bluuebunny/crossref_metadata_2025_split huggingface dataset:
Maternal Alcohol Exposure Augments Fetal Skeletal Anomalies in Diabetic Mouse Pregnancy
Authors: Rengasamy Padmanabhan , Shafiulla Mohammed 
Published: June 2021

IntroductionThere is a several‐fold increase in birth defects in diabetic pregnancy. Alcohol is an established‐teratogen in humans. Both alcohol and diabetes mellitus (DM) are independent risk factors for congenital anomalies. However, their teratogenic mechanisms are unknown. Diabetic women also might consume alcohol during pregnancy.AimOur aim was to determine the effects of potential interaction between maternal alcohol and maternal DM during gestation on fetal development.MethodsWe made pregnant mice diabetic by a single dose (IP) of the diabetogenic compound streptozotocin (STZ) administered on gestation day (GD) 2. Controls received a proportionate volume of vehicle (citrate buffer). Groups of DM and non‐DM animals received ethanol (IP) in single doses of 0.03 ml/g body weight (25% v/v ethanol in normal saline) on GD 7 or 8. One group of DM animals had a daily dose of 6–8 IU of insulin subcutaneously. We collected the fetuses on GD 18, fixed in 95% ethanol and stained their skeletons with Alizarin red‐S and Alcian blue and observed for skeletal anomalies.ResultsBoth ethanol and DM independently induced a low incidence of skeletal defects in fetuses. Ethanol injection of DM mice on GD 7 resulted in a moderate accentuation of DM‐related skeletal defects. Treatment of DM animals with ethanol on GD 8 resulted in severe skeletal defects, which affected regions of the axial and appendicular skeletons differentially. The combination treatment affected most markedly skeletal ossification in about 60% of animals. This treatment also caused an augmented frequency of skeletal variations (e.g. supernumerary ribs and sternal anomalies) and poor ossification of the bones of fore‐, and hind paws.Discussion and ConclusionGestation day 8 was particularly susceptible to augmentation of skeletal defects with ethanol treatment. These data also suggest that alcohol‐diabetes interaction affects the ossification processes of mouse embryos resulting in extensive skeletal variations and malformations.Support or Funding InformationSupported by (1) College of Medicine and Health Sciences, UAE University, Al Ain, UAE and (2) School of Medicine, University of Texas Rio Grande Valley, Edinburg, TXThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Sources of Variation in Inter‐observer Reliability Scoring of Facial Expressions Using the ChimpFACS
Authors: Molina Alexandra , Florkiewicz Brittany , Cartmill Erica 
Published: June 2021

The Facial Action Coding System (FACS) is currently the most popular systematic and standardized method of studying facial expressions in mammals. Since its original release for humans, the FACS has expanded to include primates such as chimpanzees. The FACS trains its users to analyze facial expressions by identifying individual muscle movements (called action units or AUs) that are combined to create facial expressions (or AU combinations). All researchers must pass a species‐specific certification test and are then considered reliable coders. After certification, interobserver reliability is rarely performed. However, reliability may be affected by variables like video length, apex identification, AU combination composition, and sociality effects. We analyzed the effects of these variables on reliability and propose that researchers should predetermine action units of interest for apex choice to reduce variation between observers. Two ChimpFACS certified researchers independently coded facial expressions from 30 hours of video of 17 chimpanzees (Pan troglodytes) housed at the Los Angeles Zoo. Coding reliability was then evaluated using Wexler's ratio (the method of calculating FACS inter‐observer reliability on the certification test). Preliminary results indicate that AU combination composition and sociality effects could have significant effects on coding reliability. We argue that researchers should predetermine which AUs to code as notable to facial expressions of interest and only code one individual during a time of high sociality to improve reliability in studies involving FACS coding. Employing these additional steps would improve replicability in facial expression studies, thereby strengthening research on the evolution of facial communication in primates.Support or Funding InformationThis research was supported by the National Institute of General Medical Sciences of the National Institute of Health under award number R25GM055052 awarded to T. Hasson. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This project could also not have occurred without the support of the Los Angeles Zoo.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutating the Telomere Gene
Authors: Palmero Brett , Sandler Anna , Dejneka Alexandra , Kirk Karen E 
Published: June 2021

