bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Assembly of the Mitochondrial Protein Transport Machinery
Authors: Alder Nathan , Peixoto Pablo , Modak Arnab
Published: April 2020

The vast majority of proteins that reside within mitochondria are encoded in nuclear DNA, synthesized on cytosolic ribosomes, and imported into the organelle. Within the mitochondrion reside several types of complex machineries that mediate the sorting, translocation and integration of incoming polypeptides based on their targeting information. Among these translocases, the TIM23 Complex mediates the import and biogenesis of most resident proteins, which are synthesized with cleavable amino‐terminal targeting sequences. The TIM23 Complex is a dynamic multi‐subunit assembly that serves versatile functions in mediating the transport of soluble proteins across the mitochondrial inner membrane as well as the lateral integration of membrane proteins. The multifunctional nature of this complex may arise from different modular states that it can assume based on its component subunits. Using a host of biophysical approaches, we have investigated the substrate‐ and energy‐dependent structural dynamics of the central subunit, the voltage‐gated channel Tim23. We have also analyzed the lipid interactions that mediate membrane associations with the receptor subunit, Tim50, as well as channel‐receptor interactions. To investigate the structure and function of the membrane‐sorting modular form of TIM23, we have utilized a reductionist approach in which five component subunits are purified from a heterologous expression system and reconstituted into a biomimetic model membrane. These subunits include the polytopic channel proteins Tim23 and Tim17, the Tim50 receptor, and additional membrane proteins Tim21 and Mgr2. The innovation of this system derives from the fact that multiple independently purified membrane proteins can form a functional complex spontaneously when reconstituted with the proper topology in a bilayer of synthetic lipids. This reconstituted complex provides a minimal system for the direct analysis of its channel features, its energetic requirements for mediating membrane protein integration, and the factors that mediate assembly, including lipid requirements. It also provides a highly purified system for structural analysis.

Support or Funding Information

This work was supported by NIH Grant 1R01GM113092 (to N.N.A.) and PSC‐CUNY Grant PSC‐CUNY 626860050 (to P.P.)
Constraining evolution → avoiding drug resistance: lessons from viruses
Authors: Schiffer Celia
Published: April 2020

Drug resistance negatively impacts the lives of millions of patients and costs our society billions of dollars by limiting the longevity of many of our most potent drugs. Drug resistance occurs when heterogeneity exists within a genome and selection weakens inhibitor binding but maintains the biological function of the target. Genome diversity occurs both from transcriptional infidelity and the specific activity of human APOBEC3’s.

To avoid drug resistance, the structure at atomic resolution and evolutionarily constraints on its variation is required. This rationale was derived from our lab’s experience with substrate recognition both of human APOBEC3s and of antiviral drug targets. In particular we have acquired a rich and versatile experimental dataset of viral proteases altered by the selective pressures of inhibitors. With this data we are integrating alterations in both the protein sequence and the inhibitor with changes in potency and we correlate this data to our co‐crystal structures and our strategy of parallel molecular dynamics (pMD) to elucidate specificity and the molecular mechanisms of drug resistance.

pMD is a strategy we have developed to collectively analyze a series of MD simulations of similar yet distinct molecular complexes to decipher conformational and dynamic differences responsible for changes in molecular recognition. We perform pMD simulations on complexes with varying protein sequence and ligand identity to unravel structural and dynamic properties that underlie coupled changes in molecular recognition and resistance. We have applied this pMD strategy to protein‐ligand complexes for series of natural substrates, inhibitors, and protease mutations. Combined with experimental potency data, we determine which physical properties or molecular interactions are key to molecular recognition for a given system.

These changes in structure and dynamics dictate the interdependency of molecular mechanism of resistance and areprincipals that are generally applicable to other quickly evolving diseases where drug resistance is quickly evolving.

