bluuebunny/crossref_metadata_2025_split huggingface dataset:
Brain Endothelial Cell Bioenergetics via Extracellular Flux Analysis
Authors: McDonald Cade , Drewes Lester , Blankenheim Zachary 
Published: June 2021

The neurovascular unit (NVU) is an important functional component in the central nervous system (CNS). The NVU consists of multiple cell types (endothelial cells, pericytes, astrocytes, others) working together as a restrictive blood‐tissue interface and functions to maintain homeostasis within nervous tissue in the CNS. A dysfunctional NVU is associated with many disease states, including Alzheimer's Disease, strokes, and tumors. The endothelial cell is an important barrier‐forming cell of the NVU because of its direct contact with the blood, its intercellular tight junctions, biotransforming enzymes, and asymmetric distribution of active and carrier‐mediated transporters. These properties are important for maintaining the neuro‐microenvironment and require an abundant supply of energy (ATP). Although energy metabolism has been studied in various tissues and cells, little is known about the energy producing pathways of brain endothelial cells. Therefore, using extracellular flux analysis, we characterized the bioenergetics of human brain microvascular endothelial cells (BMECs) in vitro. BMECs are primarily glycolytic and D‐glucose, pyruvate, and glutamine are preferred metabolic substrates. Inhibitors of monocarboxylate transporter (MCT1), mitochondrial pyruvate carrier, and glycolysis significantly alter ATP production, cellular respiration, and/or glycolytic rates. These findings contribute to our understanding of endothelial cell metabolic plasticity in normal and diseased brain as well as active angiogenesis during development or tumorigenesis. Understanding endothelial cell bioenergetics will be useful for future studies regarding development of therapies, that target endothelial cell energetics, for neurological disorders.Support or Funding InformationWhiteside Foundation and the Department of DefenseThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Daily acute, but not chronic, intermittent hypoxia enhances phrenic motor plasticity in chronic cervical spinal cord injury
Authors: Gonzalez‐Rothi Elisa J , Tadjalli Arash , Allen Latoya L , Ciesla Marissa C , Simon Alec , Asa Zachary , Smith Kristin , El‐Chami Mohamad , Holland Ashley , Santiago Juliet , Stefan Kelsey , Ross Ashley , Mitchell Gordon S 
Published: June 2021

Daily acute intermittent hypoxia (dAIH; 3–15 hypoxic episodes per day) is a promising therapeutic strategy to improve respiratory function after incomplete spinal cord injury by inducing spinal respiratory motor plasticity. Conversely, chronic intermittent hypoxia (CIH; >100 episodes per day) elicits multisystem pathology and is a hallmark of moderate sleep apnea, a condition highly prevalent in individuals with cervical spinal injury. In intact rats, 7 days of dAIH preconditioning enhances phrenic motor facilitation whereas it is abolished by 7 days of CIH, an effect likely due to CIH‐induced neuroinflammation. Since the impact of prolonged CIH (> 1 wk) or dAIH on the capacity for phrenic motor facilitation with chronic spinal cord injury has not been explored, we compared the effect of 28 days of normoxia (21% O2; 8hrs/day), dAIH (10, 5 min episodes of 10.5% O2 with 5 min normoxic intervals/day) or CIH (2 min hypoxic episodes with 2 min intervals; 8 hrs/day) on phrenic output in rats with chronic C2 spinal hemisection (C2Hx). Neurophysiology experiments were conducted one day after the final hypoxic exposure; bilateral phrenic nerve activity and moderate AIH‐induced phrenic long‐term facilitation (pLTF) were assessed. Ipsilateral phrenic nerve activity during baseline conditions and maximal chemoreflex activation was: 1) reduced in all injury groups vs intact rats (p<0.0001 and p<0.009, respectively); and 2) unaffected by dAIH or CIH (p>0.94 and p>0.98, respectively). No group differences in contralateral phrenic nerve activity were observed in either condition (p>0.49 and p>0.18, respectively). dAIH enhanced ipsilateral pLTF vs. all other groups (p=0.01); in contrast, CIH had no effect on pLTF (p=0.43). In conclusion, neither dAIH nor CIH had any detectable impact on phrenic nerve activity in rats with chronic C2Hx, but dAIH enhanced the capacity for additional pLTF. Further work is needed to explore: 1) mechanisms of enhanced respiratory plasticity following dAIH; 2) if detrimental effects of CIH can be resolved by treatments that mitigate inflammation; and 3) how enhanced/restored phrenic motor plasticity can be harnessed to improve breathing ability in individuals with chronic cervical spinal cord injury.Support or Funding InformationNIH: OT2OD023854, K12 HDO55929 (EGR), T32 HD055929 (LLA); Department of Defense: SCI160123 (EGR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gastrointestinal neuroimmune disruption in a mouse Gulf War Illness model
Authors: Hernandez‐Rivera Siomara , Grubišić Vladimir , Fried David , Gulbransen Brian D 
Published: June 2021