Telomeres are protein‐nucleotide sequences at the ends of chromosomes that serve to prevent the loss of genetic information during cell replication. A telomerase ribonucleoprotein complex includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Our lab has been studying the biogenesis of TER in Aspergillus nidulans, a molecule that is in part structurally similar to that of Saccharomyces cerevisiae and partly to that of mammalian cells. Research in S. cerevisiae has shown that the telomerase RNA appears to leave the nucleus and is potentially assembled in the cytoplasm. Whether TER migrates to the cytoplasm is not known in A. nidulans, but its ability to form a multi‐nucleate state known as a heterokaryon can be used to determine the localization of TER. We assay for the presence of two genetically distinct nuclei via the heterokaryon test, a molecular tool that has allowed us to deduce whether the RNA products for TER and TERT leave the nucleus. We knocked out the genes for TER and TERT, analyzed the results of the heterokaryon test, and determined if assembly of the active enzyme occurs in the nucleus or the cytoplasm. Our results suggest assembly in the nucleus, a scenario that is similar to humans but not to yeast. These results further suggest the potential of A. nidulans as a useful model organism to study the localization of telomerase components.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the association of the TrmB‐like protein OxsR to chromatin binding and oxidative stress in Haloferax volcanii
Authors: Mondragon Paula Giselle , Hwang Sungmin , Schmid Amy , Maupin‐Furlow Julie 
Published: June 2021

Archaea are single‐celled microorganisms that are well known for their ability to survive and thrive in harsh environmental conditions; however, the factors that mediate homeostasis during redox and/or oxidative stress are not fully understood this domain of life. OxsR (redox stress responsive regulator, HVO_2970) is a TrmB‐like protein of arCOG002242 that is increased several‐fold in abundance during hypochlorite stress based on quantitative proteomic analysis of the halophilic archaeon Haloferax volcanii. TrmB (transcription regulator of mal operon) family proteins are typically transcription factors that bind sugar/effector molecules and regulate the expression of carbon metabolism and transport genes. OxsR has a winged‐helix DNA binding domain but lacks the sugar/effector binding domain common to members of the TrmB family. Here we find that OxsR has a conserved cysteine residue (C24) at a predicted homodimer interface that may to be intimately associated with redox homeostasis in this archaeon. OxsR is required: i) for tolerance to oxidative stress and ii) for an increase in the level of transcripts associated with DNA repair and oxidative stress response. Current work is focused on determining how OxsR senses redox stress, binds DNA, and regulates transcript levels by use of RNAseq, ChIP‐Seq and ChIP‐PCR methods. To this end, we have prepared an RNA library for RNAseq analysis and integrated a C‐terminal hemagglutinin (HA) tag onto the oxsR gene locus for the ChIP methods. Overall, OxsR is an apparent redox sensor required for elevated transcript levels of DNA repair and oxidative stress response pathways. The distribution of OxsR homologs with conserved cysteine residue(s) in Euryarchaeota and Crenarchaeota phyla suggests this type of redox sensor is widespread in Archaea.Support or Funding InformationFunds for this project were awarded to JM‐F through the Bilateral NSF/BIO‐BBSRC program (NSF 1642283), the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences and Biosciences, Physical Biosciences Program (DOE DE‐FG02‐05ER15650) and the National Institutes of Health (NIH R01 GM57498).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cysteine‐Mediated Dimerization of the Transmembrane Domain of Mucin 16
Authors: Li Edwin , Butch Nicole , Micua Taylor 
Published: June 2021