Support or Funding Information

NIH P01 GM109767, R01 AI085051, R01AI150478
Structural Insights into TRPV Channel Gating
Authors: Moiseenkova-Bell Vera
Published: April 2020

The transient receptor potential vanilloid 5 (TRPV5) channel is a highly calcium selective ion channel that regulates systemic calcium homeostasis by acting as a critical gate for calcium reabsorption in the kidney. Human polymorphisms of this channel have exemplified the importance of TRPV5 in disorders of calcium homeostasis, but atomic level information regarding this channel had remained unknown. To address this gap, we utilized cryo‐electron microscopy (cryo‐EM) to understand the gating and modulation of TRPV5. Moreover, this newly obtained structural information coupled with structure‐based virtual screening allowed us to uncover novel inhibitors of TRPV5 and revealed unique mechanisms of TRPV5 inhibition. These studies have thus expanded the base of understanding of TRPV5 at the atomic level and laid the foundation for future rational drug design.

Support or Funding Information

NIGMS R01GM103899; NIGMS R01GM129357
Cardiolipin‐dependent Carriers
Authors: Claypool Steven , Senoo Nanami , Kandasamy Selvaraju , Ogunbona Oluwaseun , Baile Matthew , Lu YaWen
Published: April 2020

The phospholipid, cardiolipin, has pleiotropic structural and functional roles that are collectively essential for mitochondrial biology. Yet, the molecular details of how this lipid supports the structure and function of proteins and protein complexes is poorly understood. To address this property of cardiolipin, we use the mitochondrial ATP/ATP carrier as a model. Here, we have determined that cardiolipin is critical for both the tertiary and quaternary assembly of the major yeast Aac isoform, Aac2, as well as its conformation. Notably, these structural roles provided by cardiolipin are separable. Additionally, we show that multiple copies of Aac2 engage in shared complexes that are largely dependent on the presence of assembled respiratory complexes III, IV, or respiratory supercomplexes. Intriguingly, the assembly state of Aac2 is sensitive to its transport‐related conformation. Collectively, these results significantly expand our understanding of the numerous structural roles provided by cardiolipin for mitochondrial membrane proteins.

Support or Funding Information

This work was supported by grants from the National Institutes of Health (R01HL108882 to S.M.C and Biochemistry, Cellular, and Molecular Biology Program Training Grant T32GM007445 to M.G.B. and Y.L.), pre‐doctoral fellowships from the American Heart Association (15PRE24480066 to O.B.O., 10PRE3280013 to M.G.B. and 12PRE11910004 to Y.L.), and a post‐doctoral fellowship from the Uehara Memorial Foundation (to N.S.).
Getting Hedgehogs Where They Need to Go: Cleavage Activates Dispatched for Sonic Hedgehog Release
Authors: Ogden Stacey K.
Published: April 2020

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is required for Hedgehog morphogen gradient formation and signaling to long‐range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. We reveal that Dispatched is activated by Furin‐mediated cleavage at a conserved processing site in its first extracellular loop. CRISPR/Cas9 gene editing was used to generate transgenic mice harboring cleavage site mutation in the endogenous Dispatched locus. Homozygous animals show neuronal patterning phenotypes and decreased viability, confirming a requirement for Dispatched processing in vivo. As such, Furin serves as a crucial post‐translational regulator of Dispatched activity in Hedgehog‐producing cells during tissue morphogenesis.

Support or Funding Information

This research was supported by funding from the National Institute of General Medical Sciences grants R01GM114049, R35GM122546.
Roles of Notch Glycoslation in Signaling
Authors: Stanley Pamela
Published: April 2020

Notch receptors are single pass transmembrane glycoproteins with an extracellular domain (ECD) comprised of many epidermal growth factor‐like (EGF) repeats. Specific Ser and Thr residues in numerous EGF repeats have an attached O‐glycan initiated by fucose, glucose, or GlcNAc, and elongated with 1–3 more sugars. Notch receptors signal in response to the binding of Notch ligands to Notch ECD. Notch signaling regulates cell fate decisions in many organs during development and differentiation, and its dysregulation leads to developmental disorders and cancer. A point mutation that precludes the addition of a single O‐fucose glycan to EGF12 in the NOTCH1 ligand binding domain alters Notch signaling differently depending on genetic background. In a mixed genetic background a mild effect on T and B cell development is observed, whereas in the C57BL/6J background, lack of the EGF12 O‐fucose glycan is embryonic lethal. Lunatic (LFNG), Manic (MFNG) and Radical (RFNG) Fringe are GlcNAc transferases that modify Notch EGF repeats which already carry a fucose. We are investigating roles for each Fringe in T and B cell development using mice lacking two or three Fng genes. We are also identifying individual roles for Fng genes using a co‐culture assay containing OP9 stromal cells expressing one of the four Notch ligands, DLL1, DLL4, JAG1 or JAG2 and bone marrow hemopoietic cells from mice expressing all Fng genes, no Fng genes (tKO) or a single Fng gene. The combined results show that the O‐fucose glycans on NOTCH1 are key regulators of the strength of NOTCH1 signaling induced by canonical Notch ligands.

