bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiac function in a chemical toxin (organophosphate) induced model of gulf war Illness: benefit of exercise training
Authors: Machi Jacqueline Freire , Conti Filipe , Schmidt Rodrigo , Morris Mariana
Published: June 2021

Background

Gulf War Illness (GWI) is a debilitating disease characterized by a cadre of neural, immunological and autonomic symptoms. We show that exposure to a sub‐lethal dose of Sarin lead to autonomic imbalance, cardiomyopathy and central nervous system changes. Diisopropyl fluorophosphate (DFP), an analogue of Sarin (GB), can be used as surrogate agent to produce neurotoxicity and neuro‐inflammation. There is limited information on the use of DFP to produce model of GWI with emphasis on the study of cardiovascular‐autonomic dysfunctions. Consideration was given to the inclusion of exercise training (ET) as part of the treatment of cardiac dysfunction induced by organophosphate exposure.

Objective

The aim was to determine the effect of chronic exercise training to the response to DFP on autonomic and cardiovascular function.

Methods

Male C57BL/6J mice were divided into two groups (n=8/group): Sedentary (S) and trained (T) before and after DFP exposure. ET was performed on an electronic wheel at low intensity for 1 h/day, 5 days/wk. GWI model was induced by corticosterone (CORT) administered for 7 days (200 mg/l) followed by DFP (1.5 mg/kg, s.c). Cardiac function and ECG were assessed by transthoracic echocardiography (echo) under isoflurane anesthesia (2 – 2.5% on 0.8 l/min of O2).

Results

The low dose of DFP produced left ventricular dilation on sedentary group, specifically, LV dilation of end systolic area (ESA) (S: 10.34±0.86 vs S+DFP: 6.69 ±0.41 cm2). The ET ameliorated DFP induced toxicity (T: 7.99 ±0.40 vs T+DFP 7.66±0.96cm2). Cardiac Output (CO) was reduced in sedentary group after DFP (S: 20.61±2.63 vs S+DFP: 14.78±0.85 ml/min), not change was observed in the ET group, revealing the efficacy of exercise protocol (T: 16.99±1.69 vs T+DFP: 17.90±1.42 ml/min). Myocardial performance index (MPI) and E/A ratio were significantly increased in S group after DFP exposure (MPI: S: 0.52±0.03 vs S+DFP: 0.60±0.02; E/A: S: 1.29±0.09 vs S+DFP: 1.64±0.07) changes that were not observed on T group (MPI: T: 0.50±0.04 vs T+DFP: 0.48±0.03; E/A: S: 1.35±0.09 vs S+DFP: 1.56±0.10) suggesting protection against DFP. Finally sympathetic modulation (LF) was increased in S group compared initial values and T group, as well as LF/HF ratio. Vagal modulation was similar between both groups.

Conclusion

In conclusion, results demonstrate that DFP exerts a negative impact on cardiac/autonomic function. Our critical finding is that chronic exercise prevented most of the cardiovascular dysfunctions elicited by DFP, documenting the positive effects of exercise. This treatment protocol maybe equally applicable to other animal models or even humans.

Support or Funding Information

This work was funded under U.S. Department of Defense grant W81XWH‐13‐2‐0085

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Paralytic hypo‐energetic state facilitates anoxia tolerance despite ionic imbalance in adult Drosophila melanogaster
Authors: Campbell Jacob B. , Andersen Mads Kuhlmann , Overgaard Johannes , Harrison Jon F.
Published: June 2021