Mucins are proteins in the epithelial tissue that produce mucus barriers to provide moisture and also prevent harmful, foreign material from entering the cell. These proteins are heavily glycosylated, and can be secreted to the extracellular space or be embedded in the plasma membrane. The overexpression of transmembrane mucins have been correlated with the development of cancerous growths. For example, the transmembrane mucins mucin 1 (MUC1) and mucin 16 (MUC16) have been associated with breast and ovarian cancer, respectively.The role of MUC1 in tumor progression has been extensively studied. Dimerization of MUC1 leads to its trafficking to the nucleus where it can alter the expression of genes that regulate cell growth. The cysteine‐glutamine‐cysteine (CQC) motif in the cytosolic juxtamembrane domain of MUC1 is the main driving force of this dimerization, primarily through the formation of disulfide bonds. Contrary to MUC1, there is not much information regarding the possible intracellular role of MUC16 in tumor formation and progression. Similar to MUC1, MUC16 also has two cysteine residues at or near the juxtamembrane domain. Furthermore, recent studies have observed the presence of MUC16 in the nucleus. Thus, we hypothesized that there may be a similarity in the mechanism of trafficking of MUC1 with that of MUC16.We are using the ToxR activity assay to assess the dimerization of the transmembrane domains of MUC1 and MUC16 with and without the juxtamembrane cysteines. Our preliminary results show that, in the presence of the cysteine residues, the transmembrane domain of MUC16 dimerizes as strongly as that of MUC1. Our results also show that the strength of dimerization, in both MUC1 and MUC16 are associated with the formation of disulfide bonds. These results suggest a possible mechanism of MUC16 nuclear trafficking, similar to that of MUC1, involving cysteine‐mediated dimerization.Support or Funding InformationNicholas & Susan Nicholaides Research Fellowship, and the SJU Summer Scholars ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Superposition of Macromolecular Electron‐Density Maps in X‐ray Solution Scattering (SAXS)
Authors: Nguyen Nhan D , Grant Thomas D 
Published: June 2021

Understanding the structure of biological macromolecules is important for determining their functions and cellular activities. With the growth of computational techniques in crystallography, X‐ray Solution Scattering Data (SAXS) becomes more applicable to reconstruct a wide range of 3D biomolecular structures from a low‐resolution profile by its easier use and simpler sample preparation. A novel program called (DENsity from Solution Scattering) resolves these disadvantages of low‐resolution data and retrieves 3D structural information of the complexes in solution. Due to containing the lack of information of 1D scattering data, these SAXS profiles require more modeling procedures and novel iterative algorithm to retrieve desired macromolecular structures. Our lab's previous publication on Nature elucidates the modeling algorithm using the Fast Fourier Transform to calculate and reconstruct 3D electron density maps of each profile. To obtain better 3D maps of macromolecules, we face another challenge of averaging method. The averaging step involves principal axis alignment, enantiomer selection, overlap score optimization among multiple (20) electron density maps. The updated version has been worked out by minimizing interpolation overuse and independently running with new Python codes that no longer require heavy EMAN2 installation as previous for the reconstruction purpose.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of Specificity and Stability in the Folding Trajectory of Caspase
Authors: Shrestha Suman , Clark Clay 
Published: June 2021

Proteins must fold properly and efficiently to accomplish cellular functions. Over the course of evolution, the energy landscape must be maintained such that the protein folds in the right conformation, however, how protein's energy landscape is maintained or altered is unclear. To study how a protein's energy landscape changed over time, we characterized the folding trajectories of caspase homologs and their common ancestor. Caspases are an ancient class of cysteinyl proteases that control apoptosis, responsible for cell differentiation, and maintain cellular homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because substrate specificity and conformational selection are retained through hundreds of millions of years of evolution. Some regulatory features are ancient, and therefore common, while other features are modern and cluster‐specific. In this study, we performed enzyme kinetic assay using synthetic tetrapeptides and substrate phage display using peptide library on ancestral reconstructed and modern proteins to study the evolution of substrate specificity. In addition, we examined urea‐induced equilibrium unfolding properties to evaluate thermodynamic stability and folding pathways. We found that common ancestor of effector caspases showed promiscuity over substrates (D/L/I/VXXD) which is specified later on in modern proteins in different lineages; caspase‐3 and ‐7 prefers DXXD whereas, caspase‐6 prefers VXXD. On the other hand, equilibrium unfolding result suggests all the human effector caspases unfolds via four‐state process; the native dimer (N2) undergoes an isomerization to a dimeric intermediate (I2) and to a monomeric intermediate (I) before completely unfolded (U). Overall, the data show that the folding landscape was established in the early ancestor (>650 MYA) and enzyme specificity evolved from the common landscape. A discrepancy in the overall ΔG0conf, in the folding process between extant caspases, may be due to subsequent mutations in the subfamily.Support or Funding InformationDepartment of Biology, University of Texas at ArlingtonThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Evolution and Mechanism of Enzyme Specificity and Stability of Caspase‐3
Authors: Yao Liqi , Clark Clay 
Published: June 2021