Support or Funding Information

National Institutes of Health grant RO1 GM106417National Institutes of Health grant PO1 CA13330
Watching a Fine‐tuned Molecular Machine at Work: Structural and Functional Studies of the 26S Proteasome
Authors: Martin Andreas , Jonsson Erik , Greene Eric , Lopez-Alfonzo Erika , Htet Zaw , Gates Stephanie
Published: April 2020

The degradation of ubiquitin‐tagged proteins by the 26S proteasome is a highly complex process with several well‐coordinated sub‐steps, including ubiquitin recognition, deubiquitination, substrate engagement by the AAA+ motor, mechanical unfolding, translocation, and proteolytic cleavage. Our recently developed FRET‐ and fluorescence‐based assays, relying on the labeling of proteasome‐incorporated unnatural amino acids, allow us to dissect the kinetics and coordination of individual processing steps, and study the coupled conformational changes of the proteasome. Single‐molecule measurements provide exciting details about the conformational dynamics of the proteasomal regulatory particle and how ubiquitin binding, substrate engagement by the AAA+ motor, or the presence of certain receptor subunits affect the switching between substrate‐free and substrate‐processing states, which are furthermore controlled through specific interactions between the ATPase hexamer and the proteasomal lid subcomplex.

FRET‐based assays reveal how substrates are propelled through the central channel of the proteasome and progress through the various stages of degradation, including a translocation stall during deubiquitination and the backtracking of substrate upon failure to remove ubiquitin chains prior to substrate entry into the AAA+ motor. Our mutational studies thereby provide important new insight into the mechanisms of substrate engagement and the contributions of individual ATPase subunits depending on their position in the vertical spiral‐staircase arrangement of the AAA+ hexamer. By using substrates with various architectures, we also gained a better understanding of how a substrate’s geometry and ubiquitin modifications affect the degradation rate, and how the proteasome may prioritize its substrates in the cell.

Support or Funding Information

Howard Hughes Medical Institute; NIH‐NIGMS (R01‐GM094497)
tRNA Quality Control: Mechanisms, Evolution, and Implications for Human Disease
Authors: Phizicky Eric
Published: April 2020

In all organisms tRNAs are subject to numerous post transcriptional modifications, imparting them with properties to accurately and efficiently execute their critical roles in translation. Modifications in and around the anticodon loop generally affect translation, whereas modifications in the tRNA body often affect tRNA folding or stability. Lack of modifications frequently results in growth and translation defects in the budding yeast Saccharomyces cerevisiae and in neurological disorders or mitochondrial diseases in humans, but the mechanisms by which these defects occur are in many cases not known. Our lab focus is to understand the exact roles of selected modifications implicated in human disease, by genetic and biochemical analysis of their roles in S. cerevisiae and in the evolutionarily distant fission yeast, Schizosaccharomyces pombe, to understand if and to what extent these roles have evolved in different eukaryotes, including humans. We will present results describing the conservation and evolution of the roles of two modifications, highlighting interactions with a widely conserved stress response pathway.