Oxygen limitation plays a key role in many pathologies, and yet we still lack a fundamental understanding of the mechanisms responsible for variation in anoxia tolerance. Most vertebrate studies suggest that anoxia tolerance involves the ability to maintain cellular ATP despite the loss of aerobic metabolism. However, insects such as adult D. melanogaster are able to survive long periods of anoxia (LT50: ~8 h) in a hypo‐energetic state characterized by low [ATP]. In this study, we tested for possible mechanisms that allow D. melanogaster adults to survive long periods of anoxia. The adults are paralyzed within 30 sec and after two hours of anoxia, ATP was 3% of normal, [K+]o increased approximately threefold, pH dropped 1 unit, but survival was 100%. With 0.5–6 h of anoxia exposure the live adults maintained low but constant ATP levels while [K+]o and pH continued to change. When these animals were returned to normoxia they could restore hemolymph [K+]o and subsequent movement. However, with longer durations of anoxia, ATP levels decreased and [K+]o rose further, and both of these variables correlated tightly with decreased survival. This response contrasts with the anoxia‐sensitive larval stage (LT50: ~1 h). During anoxia larvae attempt escape for up to 30 min and after two hours of anoxia, ATP was <1% of resting, hemolymph [K+]o increased by 50%, hemolymph pH fell by 1 unit, and survival was zero. The superior anoxia tolerance of adult D. melanogaster appears to be due to the capacity to maintain a paralytic hypometabolic energy state with low but non‐zero ATP levels, and to be able to tolerate extreme extracellular ionic variability. This study suggests that a focus of research in anoxia‐tolerance should be the mechanisms by which animals can survive and quickly recover from such energetic and ionic conditions.

Support or Funding Information

Supported by NSF IOS1256745.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long‐term Delivery of “Low Dose” Repetitive Intermittent Hypoxia is Not Associated with Detectable Pathology
Authors: Gonzalez‐Rothi Elisa Janine , Allen Latoya A. , Santiago‐Moreno Juan , Ciesla Marissa C. , Asa Zachary A. , Smith Kristin N. , Tadjalli Arash , Perim Raphael , Santiago Juliet V. , Holland Ashley E. , Stefan Kelsey A. , Ross Ashley , Satriotomo Irawan , Kelly Mia N. , Simon Alec K. , Poirier Amy E. , Seven Yasin B. , Yarrow Joshua F. , Mitchell Gordon S.
Published: June 2021

Repetitive acute intermittent hypoxia (rAIH) is a promising therapeutic strategy to induce spinal plasticity and improve respiratory and non‐respiratory function after chronic, incomplete spinal cord injury. Although brief rAIH exposures (< 2 weeks) have proven safe in rodent models and humans with cSCI, the safety profile of prolonged rAIH exposures is unknown. This is an essential step in the translation of rAIH to treat humans with respiratory and non‐respiratory paralysis due to injury and/or disease. Here we report an absence of detectable respiratory, cardiovascular, neurological, renal, liver and osteological pathologies after prolonged rAIH (3 months) in rats. Beginning 8 weeks post‐injury spinally intact rats, or rats with C2 hemisections (C2Hx) were treated 4 times per week for 3 months with: 1) rAIH (10, 5 min episodes of 10.5% O2; 5 min normoxic intervals) versus 2) sham normoxia. Whole body plethysmography was conducted following the exposures to assess respiratory function in spontaneously breathing rats. To assess cardiovascular pathology: 1) arterial blood pressure was assessed in urethane anesthetized rats, and 2) the heart and aorta were processed for histological analyses. To assess neurological pathology, the cortex, hippocampus and cervical spinal cord were harvested and histology was used to assess possible gliosis and/or cell death. The liver and kidney were processed for histological analyses, and all tissues are being evaluated by a board certified veterinary pathologist. Lastly changes in bone density were evaluated in the left and right humerus bones using microcomputed tomography. No differences in respiratory or cardiovascular function were observed following rAIH exposures (n = 8–12 per group; all P > 0.50), nor did we observe any evidence of pathology in the heart, aorta, liver or kidney. We found no evidence of hippocampal cell death and/or reactive gliosis based on cressyl violet, NeuN, cd11b and GFAP staining. Bone density analyses are pending, but preliminary results indicate significant bone loss only as a result of C2Hx, but not rAIH. These results add to a building body of evidence that prolonged, “low dose” rAIH is a safe, simple and effective means to improve respiratory and non‐respiratory motor function after chronic spinal cord injury.

