diseases. The aims of the study were to determine if eHsp70 levels in adult patients with septic shock could be a prognostic marker and if it correlated with APACHE II and SOFA scores and the inflammatory cytokines response.MethodsWe performed an observational, longitudinal, prospective, cohort study. Adult patients with septic shock admitted to the Intensive Care Unit were included. eHsp70 levels were measured in sera by ELISA, APACHE and SOFA were determined on ICU admission and every 2 days until death or discharge for improvement. Serum cytokines were measured at the admission and discharge with flow cytometry.ResultsNinety patients were included, 53 men and 37 women. Fifty percent (45) died. Higher APACHE II and SOFA scores, were found on those who died compared to survivors. Ninety‐one percent of patients who had persistent levels above 500 pg/ml of eHsp70 up to days 14 and 16 of hospitalization died; eHsp70 above 600 pg/ml was related with APACHE score higher than 30 and SOFA score higher than 15 and correlated with deaths. IL‐6, IL‐8 and IL‐18 were higher in deceased patients. eHsp70 persistent levels above 500 pg/ml could be related with mortality.ConclusionsPatients with APACHE II score higher than 30 and SOFA score higher than 15 correlated with eHsp70 above 600 pg/ml and a higher proinflammatory response in the death group. eHsp70 could be a severity marker in patients with septic shock.Support or Funding InformationThis work was supported by Federal Funds from the Departamento de Investigación del Hospital General de México “Dr. Eduardo Liceaga”
Proximate Analysis, Phytochemical Screening and Evaluation of Acute and Sub‐chronic Toxicity of Methanol Extract of Ficus exasperata
Authors: OSAGIE ODODO AUGUSTINE , AGUEBOR-OGIE BOBBY NOGIOMWAN 
Published: April 2020

Herbal medicines are the most common available healthcare remedy in most African nations. The use of medicinal plants dated back to centuries ago due to their easy availability. Ficus exasperata is an ethno‐medicinal herb used in Nigeria. The medicinal properties are known to be attributed to the presence of some nutritional and bioactive components. Their use as analgesics, diuretics and anti‐microbial prompted this investigation. This study evaluates the nutritional and phyto‐chemical content, as well as the safety of the methanol extract of Ficus exasperata leaves on albino wistar rats. Proximate analysis and phytochemical screening were conducted using standard methods. GC‐MS analysis was further used to profile small and volatile compounds. The acute and sub‐chronic toxicity study was performed using the modified Lorke’s method. The acute toxicity study was observed for 7 days for any sign of toxicity and mortality while the sub‐chronic toxicity study was observed for 28 days after which some hematological and biochemical parameters were determined. The result from proximate or nutritional analysis showed that nitrogen free extract (39.13±0.09) was highest followed by crude protein (27.13±0.09), ash content (12.50± 0.13), moisture content (8.25±0.17), ether extract (8.00±0.08) and crude fiber (5.00±0.10). The phytochemical analysis revealed the presence of some secondary metabolites: saponins, alkaloids, tannins, terpenoids, phenols, flavonoids, glycosides, steroids and anthocyanins. The GC‐MS analysis revealed the presence of the following phyto‐compounds: Hexadecanoic acid methyl ester, 9‐Octadecenoic acid, Methyl stearate, 7‐Octadecenoic acid methyl ester and Oleic Acid. The
acute toxicity study showed that the methanol extract of Ficus exasperata leaves is safe even at the highest dose tested (5000 mg/kg body weight). No behavioral changes and gross pathological
abnormalities were observed. The sub‐chronic toxicity study at the extract doses of 200, 250 and 500mg/kg body weight showed no observed changes in the extract treated animal’s body weight, behaviour and mental
alertness with no mortality recorded. The levels of white blood cells (WBC), packed cell volume (PCV), platelets, monocyte and granulocyte were not significantly altered, whereas a significant change (p< 0.05) of lymphocyte count was observed in treatment groups compared to the control group. There was no significant change in the levels of serum concentration of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea. These preliminary results suggest that the methanolic leaf extract of Ficus exasperata contained both nutritional and bioactive substances and was not likely to produce severe toxicological effects on biological system when administered at normal therapeutic doses.Support or Funding InformationSelf