Support or Funding Information

NIH NIGMS grant GM052347
Teaching Biochemistry in Context
Authors: Dries Daniel Robert
Published: April 2020

21st‐century biochemistry and molecular biology (BMB) education has recognized that the breadth of knowledge within the discipline is too expansive to expect students to retain the entirety of BMB content. Instead, BMB education has prioritized a competency‐based approach over a content‐focused curriculum. Examples of such demand include AAAS’s Vision and Change (https://visionandchange.org/) and ASBMB’s Foundational Concepts and Skills (https://www.asbmb.org/education/teachingstrategies/foundationalconcepts/). To meet the demand of a competency‐based curriculum, the sophomore‐level introductory biochemistry course at Juniata College uses as the primary course materials the scientific literature around a central theme (acetylcholine neurotransmission). The curriculum begins with a 16th‐century text and culminates in modern scientific work. Students work in teams to interrogate the scientific literature by (i) developing specific questions, (ii) using resources to find answers to those questions, (iii) communicating those answers with the peers in their team, and (iv) using the answers to interpret data and figures in the scientific papers. Attitudinal assessment of this social constructivist approach to BMB education revealed outcomes consistent with inclusivity and self‐efficacy, including gains in affect with respect to teamwork, curiosity, and scientific literacy. The narrative aspect of the course has led to several collaborative spin‐off projects including the translation of a 19th‐century medical text and an independent science education project. The design of this course stretches BMB education beyond the limits of traditional content acquisition and into new and exciting ways of engaging students and encouraging them to take control of their own education.
Reimagining STEM Education to Help Underrepresented Students Thrive in the Classroom
Authors: Jones Shannon Z.
Published: April 2020

This presentation will highlight a novel, interdisciplinary course for students interested in pursuing an undergraduate degree in STEM. Science, Math, and Research Training (SMART) is a year‐long course that provides first‐year students with a strong foundation in biology, chemistry, calculus, and computer science. These disciplines are integrated in a manner that allows students to develop problem solving skills and to appreciate the value of collaboration and communication across multiple STEM disciplines. SMART is a thematic course that focuses on important and timely issues such as antibiotic resistance and infectious disease. In order to provide this type of interdisciplinary training for students, the topics covered during the course of each semester must be limited. This presentation will discuss the means by which the content that is covered in a traditional introductory science course has been stripped down and limited to the most important, foundational concepts. The purpose of this redesign was to provide students with foundational knowledge of science, but also focus on the development of critical thinking and scientific writing skills. The removal of traditional content also allows the instructors to focus on metacognition to allow students to assess their own learning needs. Our goal is for our students to feel empowered to take charge of their own learning and to determine, early on in their scientific career, the strategies that work best for their learning. Furthermore, this course redesign allows for instructors to implement high impact classroom practices as well as inclusive pedagogy techniques that guarantee the success of all students, regardless of their background and preparation in high school. SMART is geared primarily to first generation college students, student from underrepresented backgrounds, and students with a low socioeconomic status. Helping these students develop a sense of belonging and self‐efficacy in STEM is a primary goal of this program.
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 Information

NIHGM20501‐45
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 Information

Work 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 Information

NIH 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 Information

This 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

Objective

To investigate the effects of chlorogenic acid on serum nuclear factor κB (NF‐κB) and motor function of rats with traumatic spinal cord injury.

Methods

Forty‐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.

Results

The 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).

Conclusion

Chlorogenic 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 Information

This 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 Information

This 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.).
Bilirubin: A Fat Busting Metabolic Hormone?
Authors: Gordon Darren Mikael , Mizzuri Scott , McCullumsmith Robert , Stec David , Hinds Terry D.
Published: April 2020

Obesity is an epidemic in the United States, which has led to an increased prevalence of insulin‐resistant type II diabetes and heart disease, heightening patient morbidity and mortality. Current treatments of obesity are not sufficient, often requiring combinations for beneficial results, and most have the negative side effect of increasing blood pressure. It has been recently shown that bilirubin, a red blood cell metabolite that at very high levels is commonly associated with liver pathology, prevents cardiovascular disease, diabetes, and obesity at modest levels. We have found that bilirubin induces transcriptional activity via the fat‐burning nuclear receptor, peroxisome proliferator‐activated receptor α (PPARα). RNA‐sequencing in HepG2 hepatocytes showed that bilirubin‐induced transcriptome responses that are predominantly PPARα‐dependent (~95%). From our investigations, we were able to demonstrate a preference for binding of bilirubin to PPARα compared to the other PPAR isoforms (gamma and delta). Utilizing a completely new fluorescent technique established by our lab, we found that bilirubin directly binds to the ligand‐binding domain (LBD) of PPARα. As measured by Pamgene Pamstation Nuclear Hormone Receptor (NHR) chip analysis, we also found that bilirubin functions as a ligand for PPARα and induces the recruitment and dissociation of coregulators that regulate gene function. Subsequent chromatin immunoprecipitation assays showed that bilirubin drives PPARα to the promoters of genes involved in mitochondrial respiration, including the uncoupling protein 1 (Ucp1) and carnitine palmitoyltransferase I A (Cpt1a). We affirmed that the effect of bilirubin on mitochondrial function, analyzed by oxygen consumption rate (OCR) using Seahorse analysis, was higher in the cells that expressed PPARα, but not PPARg2. These findings aid in defining the role of bilirubin in altering the metabolic function and, more specifically, through the regulation of mitochondrial‐mediated mechanisms. The investigation of the bilirubin‐PPARα axis reveals a newfound position of bilirubin as a metabolic hormone mediating protection from lipid accumulation and subsequent metabolic dysfunction.
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 Information