Support or Funding Information

DoD SC130298 CDMRP; NIH R01 HL69064 and OT2OD023854 (SPARC); NIH T32 HD043730 (LLA), NIH K12 HD055929 (EGR); and the McKnight Brain Institute.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating a novel regulation on a checkpoint protein Sda that is essential for biofilm formation and sporulation in Bacillus subtilis
Authors: He Yinghao , Gozzi Kevin , Qin Yuxuan , Chai Yunrong
Published: June 2021

Bacteria have evolved various protection mechanisms, such as biofilm formation and sporulation, to help them adapt to and survive extreme environments. In the soil bacterium Bacillus subtilis, Sda is a “checkpoint” protein that plays a critical role in sporulation and biofilm formation by regulating the phosphotransfer from the histidine kinase KinA that culminates in phosphorylation of the transcription factor Spo0A, a master regulator for both sporulation and biofilm formation. Recent studies have shown a serine codon hierarchy‐based novel signaling mechanism for biofilm formation in B. subtilis. Interestingly, the sda gene has a much higher percentage of serine codons (12%) compared to the genome average (5%) in B. subtilis and a biased distribution of different serine codons. We hypothesize that the serine codon hierarchybased signaling mechanism also regulates Sda in that translation of Sda will slow down during nutrient limitation at the onset of biofilm formation and sporulation. To test our hypothesis, we constructed six Sda point mutants with synonymous substitutions in three TCN serine codons. We designed biofilm and sporulation competition assays to determine the impact of those synonymous changes in serine codons. We found that the Sda mutant with AGC substitutions outcompeted other mutants and became the dominant strain after several rounds of competition in both biofilm and sporulation assays. Our data indicates that synonymously switching serine codons in sda gene can delay biofilm formation and sporulation. This study suggests that this codon‐based serine sensing mechanism may be widely present in B. subtilis.

Support or Funding Information

National Science Foundation

Northeastern University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the Substrate Proteins of E3 Ubiquitin Ligase by Orthogonal Ubiquitin Transfer (OUT)
Authors: Yin Jun , Wang Yiyang , Bhuripanyo Karan , Chen Geng , Zhou Li , Liu Ruochuan , Zhou Han
Published: June 2021

Ubiquitin (UB) is transferred through an E1‐E2‐E3 cascade to cellular proteins and encodes diverse signals such as proteasome degradation, subcellular trafficking, interacting with other proteins, and enzyme catalysis. The 2 E1s, 50 E2s, and more than 600 E3s constitute a complex network of UB transfer and make it a significant challenge to identify the substrates of a specific E3. Here, we developed a method that we called “orthogonal UB transfer (OUT)” to identify the direct ubiquitination targets of an E3. In this approach, an engineered UB (xUB) is exclusively transferred through an engineered cascade of xE1‐xE2‐xE3 to the substrates of a specific E3 (“x” designates engineered enzymes that are free of cross‐reactivities with their native partners). We used phage and yeast cell surface display to construct the OUT cascades of HECT E3 E6AP, U‐box E3s E4B and CHIP, and Ring‐between‐Ring (RBR) E3s HHARI and Parkin to profile their substrates. We found key signaling proteins such as kinases, phosphatases, and methyltransferases are ubiquitinated by these E3s. Based on the substrate profiles from the OUT screen, we discovered new pathways to regulate protein ubiquitination and degradation in the cell. Overall, we validated OUT as a powerful proteomic platform to identify the substrates of an E3 and map its position in the cell signaling networks.