Gulf War Illness (GWI) is a chronic disorder characterized by a spectrum of six symptoms that include gastrointestinal disorders. Exposure to the anti‐nerve gas drug pyridostigmine bromide (PB) is linked with the development of GWI, but the exact mechanisms remain unclear. We hypothesize that PB disrupts gut functions by creating persistent neuroinflammation within the enteric nervous system (ENS). We tested the effects of PB in vivo by exposing male and female mice to 9 μg/mL or 90 μg/mL PB for 7 and 30 days and subsequently assessing gut function using in vivo and ex vivo tests of colonic motility and barrier function. Neurochemistry of the ENS was assessed by immunohistochemistry and immune responses were studied using multiplex cytokine and chemokine arrays in the gut and brain. Acutely exposing whole mount preparations of myenteric plexus to PB drove calcium responses in enteric glia (181.0%, p<0.001). In vivo, exposure to PB acutely increased fecal pellet output (44.3%, p<0.05) and increased fecal fluid content (13.2%, p<0.05) in male mice, but decreased fecal pellet output (62.5%, p<0.05) and reduced fecal fluid content (18.1%, p<0.05) in females. Regardless of sex, PB treatment altered neuromuscular control, slower colonic bead expulsion (male 504.4%, p<0.001; female 275.8%, p<0.01), and reduced colon length (male 9.4%, p<0.05; female 9.9%, p<0.05). PB also drove enteric neurodegeneration in male mice (15.5% neuronal loss, p<0.05) and increased the proportion of excitatory enteric neurons (by 67.7% in 90 μg/mL group, p<0.05) in females. Despite having no effect on colonic permeability, exposure to PB caused major shifts in the expression of pro‐inflammatory cytokines and chemokines in the colon and brain that suggest immunosuppressive effects. Interestingly, immune disruption was still evident in the colon and brain of female animals at one month following exposure to PB. Our results show that the paradigm of PB exposure experienced by veterans of the Persian Gulf War contributes to long‐lasting pathophysiology by driving enteric neuroinflammation, promoting immunosuppression, and altering functional anatomy of the colon in a sex‐dependent manner.Support or Funding InformationDepartment of Defense (DOD) W81XWH1610631This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using dual labeling of thymidine analogs to investigate cell cycle dynamics in the regenerating salamander limb
Authors: Jeon Eun Kyung , Martinez Andy , Monaghan James R. 
Published: June 2021

For almost two centuries, scientists have observed that most forms of regeneration fail to regrow in the absence of nerves, but at the cellular level, how the nerves regulate this phenomenon is still unknown. Understanding how cell cycle kinetics change when the nerve supply is cut would help us understand what role nerves play during regeneration. We used dual labeling of cells using thymidine analogs, 5‐bromo‐2′‐deoxyuridine (BrdU) and 5‐ethynyl‐2‐deoxyuridine (EdU), to quantify the changes in the cell cycle in the denervated regenerating limb of axolotl salamanders. Eight larval axolotls were denervated in their left limbs 12 days after amputation and injected with BrdU and EdU at 24 hour intervals. 72 hours after denervation, the limbs were collected and stained for DNA synthesis (S), mitosis (M), and cell death markers. The duration of S phase of the cell cycle shortened in the actively proliferating cells of denervated regenerating limb almost by half compared to those of the innervated regenerating limb. The total cell cycle lengths differed by about 10%, which suggests that the growth phases (G1 and G2) may have increased in length in the denervated limb. When the nerve supply was cut, less cells returned to cell cycle. Our results suggest that the nerves may regulate the duration of S phase. Future analysis of upregulated and downregulated proteins such as transcription factors during cell proliferation in denervated limb blastema may suggest what pathways are causing the observed changes in the cell cycle.Support or Funding InformationThis project was supported by Summer Scholars Independent Research Fellowship from the Office of Undergraduate Research and Fellowships and NSF grants 1558017 and 1656429.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome‐wide Discovery of Rare Riboswitches in Bacteria
Authors: Arachchilage Gayan Mirihana , Atilho Ruben , Stav Shira , Higgs Gadareth , Breaker Ronald 
Published: June 2021

