bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identifying genes required for the use of p‐coumarate in coenzyme Q biosynthesis in Saccharomyces cerevisiae
Authors: Nag Anish , Li Yubing , Basset Gilles , Kwon Ohyun , Clarke Catherine 
Published: June 2021

Coenzyme Q (ubiquinone or Q) is a redox active lipid molecule, with a fully substituted benzoquinone ring and a polyisoprenoid tail, containing up to ten isoprene units. Q is an essential component of the mitochondrial respiratory electron transport chain. Q deficiency has been directly as well as indirectly linked to a number of neurological, muscular, and cardiovascular disorders in humans that include encephalomyopathy and cerebellar ataxia, both of which can be fatal. The exact biosynthetic pathway in eukaryotes has still not been completely characterized. Traditionally, 4‐hydroxybenzoic acid (4‐HB) has been known as the ring precursor for Q biosynthesis in eukaryotes. In addition to 4‐HB, recent reports have revealed additional phenolic compounds, such as resveratrol, p‐coumaric acid, and kaempferol to be novel compounds that can enhance Q content in mammalian cells, and to act as Q ring precursors. It has been hypothesized that these phenolic compounds may be converted to 4‐HB in living systems. Evidence has shown that S. cerevisiae uses tyrosine as precursor for 4‐HB biosynthesis. In particular, p‐coumarate, which was shown to increase Q levels in yeast, could be a potential intermediate in the biosynthesis of 4‐HB from tyrosine, through a possible beta‐oxidative pathway. However, it is also possible that the role of p‐coumarate might be independent of tyrosine, and there may exist multiple pathways of 4‐HB biosynthesis. Our efforts focus on investigating the roles of both tyrosine and p‐coumarate in the biosynthesis of Q in yeast. A number of yeast null mutants were tested in order to shed further insight into the use of p‐coumarate and tyrosine in Q biosynthesis. The yeast mutants are grown in the presence of 13C6‐tyrosine and 13C6‐p‐coumarate, and in the content of 13C6 –labelled Q and unlabelled Q is determined for WT controls and the null yeast mutants. Thus we can identify genes required for the metabolism of natural products into ring‐precursors of Q and, thereby yield insights into modulating Q biosynthesis.Support or Funding InformationNational Science Foundation (NSF MCB‐1330803)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Patient‐Specific Mechanisms of Lafora Disease Mutations in the Human Glycogen Phosphatase
Authors: Gentry Matthew , Brewer M. Kathryn , Raththagala Madushi , Wayne Jeremiah , Kooi Craig Vander 
Published: June 2021

Lafora disease (LD) is a fatal neurodegenerative epilepsy caused by defects in glycogen metabolism. Classically, LD manifests in adolescence with progressive myoclonus epilepsy, rapid neurodegeneration and death 10 years after onset. Insoluble inclusions of abnormal glycogen known as Lafora bodies (LBs) are found in the brain and other tissues of Lafora patients. Genetic ablation of glycogen synthesis eliminates LBs and rescues LD in mice, indicating LBs are the pathological cause of LD.50% of LD cases are caused by recessive mutations in the Epilepsy Progressive Myoclonus 2A (EPM2A) gene encoding laforin, the glycogen phosphatase. Laforin is a bimodular protein containing a carbohydrate binding module (CBM) and a dual specificity phosphatase domain. In the absence of laforin, glycogen becomes hyperphosphorylated and abberantly branched, leading to LB formation. >90 distinct missense, nonsense, and frameshift mutations have been described in LD patients. We previously determined the crystal structure of laforin and defined the structural basis of its function.The objective of this study is to define the molecular defect(s) of >30 pathogenic mutations from LD patients and determine why some are linked to milder phenotypes.Using the laforin crystal structure and a variety of cellular, biochemical, and biophysical tools, we discovered a hierarchy of mutations that differentially affect aspects of laforin function. Specifically, we discovered that a large group of pathogenic mutations cluster in the CBM, leading to destabilization, domain decoupling, and reduced protein‐protein interactions. Further, a mutation associated with an extremely mild case of LD impairs dimerization and preferential binding to abnormal glucan substrates.Our study reveals the importance of the CBM for both stability and glucan binding, defines the atomic‐level effects of patient mutations, and provides a biochemical explanation for late‐onset phenotypes. Our work not only provides a framework for defining novel LD mutations and understanding patient‐specific disease progression, but also deepens our understanding of the enzymatic regulation of glycogen.Support or Funding InformationThis work was supported by NIH Grants R01 NS070899 (M.S.G.), P01 NS097197 (M.S.G.), and F31 NS093892 (M.K.B.); NSF Grant IIA‐1355438 (M.S.G.); Mizutani Foundation for Glycoscience Award (M.S.G.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resuscitation Fluids Modulate Sterile Inflammation in a Rodent Model of Hemorrhagic Shock
Authors: Torres Luciana N. , Salgado Christi L. , Dubick Michael A. , Torres Filho Ivo P. 
Published: June 2021