Support or Funding Information

This work is supported by the National Institutes of Health (GM104498) and the National Science Foundation (1420193 and 1710460).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Determination of Inteins from Halophilic Organisms
Authors: Reidy Deirdre A.
Published: June 2021

Protein splicing is a post‐translational process by which an intervening polypeptide, or intein, can excise itself and ligate the flanking polypeptides, or exteins. Two extreme halophilic archaea, Halobacterium salinarum (Hsa) and Haloquadratum walsbyi (Hwa), have proteins with inteins. Previous work in the lab demonstrated that splicing of the Hsa DNA Polymerase II (Polll) intein is salt‐dependent. In order to solve the structure of the intein, we have begun to screen for and optimize crystallization conditions. Structural studies will be done by x‐ray crystallography and/or high resolution NMR. We plan to do similar work with Hsa Cell Division Control Protein 21 (Cdc21) and Hwa Gyrase B (GyrB) to determine the structures of the inteins. Previous work demonstrated that the splicing of the Hsa Cdc21, like Hsa PolII, is salt‐dependent. In contrast, the Hwa GyrB intein is likely to have been a recent invader of the haloarchaea and has been shown to splice efficiently in E. coli without salt incubation. We are interested in determining the structure of these inteins from haloarchaea using the methods described.

Support or Funding Information

This work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM), the Dreyfus Foundation (KVM), and William F. McCall Jr. (DAR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Homing Endonuclease and Protein Splicing Activity of Inteins from Extreme Thermophiles
Authors: Comeau Hannah Y. , Gomez Alvin V. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational modification by which intervening polypeptides, or inteins, catalyze their own excision from two flanking polypeptides, or exteins, concomitant with the ligation of the exteins. Many inteins contain a homing endonuclease domain, which can facilitate intein homing by catalyzing double strand breaks in intein‐less extein alleles. We studied the in vitro protein splicing and endonuclease activity of homing endonuclease‐containing inteins that interrupt the DNA Polymerase II (PolII) from the deep‐sea thermophiles Thermococcus kodakarensis (Tko) and Thermococcus barophilus (Tba). It was found that the Tba and Tko inteins can be purified as unspliced precursors and induced to splice in vitro at temperatures greater than 37°C. Additionally, when incubated at pressures up to 1500atm, the Tba and Tko inteins continue to exhibit splicing activity. Tba and Tko inteins also have temperature‐dependent endonuclease activity; the Tba intein is enzymatically active at pressures up to 1000atm. Future work will focus on identifying the active site residues of the homing endonuclease within the Tba and Tko inteins as well as determining the target sequence specificity of the enzyme. We also will determine if there is a difference in homing endonuclease activity between excised inteins and unsplicing intein precursors.

Support or Funding Information

This work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analyzing Protein Kinase C Domain Interactions with the Formin SepA
Authors: Olsen Elisabet , Betton Brianna , Atiq Zainab , Singh Ashmeet , Campbell Lynsey , Parish Paul C. , Hill Terry , Jackson‐Hayes Loretta
Published: June 2021

This research focuses on understanding polarized growth in filamentous fungi. In the model organism Aspergillus nidulans and other filamentous fungi, polarized growth is focused at both the hyphal tip and septa, and growth at both areas involves deposition of cell wall material. To better understand polarized growth, we study proteins that localize to growing hyphal tips and forming septa. In A. nidulans, protein kinase C (PkcA) is involved in the organism's cell wall stress response, and our previous work uncovered a protein‐protein interaction between PkcA and the formin SepA. We also illustrated complementary roles of the two proteins in growth and cell wall integrity. Both PkcA and SepA localize to hyphal tips and septation sites. Using bimolecular fluorescence complementation, we found that SepA and PkcA physically interact at both sub‐cellular locations, and that this interaction occurs through amino acid residues within the C‐terminal half of SepA. Previously, we showed using a series of fluorescence‐labeled PkcA truncated proteins that two regions of PkcA are responsible for PkcA localizing to hyphal tips and septation sites. The first region is a 10 amino‐acid sequence near the carboxyl end of the C2 domain that is required for localization to hyphal tips and septation sites, while the second region is between the C2 and C1B domain, and is sufficient for localization to septation sites only. Here we present our progress towards determining which PkcA domains are involved in its interaction with SepA by using the previously published PkcA truncations in Gal4‐based yeast two hybrid assays. These results help to shed light on the mechanism underlying SepA's and PkcA's complementary involvement in polarized growth.