Riboswitches are noncoding RNAs (ncRNAs) that directly sense specific ligands and regulate various metabolic and physiological pathways. More than 40 different classes of riboswitches have been experimentally validated, and hundreds of additional novel riboswitch classes are predicted to remain to be discovered and analyzed. We are exploiting an improved computational pipeline that can be used to comprehensively uncover novel structured ncRNAs in bacteria, including rare riboswitches. Comprehensive analysis of long and GC‐rich intergenic regions in five different bacterial genomes yielded nearly 70 novel genetic element candidates, including 30 putative ncRNA motifs. Among these novel ncRNA classes, five are considered strong riboswitch candidates. One of the riboswitch candidates, called the thiS motif, has now been experimentally proven to regulate thiamin biosynthesis by directly sensing 2‐methyl‐4‐amino‐5‐hydroxymethylpyrimidine pyrophosphate (HMP‐PP). The mechanistic studies of this HMP‐PP riboswitch demonstrate that the nascent RNA transcript undergoes a conformational change from an ‘on’ state to an ‘off’ state during the transcription process. HMP‐PP binding stabilizes the riboswitch aptamer in its ‘on’ conformation. Experimental validation of the HMP‐PP riboswitch also indicates that the existing bioinformatics pipeline can now be exploited to discover the vast majority of structured ncRNAs hidden among hundreds of sequenced bacterial genomes.Support or Funding InformationHigh‐performance computing at the Yale Center for Research Computing is supported in part by Yale University and by NIH S10RR029676. R.M.A was supported by the National Science Foundation Graduate Research Fellowship Program (DGE1122492). This work was supported by the National Institutes of Health grants (GM022778, DE022340) to R.R.B. Research in the Breaker laboratory is also supported by the Howard Hughes Medical Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Mechanism of Action of Membrane Fusion Protein IncA Using Molecular Dynamics Simulations
Authors: Greco Deanna L. , Bryer Alexander J. , Perilla Juan R. 
Published: June 2021

Infecting millions of people each year, Chlamydia trachomatis is currently the leading cause of non‐congenital blindness and pelvic inflammatory disease. Incapable of replicating on its own, this bacterium survives by entering a host cell through endocytosis and subsequently modifying the vacuole in which it is enclosed to allow for replication. The newly formed replicative compartment, referred to as an inclusion, has numerous inclusion membrane proteins (Inc proteins) that promote growth and maintenance of the developing C. trachomatis bodies. One Inc in particular, known as IncA, exponentially enhances growth of C. trachomatis by initiating the homotypic fusion of multiple inclusions within a host cell. Although the structure and significance of IncA are known, its mechanism of action remains a mystery. The present research aims to elucidate the structure‐function relationship of IncA; offering insight into fusion protein functionality and potential targets for drug development. Analysis of IncA was accomplished by running Constant pH Molecular Dynamics (CpH MD) simulations with a refined focus on aspartic acids, glutamic acids, and histidines. Similar to conventional MD, CpH MD creates conditions that mimic the natural environment of IncA but with the additional capability to explore protonation states of residues. By running the CpH MD system across a range of pHs, the necessary data are generated to determine dissociation constants for specific residues. Combining proton dissociation with tests examining the solvent accessibility of IncA residues, results suggested that there is a correlation between accessibility of a residue and its proton dissociation. Further analysis of residue fluctuation at varying pHs shows that there appears to be a relationship between a residue's protonation state and the degree to which that residue fluctuated throughout a simulation. These findings demonstrate that the location of titratable residues influence the activity of IncA and furthermore, that IncA's titratable residues affect inclusion membrane fusion.Support or Funding InformationNational Science Foundation, Grant No. 1560325.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Molecular Mechanisms of Antibiotic Resistance in Acinetobacter baumannii
Authors: Whitehead Briana , Robinson Kyra , Alexander Heidi , Marshall Dana , Damo Steven 
Published: June 2021