Sterile inflammation may trigger an array of danger‐associated molecular patterns and activation of the vascular endothelium, leading to loss of vascular barrier, microvascular leakage, coagulation dysfunction, leucocyte extravasation and organ injury. In our rat hemorrhage model, we have demonstrated previously elevated cytokine levels in liver, kidney and lung along with circulating proteases and endothelial dysfunction following hemorrhagic shock (HS) and/or fluid resuscitation. We hypothesized that normal saline (NS) resuscitation after HS may sustain sterile inflammation by increasing leukocyte influx to tissues (transmigration) and net leukocyte recruitment (overall efficiency), which results in loss of barrier integrity and tissue injury, compared to HS alone or blood products. Anesthetized rats (n= 5–7/group) were bled 40% of blood volume and either left untreated (HS ONLY) for over 3h or resuscitated with NS, 45 ml/kg, or 5% Albumin (ALB), 15ml/kg. Shams rats (control) were subject to all procedures, except hemorrhage or resuscitation. Intravital microscopy was performed in over 100 cremaster venules to determine leukocyte transmigration and overall efficiency as well as blood flow, wall shear rate (WSR), endothelial glycocalyx thickness and vascular permeability. Post‐shock or post‐resuscitation blood and tissue samples were collected over 3h from onset of hemorrhage. Our HS model alone decreased blood flow and WSR more than 60% (vs. sham, P<0.05), and increased glycocalyx shedding, permeability as well as transmigrated leukocytes (vs. sham and ALB, P<0.05). High levels of transmigration post‐shock was sustained after NS (883 ± 433 transmigrated cells/mm2, vs. sham, P<0.05). Compared to HEM ONLY, IL‐1β, IL‐6 and TNF‐α levels in kidney were 2–3‐fold higher in NS group, as well as blood flow and WSR, glycocalyx shedding, permeability (P<0.05). The highest overall efficiency post‐shock was observed in NS group (224 ± 117 transmigrated cells/mm2 per 1,000 leukocytes/μL) vs. HS ONLY (164 ± 94, P=0.04), sham (113 ± 50, P<0.001) and albumin group. In contrast, ALB normalized glycocalyx, venular blood flow and WSR, as well as restored barrier function and decreased inflammation to sham levels, compared to HEM ONLY. Fluid administration presents a therapeutic conflict between hemodynamics stabilization and detrimental effects on the endothelial and microvascular function. Iatrogenic increase in leukocyte recruitment and migration into tissues after NS sustained sterile inflammation after hemorrhagic shock and thus presents some clinically relevant deleterious effects.Support or Funding InformationUSArmy Medical Research Materiel CommandThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Exogenous Ketone Supplements on Latency to CNS Oxygen Toxicity Seizures in Middle‐Aged Rats
Authors: Ari Csilla , Koutnik Andrew P. , DeBlasi Janine , Landon Carol , Vallas John , Bharwani Sahil , Dean Jay B. , D'Agostino Dominic P. 
Published: June 2021

Central nervous system oxygen toxicity (CNSOT) seizures manifest as tonic‐clonic seizures and are a limitation of hyperbaric oxygen therapy (HBOT) and Navy SEAL divers breathing 100% oxygen using closed circuit rebreathers. Previous studies showed that ketone ester (1,3‐butanediol acetoacetate diester, KE) delayed latency to seizures in 3 months old Sprague‐Dawley rats. Our present study explores the effect of exogenous ketone supplements on CNSOT seizures across different ketone formulations and dosages in 18‐month‐old Sprague‐Dawley rats that resemble middle age in humans. Rats received one dose of the ketogenic agent by oral gavage 60 minutes before a dive. Treatment groups included control (water), 1,3‐butanediol acetoacetate diester (KE 10g/kg, KE/2 5g/kg), KE+MCT (KE 5g/kg + medium chain triglyceride 5g/kg) and KS+MCT (CaNaBHB 5g/kg + MCT 5g/kg). To simulate a dive, rats were placed on 100% oxygen followed by gas compression to 5 atmospheres absolute (ATA) in a hyperbaric chamber and remained at 5 ATA until exhibiting CNSOT seizures, defined by tonic‐clonic movements. Upon seizure presentation, experiments were immediately terminated and the chamber was decompressed at a rate of 1 ATA/min. A small sample of blood was drawn and tested immediately for glucose and R‐beta‐hydroxybutyrate (βHB) levels. While βHB levels were significantly elevated in all aged‐rat treatment groups, compared to control, the latency to seizures was significantly delayed only in KE (p=0.0003), KE/2 (p=0.023) and KE+MCT (p=0.028) groups. In these groups, the severity of seizures was less than control. The latency to seizures in 18 months old rats were delayed by 179% in KE, 219% in KE+MCT and 55% in KE/2 groups, while only by 29% in KS+MCT group. Previous studies indicate that 3‐month‐old Sprague‐Dawley rats presented 574 ± 116% latency to seizure compared to control indicating age related influence on latency to seizure. Glucose levels were not significantly different from the control group. We can conclude that middle‐aged rats demonstrated increased latency to CNSOT seizures, although at a different rate than younger rats, in response to ketogenic supplements and their combinations, while elevating βHB levels.Support or Funding InformationThis work was supported by ONR Grant N000141310062 (DPD), ONR, Undersea Medicine Program (N000140710890, JBD), ONR, Undersea Medicine Program (N000141612537, JBD).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Late‐Migrating Neural Crest Cells in Turtle Embryos Display Strong Osteogenic Potential
Authors: Martin Seth , Spengler Jen , McLennan Rebecca , Kulesa Paul , Gilbert Scott , Cebra‐Thomas Judith 
Published: June 2021