Support or Funding Information

National Science Foundation Grant 1615192 (Loretta Jackson‐Hayes, PI)

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 Intersection of Learning Progressions and Concept Inventories: Assessment of Student Understanding of Acid/base Concepts
Authors: Wolfson Adele J. , Reed Charlotte R. , Mercer Allison M. , Sutheimer Susan , Lewis Jennifer E.
Published: June 2021

Learning progressions are descriptions of students' growing sophistication in their understanding of a particular construct through a curricular sequence. Learning progressions are particularly useful for organizing complex constructs where students do not necessarily connect concepts as taught in different courses. One such complex construct is acid/base chemistry, which is a vital underpinning to much of biochemistry. We have developed a hypothetical learning progression for student comprehension of acid/base chemistry based on empirical studies. Learning progressions are only useful, however, if we can assess students' arrival at particular steps along the progression. To that end, we have compared the types of questions asked on concept inventories in chemistry, biology, and biochemistry, as well as common standardized tests. We find that none of these tests or surveys probes students' mastery of acid/base chemistry beyond the most elementary level. Therefore, there is a need for new assessment tools that target student understanding of the complex problems and relationships addressed in upper division chemistry and biochemistry courses.

Support or Funding Information

Supported by an award from the National Science Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of Membrane Biogenesis
Authors: Naik Akshata Ramesh , Kuhn Eric R. , Lewis Kenneth T. , Kokotovich Keith M. , Maddipati Krishna R. , Chen Xuequn , Hörber Heinrich , Taatjes Douglas J. , Potoff Jeffrey J. , Jena Bhanu P.
Published: June 2021

Biological membrane in addition to delimiting the cell and its intracellular compartments, govern a wide range of life processes including ion transport, cell division, development, growth, and migration. The addition of new membrane to existing cellular membranes may involve fusion of preformed proteoliposomes and or the incorporation of intracellular free lipids and proteins. It is reported that supported lipid bilayers with adhered lipid vesicles when laterally stretched, expand due to fusion of the adhered vesicles. Mathematical modeling of the plasma membrane suggests trafficking of lipids into stretched membranes to relieve tension. Cell swelling has been reported during mitosis and mechanical forces are implicated in morphogenesis and development. However, the process of addition of new membrane to preexisting membranes in live cells is poorly understood. Here we report in live cells, that in addition to membrane stretch, composition of the existing membrane dictates membrane growth through the selective incorporation of both proteins and lipids.

Support or Funding Information

Work presented in this article was supported in part by the National Science Foundation grants EB00303, CBET1066661 (BPJ); the WSU Interdisciplinary Biomedical Systems Fellow‐ship and Graduate Research Assistantship (ARN), the WSU Interdisciplinary Biomedical Systems Fellow‐ship and Graduate Research Assistantship (KTL). All authors critically analyzed results and proof read the manuscript. The authors declare no competing financial interests or conflicts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human COQ10A and COQ10B are distinct putative StART domain proteins that restore Q biosynthesis and function in yeast
Authors: Tsui Hui S. , Pham Nguyen V. , Fernández‐Del‐Río Lucía , Amer Brendan R. , Bradley Michelle C. , Clubb Robert T. , Clarke Catherine F.
Published: June 2021