NSF‐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

Background

Septic 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.

Methods

We 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.

Results

Ninety 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.

Conclusions

Patients 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 Information

This 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 Information

Self
Adrenergic activation of cardiac preganglionic neurons in the Nucleus Ambiguus
Authors: Aiba Isamu
Published: April 2020

Cholinergic neurons within the nucleus ambiguus (NAm) are the major parasympathetic preganglionic neurons innervating heart. This study characterized the excitability of NAm neurons using in vitro acute brainstem slice preparations obtained from young adult mice. Cholinergic premotor NA neurons were identified based on their expression of tdTomato fluorescence in the Chat‐Cre:floxed‐tdTomato mouse line. Retrograde labeling of pericardiac axon terminals study confirmed cholinergic NAm neurons with large soma as preganglionic cells. In current‐clamp recordings, a brief hyperpolarizing current evoked rebound action potentials (APs). Low voltage‐activated, T‐type calcium current (ICaT) is required for this rebound discharge pattern of NAm neurons. Voltage‐clamp recordings detected a significant T‐current in NA neurons, and quantitative in situ hybridization analysis identified Cav3.1 and 3.2 channels as the major contributors. These preganglionic neurons are strongly activated by adrenergic agonists in vitro. Bath application of adrenergic agonists, epinephrine (10 μM) and norepinephrine (20 μM), induced spontaneous action potentials in these neurons. Inhibition of synaptic ionotropic receptors did not prevent the adrenergic activation, suggesting a minor contribution of synaptic mechanism. Instead, adrenergic stimulation significantly increased the intrinsic excitability of NA neurons; input resistance of NA neurons was significantly increased and single evoked rebound discharges (normally composed of 1–2 APs) converted to regenerative oscillations with burst of >10APs lasting up to a few seconds. Pharmacological tests suggest that persistent sodium current is an important contributor to the activated action potential generation. Together this study identified basic intrinsic membrane excitability mechanism of NAm and its activation by adrenergic agonists.

Support or Funding Information

U01 NS090340 (JLN) AHA Career development award (IA)
Characterization of Modified‐DNA Polymerase Fidelity
Authors: Seto Madison K. , Barrett Susanna E. , Weiden Aurora G. , Leconte Aaron M.
Published: April 2020

Recently, several modified‐DNA (M‐DNA) polymerases have been identified which can synthesize long M‐DNAs. While these discoveries have enabled new applications of M‐DNA, these enzymes typically have poor fidelity, possessing error rates several orders of magnitude worse than native enzymes. However, to date, efforts to both quantify error rate, as well as experimental approaches towards understanding the origin of the poor fidelity of M‐DNA polymerases, have been limited. Here, we use a high‐throughput sequencing assay to characterize the error rate of leading M‐DNA polymerases, which showed that our enzyme, P1, has an error rate 1.6 times lower than SFM4‐6, the most frequently used Taq mutant for 2′M‐DNA synthesis. We also characterize the importance of individual mutations on fidelity, beginning to experimentally address the origins of fidelity, and have found that reverting even one mutation in a mutant yielded a fidelity that was 2.4 times greater than its parent, which will help us improve future M‐DNA polymerase rational design.

Support or Funding Information

National Science Foundation