Turtle plastron bones develop by intramembranous ossification, suggesting that they are derived, like the facial bones, from neural crest cells. Using cell‐labeling and neural tube explant cultures, we have shown that cells expressing neural crest markers emerge from the trunk neural tube in the turtle Trachemys scripta in two migratory phases over a greatly extended period. These findings were confirmed by electroporation of plasmids driving expression of fluorescent proteins. The early phase of migration (comparable to the period of neural crest cell (NCC) migration in other amniotes) extends until stage G10–11. These cells give rise to typical NCC derivatives. The NCCs that emerge late (beginning in stage G15–16 turtle embryos, well beyond the stage of neural crest migration in chick or mouse embryos) appear to migrate ventrally to form an ectomesenchymal dermis that gives rise to the bones of the plastron. Thus, there appear to be two distinct migratory phases in vivo. To test the hypothesis that the second wave of trunk NCCs in turtle embryos is capable of differentiating into bone, T. scripta neural tubes were cultured. The resulting NCCs were allowed to differentiate, and the differentiated cell types produced were analyzed by immunofluorescence. A subset of the cultures produced melanocytes, a typical trunk NCC‐derived cell type, while virtually all of the cultures produced a substantial number of osteoblasts. Our results suggest that the late trunk NCCs are predisposed to differentiate into osteoblasts, and thus provide good candidates for the cells that form the plastron. Craniosynostosis is a common human developmental deformity involving premature fusion of the calvarial sutures between the bones of the skull. A better understanding of intramembranous ossification, and analysis of an enriched population of osteogenic NCCs, could result in improved treatment options.Support or Funding InformationNational Science Foundation, Millersville University, and Stowers InstituteThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lateral growth in the basicranium: We drift, they drive
Authors: Smith Tim D. , DeLeon Valerie B. 
Published: June 2021

Basicranial synchondroses are crucial for elongation of the length of the skull and may contribute to basicranial angulation. Most studies have focused on midline synchondroses (e.g., sphenooccipital). More recently, however, evidence has emerged suggesting an important role for lateral basicranial regions in maintaining the functional spatial relationship with the facial skeleton. Numerous bilateral basicranial joints (both sutures and synchondroses) remain patent after birth, but few comparative studies have addressed them. Their role as pacemakers, if any, in changes in basicranial width or flexion is beyond even speculation. Using histology and μm‐CT, we examined lateral basicranial joints in four species of perinatal primates, including two monkeys and one lemur, and one bushbaby. Primates have a basic similarity in joints of the anterior and posterior cranial fossae. However, one cartilaginous growth center of the middle cranial fossa differs between higher primate groupings. The alisphenoid‐basisphenoid synchondrosis is present bilaterally in perinatal lemurs and bushbabies and is an active, bipolar growth center, with chondrocytes proliferating transversely. In contrast, this joint is fused in both monkey species, and the synchondrosis is not described in the literature on human newborns. Our pilot data indicate a dichotomy among primates: bilateral synchondroses are fused earlier in anthropoids compared to strepsirrhines. This indicates an earlier loss of intrinsic (i.e., “pacemaker”) growth within the lateral basicranium of anthropoids. As a result, we predict that anthropoids exhibit relatively more evidence for endocranial bone modeling (“drift”) at birth compared to strepsirrhines, most especially within the middle cranial fossa, which lacks active growth plates in anthropoids.Support or Funding InformationBCS‐1231350; BCS‐1231717; BCS‐1728263; NSF # BCS‐0959438This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Initially Disordered, Reflectin Assembly Tunably and Reversibly Drives Biophotonic Color
Authors: Levenson Robert , Bracken Colton , Sharma Cristian , Santos Jerome , Arata Claire , Morse Daniel E. 
Published: June 2021