Coenzyme Q (ubiquinone or Q) functions as an essential redox‐active lipid in respiratory electron and proton transport in cellular energy metabolism, and it is an important lipid‐soluble antioxidant in cellular membranes. In Saccharomyces cerevisiae, proteins Coq3‐Coq9, as well as Coq11, assemble into a multi‐subunit protein complex called the CoQ‐synthome, which is required for Q biosynthesis. Coq10, a putative steroidogenic acute regulatory (StAR)‐related lipid transfer (StART) domain protein is not a member of the CoQ‐synthome, but is required for the proper assembly of the CoQ‐synthome, efficient de novo Q biosynthesis during early‐log phase, and the function of Q in respiration and as an antioxidant. Humans possess two isoforms, namely COQ10A and COQ10B. Based on the RNA‐seq data from NCBI, COQ10A is predominantly expressed in heart while COQ10B is present in all tissues, suggesting that COQ10B is probably serving a more general role. Previous studies have shown rescue of S. cerevisiae coq10Δ respiration deficient phenotype by expression of human COQ10A. Here we present new evidence showing rescue of S. cerevisiae coq10Δ by expression of either the human COQ10A or COQ10B homolog, as determined by restoration of respiratory growth on non‐fermentable carbon sources, de novo Q biosynthesis, as well as restoration of the CoQ‐synthome.

Support or Funding Information

This research was supported by NSF MCB‐1330803 and Ruth L. Kirschstein National Research Service Award GM007185.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

National 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 Information

This 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 Information

USArmy Medical Research Materiel Command

This 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 Information

This 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 Information

National Science Foundation, Millersville University, and Stowers Institute

This 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 Information

BCS‐1231350; BCS‐1231717; BCS‐1728263; NSF # BCS‐0959438

This 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 Information

This 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 Information

NSF MCB 14‐11734

This 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 Information

This 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.
Examining Fmoc‐amino acid derivatives as selective inhibitors of butyrylcholinesterase
Authors: Ramirez Jennifer , Gonzalez Jeannette , Schwans Jason
Published: June 2021

The design of enzyme inhibitors to act as drugs or as substrates for biocatalysts remains a challenge in biochemistry, pharmacology, and biotechnology. To mitigate the effects of Alzheimer's disease (AD), a neurodegenerative disease characterized by progressive memory loss and the sixth leading cause of death in the United States, numerous enzyme inhibitors have been developed. Activity of the enzyme butyrylcholinesterase (BChE) increases in Alzheimer's patients, and this increase is suggested to lead to a depletion of the neurotransmitter, acetylcholine. To counter this increase in activity BChE‐specific inhibitors have been developed to manage the progression of AD. We hypothesized 9‐fluorenylmethyloxycarbonyl (Fmoc) amino acids could provide a new class of inhibitors, as these compounds features found in BChE inhibitors currently available as pharmaceuticals. The readily available chemistry makes amino acids an attractive scaffold for identifying BChE inhibitors. We found Fmoc‐Leu‐O− as an effective and selective BChE inhibitor with an inhibition constant (KI) value of 115 μM. A series of Fmoc‐Leu‐O− derivatives with side chain modifications were then evaluated. The Fmoc‐amino acids were purchased or synthesized from the corresponding amino acid. The KI value for each compound was determined using kinetics assays and UV‐absorbance spectroscopy. For the four Fmoc‐Leu‐O− derivatives tested, Fmoc‐neopentylglycine‐O− showed the greatest potency with an inhibition constant of 36 μM, ~three‐fold lower than the original compounds. A series of Fmocdi‐, tri‐, tetra‐, and pentapeptides were synthesized by solid phase peptide synthesis. The products were purified by HPLC and characterized by NMR and mass spectrometry. Our initial results identified the dipeptide (Fmoc‐Leu‐Leu‐O−) as an effective inhibitor with KI value of 28 μM. We are currently investigating if longer Fmoc‐Leu‐O− peptides and other Fmoc‐peptides are potent and selective BChE inhibitors. The results explore a new class of potent and selective BChE inhibitors and may identify features important for binding in the enzyme active site.

Support or Funding Information

This project was supported by the National Institute of General Medical Sciences, National Institutes of Health (NIH) under award numbers T34GM008074, UL1GM118979, and RL5GM118978, and the National Science Foundation (NSF; MRI CHE‐1337559). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or NSF.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.