The threat of antibiotic resistance has increasingly become a global issue because of the gradual emergence of multi‐drug resistant (MDR) pathogens. Identifying mechanisms of resistance as well as characterization of attributes deemed beneficial for the survival of the pathogen is vital in contributing to the development of new therapeutics. Acinetobacter baumannii is a gram‐negative opportunistic, nosocomial MDR pathogen that primarily targets immunocompromised patients in medical care facilities around the world. From an assembled bank of clinical isolates of A. baumannii, there is a diverse spectrum of antibiotic resistance patterns, cell motility and biofilm formation represented. Initial RNA sequencing studies implicated the importance of the Gcn5‐related N‐acetyltransferases (GNATs) in facilitating A baumannii resistance to the aminoglycoside class of antibiotics. We aim to determine the role of GNAT proteins to antibiotic resistance in A. baumannii as well as identify which physiological processes are associated with antibiotic resistance. GNATs belong to a superfamily of enzymes that are found in all domains of life and are involved various functions including acetylation of a broad spectrum of substrates and, often times, conferring resistance to antibiotics. We performed structural and functional experiments to understand the mechanisms causing the resistant nature of A. baumannii. Preliminary crystallization studies of the enzyme GNAT 2199 yielded 4 initial conditions suitable for optimal crystal growth. We also conducted a series of enzyme activity assays which revealed that GNATs acetylate various clinically used aminoglycosides. seeking to reveal the natural aminoglycoside substrate for GNAT 2199. Furthermore, cell motility and biofilm assays were used to determine the physiological processes attributed to antibiotic resistance of A. baumannii isolates. Future work entails optimization of the crystallization conditions to produce samples suitable for X‐ray diffraction studies. This will allow us to develop GNAT superfamily structure‐function relationships as well as identify physiological processes of A. baumannii that contribute to antibiotic resistance. In total, this information will be useful for initiating new strategies to control the spread of this bacteria as well as developing new therapeutics to effectively combat this MDR pathogen.Support or Funding InformationNSF No. HRD‐1547757This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and thermal stability of a coral metabolic enzyme
Authors: Wolfe Jacob A , Qian Yiwen , Perez Astrid M , Schermerhorn Janse T , Cela Bekim , Kalinowski Cody R , Largoza Garrett E , Fields Peter A , Brandt Gabriel 
Published: June 2021

Increased ocean temperatures can lead to coral bleaching, as the symbiotic relationship between coral and photosynthetic algae breaks down. The molecular details of this temperature‐dependent disruption are not well understood. Here, we investigate the thermal stability of a critical metabolic enzyme, glyceraldehyde‐3‐phosphate dehydrogenase, from the stony coral Acropora millepora. Binding of the cofactor NAD increases the melting temperature of the enzyme by fully 10 °C. In order to identify the basis for this stabilization, we then determined the crystal structure of the cofactor‐enzyme complex (2.4 Å, PDB ID 6DFZ). This structure is the first of any protein from coral of this genus. The occupancy of NAD across the four monomers of the tetrameric enzyme varies, allowing us to compare the response of the polypeptide structure to the presence of the stabilizing cofactor. We propose that these regions of difference represent areas of the protein important to its thermal stability.Support or Funding InformationNSF IOS‐1654249This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PDB Structure Data Impacted Discovery and Development of Recently FDA‐Approved Drugs
Authors: Zardecki Christine , Westbrook John , Burley Stephen K. 
Published: June 2021