Reflectin proteins are found in the reflective Bragg lamellae in skin cells of certain squids, where they tunably control color for camouflage and communication. Initially disordered in the monomeric form, the reflectins have unusual amino acid compositions and are block copolymers consisting of polyampholytic canonical repeats interspersed with cationic linkers. In vivo, phosphorylation of the cationic linkers induces condensation, secondary folding and hierarchical assembly of the reflectins with concomitant emergence of tunable iridescence. We analyzed the folding and assembly of recombinant phospho‐mimetic and deletion mutants of reflectin, using pH titration as a surrogate for in vivo phosphorylation. Results of dynamic light scattering, transmission electron microscopy, fluorescence, circular dichroism and x‐ray scattering reveal that neutralization of the cationic linkers progressively and reversibly drives the reflectins to form monodisperse spherical assemblies of reproducible size. These analyses reveal a strongly predictive relationship between net charge density and size of the assembled reflectin multimers, with the switch appearing to be equally distributed across the spatially segregated linkers. Accompanying the progressive neutralization of Coulombic repulsion of the linkers, assembly appears to be driven by entropically driven folding of the canonical repeats into amphiphilic secondary structures that are likely to enable hierarchical assembly. These results suggest a model for tunable reflectin assembly and provide insight into the behavior of this unique, initially disordered protein.Support or Funding InformationThis research was supported by the U.S. Department of Energy, U.S. Army Research Office, and Institute of Collaborative Biotechnologies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The intrinsically disordered PsbO subunit of Photosystem II: structure and role in photosynthetic water oxidation
Authors: Barry Bridgette Anne , Brahmachari Udita , Obi Chiagoziem E. , He Jiayuan Nancy 
Published: June 2021

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the oxidation of water into molecular oxygen at a Mn4CaO5 cluster. The largest extrinsic subunit of PSII, PsbO, is required for efficient oxygen evolution. PsbO maintains the normal function of the oxygen evolution cluster (OEC). Removal of PsbO leads to loss of oxygen evolution activity, and reconstitution restores activity (1). PsbO has been proposed to be involved in proton transfer through a hydrogen‐bonding network that extends from the metal cluster to the lumen (2). PsbO is observed to form a beta barrel with long loop regions, when bound either to the cyanobacterial (3) or plant PSII reaction center (4). However, the solution structure of intact PsbO, containing all potentially flexible loop regions, is not yet known (5). When released from the PSII, either by urea treatment or calcium chloride washing, or when produced recombinantly, PsbO exhibits a low pI, an exaggerated size estimate on SDS‐PAGE and gel filtration, thermostability, and a high content of disordered structure. Other characteristics of intrinsically disordered regions are also observed in PsbO, including low sequence complexity and enrichment in polar and charged residues. In addition, recombinant, calcium‐released, and urea‐released PsbO all exhibit different amide I infrared line shapes (6, 7), although all these preparations reconstitute activity in PSII. Here, we test the hypothesis that PsbO is an intrinsically disordered subunit and that its disordered, flexible nature promotes photosynthetic water oxidation. Recent progress will be discussed.Support or Funding InformationNSF MCB 14‐11734This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NMR Structure‐Based Optimization of Staphylococcus aureus Sortase A Pyridazinone Inhibitors
Authors: Sue Christopher Kenji , Clubb Robert , Amer Brendan , Chan Albert 
Published: June 2021

Staphylococcus aureus is a leading cause of hospital‐acquired infections in the USA and is a major health concern as methicillin‐resistant S. aureus and other antibiotic‐resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti‐infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure‐based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone‐based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second‐generation analogues that are ~70‐fold more potent than the lead molecule. These optimized molecules exhibit broad‐spectrum activity against other types of class A sortases, have reduced cytotoxicity, and impair SrtA‐mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogues are attractive candidates for further development into anti‐infective agents, and highlights the utility of employing NMR spectroscopy and solubility‐optimized small molecules in structure‐based drug discovery.Support or Funding InformationThis research was supported by funding from the National Institutes of Health (AI52217 to R.T.C. and M.E.J.; and GM31749 to J.A.M.). A.H.C. was supported by the UCLA Chemistry Biology Interface Training program (NIH T32GM008496). B.R.A was supported by the UCLA‐MBI Whitcome Pre‐Doctoral Training Grant. Additional support at UCSD has been provided by the National Science Foundation, the Howard Hughes Medical Institute, the Center for Theoretical Biological Physics, the National Biomedical Computation Resource, and the NSF Supercomputer Centers.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.