Protein Data Bank (PDB) was established in 1971 as the 1st open access digital data resource in biology and medicine. Today, the PDB currently houses >145,000 atomic level biomolecular structures determined by crystallography, NMR spectroscopy, and 3D electron microscopy. Managed by the Worldwide Protein Data Bank partnership (wwPDB; wwpdb.org), PDB data are used by researchers and educators to understand the role molecules play in health and disease of humans, animals, and plants, food and energy production, and other topics of concern to global prosperity and sustainability.RCSB PDB operates the US data center for the global PDB archive, and makes PDB data available at no charge to all data consumers without limitations on usage. During calendar 2017, >679 million structure data files were downloaded from the PDB by Data Consumers working worldwide. RCSB PDB served >1 million RCSB.org users worldwide with PDB data integrated with ~40 external data resources providing rich structural views of fundamental biology, biomedicine, and energy sciences, and supported >600,000 PDB101.rcsb.org educational website users around the globe.RCSB PDB examined the impact of PDB archiving and open access availability on the discovery of 210 new molecular entities (NMEs or drugs) approved by the United States (US) Food and Drug Administration (FDA) between 2010 and 2016. Biological targets for 90% of NMEs are known. Approximately 6,000 PDB structures containing one of these drug targets and/or one approved NME were identified, providing 85% coverage of known targets and 88% coverage of 2010–2016 approved NMEs. Coverage was comparable across all therapeutic areas. Most structures were both published and freely accessible from the PDB long before the NMEs were approved. Additional analyses revealed these PDB structures were cited by more than 200,000 PubMed‐indexed publications reporting pre‐competitive research on the would‐be drug targets. PDB structures and citing publications contributed substantially to understanding of the target biology, and led to biopharmaceutical company investment in successful drug discovery/development programs.Overall, access to PDB data contribute to patent applications, drug discovery and development, publication of scientific studies, innovations that can lead to new product development and company formation, and STEM education.Support or Funding InformationRCSB PDB is funded by a grant (DBI‐1338415) from the National Science Foundation, the National Institutes of Health, and the US Department of Energy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Salt Bridge between α4 and α5 Helices Drives Differences in Flexibility and Potency of Inhibition among Regulator of G‐protein Signaling (RGS) Proteins
Authors: Shaw Vincent S , Quinn Josiah A , Mohammadi Mohammadjavad , Vashisth Harish , Neubig Richard R 
Published: June 2021

Regulators of G‐protein Signaling (RGS) proteins accelerate the rate of GTP hydrolysis, decreasing G‐protein signaling. Because there are many RGS isoforms with distinct tissue expressions, selective inhibition of RGS action may allow improved tissue specificity over global GPCR agonist application. RGS inhibitors discovered to date act by covalent modification of cysteine residues, and are specific for RGS4. However, single‐cysteine RGS mutants vary widely in their potency of inhibition by thiadiazolidinone (TDZD) inhibitors, with RGS19 being most potently inhibited. We have previously demonstrated a correlation between RGS protein flexibility and potency of inhibition by TDZDs. We hypothesized that differences in a salt bridge‐forming residue between the α4 and α5 helices drives differences in flexibility and potency of inhibition among RGS proteins.An acidic residue on the α4 helix that takes part in an α4‐α5 salt bride network is shared by RGS4 and RGS8 (D90 and E84 respectively), but not by RGS19 (L118). Upon mutation of RGS19 to induce formation of this salt bridge (L118D), the protein became 7°C more stable than WT, as measured by DSF. A mutation that removed this salt bridge in other isoforms caused an increase in melt temperature of 5°C in RGS4 (D90L) and a decrease in melt temperature of 8°C in RGS8 (E84L). Hydrogen‐deuterium exchange was used to evaluate the flexibility of mutant proteins. The α4 helix, which contains the mutation site, was found to be protected from deuterium exchange on addition of a salt bridge (RGS19 L118D), but have higher exchange compared to WT in both mutants where a salt bridge was removed (RGS4 D90L and RGS8 E84L). Changes in the α4 salt bridge‐forming residue also induced changes in the potency of inhibition of Gα binding by TDZD inhibitor CCG‐50014. RGS19 L118D had a lower potency of inhibition than WT RGS19, while mutations at RGS4 D90L and RGS8 E85L increased potency of inhibition compared to WT proteins. Together, these results show that differences in an α4 salt bridge‐forming residue are responsible for flexibility differences among RGS isoforms, and demonstrate a causal relationship between RGS protein flexibility and potency of TDZD inhibitors.Support or Funding InformationNSF grant 1507588This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.