The caspase family of proteases is an excellent model when analyzing protein evolution. Caspases are in a large family, and proteins evolved from a common ancestor. Caspases in the apoptotic initiator subfamily form a stable monomer, and their activities result from dimerization. Apoptotic effector caspases, however, can only form stable dimers. Urea folding/unfolding studies showed that human caspase‐3, an effector caspase, folds by a four‐state equilibrium process in which the native dimer (N2), unfolds through a non‐active dimeric intermediate (I2), a non‐native monomeric state (I) and an unfolded monomeric state (U). We compared the folding process of caspases from other species in order to examine whether the folding mechanism is conserved in other species. We show that zebrafish caspase‐3b also folds by the four‐state model. In addition, the human caspase‐3 dimer is stabilized by several salt bridges across the dimer interface, and dimerization is sensitive to pH due to the presence of two histidine residues in the salt bridges. In contrast, zebrafish caspase‐3a and ‐3b do not use histidines in the salt bridges, so the zebrafish dimers appear less sensitive to pH.Following evolution of the dimer, mutations in effector caspases resulted in changes in substrate specificity. The caspase‐6 subfamily shows preference to valine at the P4 position, while caspase‐3 shows preference to aspartate at the P4 position. However, recent research shows that zebrafish caspase‐3a has relaxed specificity and allows either valine and aspartate at the p4 position. In contrast, the closely related zebrafish caspase‐3b is similar to human caspase‐3 with ~100‐fold preference for D over V at P4. Our data shows that there are two ways to change enzyme specificity. The first one is through improving H‐bonds to the P4 aspartate by decreasing the H‐bond distance between carboxyl group in P4 and asparagine from active site loop 3. A second mechanism that results in selection of D over V is by including a polar cap near the S4 site. This mechanism is observed in caspase‐3b from zebrafish and appears to be a general strategy from the scaffold of the common ancestor. We show that mutations that break the salt bridge in human caspase‐3 results in relaxed specificity compared to wild type protein.In conclusion, protein conformational ensemble and enzyme specificity are two important ways to regulate caspase activity. Gaining the knowledge with regards to the mechanisms with which these proteins and their related families evolved may assist in uncovering how and why the various functions evolved in these proteins.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 Novel Inexpensive Media for the Growth of Escherichia coli Expressing a Recombinant Lipase
Authors: Okpuzor JOY , Bello Adebayo J , Igwo‐Eziekpe Miriam , Toye Voke , Adewole Adeniyi R , Agbabiaje Olayinka 
Published: June 2021

There is an ever increasing demand for industrial enzymes for different applications to satisfy the needs of the world's ever‐growing population. Nigeria currently is at a precipice and urgent measures are needed for self‐sustainability in a distressed economy for most of her industrial processes. The progression of synthetic biology techniques has been a continuous driving force for sustainable development on industrial enzymes. Lipases (EC 3.1.1.3) are important biocatalysts due to their utility in various industrial applications. For industrial processes, it is necessary to define the best set of conditions for cell growth, protein induction and recovery. This work therefore investigates the conditions necessary for optimum expression of novel recombinant lipase (sourced originally from marine sponge Haliclona simulans metagenome) in Escherichia coli.A codon‐optimized gene that codes for lipase Lpc53E1, a member of the group VIII family of lipases from Haliclona simulans was heterologously expressed in E. coli by induction of graded concentrations (0.1 mM, 0.25 mM, 0.5 mM and 1 mM) of Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) when the cell's density (OD600) reached 0.4, 0.5, 0.6 and 0.7 accordingly. Analysis of the SDS‐PAGE revealed the presence of lipase with an approximate molecular weight (MW) of 44 kDa. The recombinant lipase exhibited a higher halo‐tolerance and thermostable properties up to 60°C when the cell density reached 0.7 OD600 after induction with 0.5 mM IPTG. A novel and newly formulated inexpensive media for cell growth of the E. coli culture showed distinct colonies comparable to the LB control. This suggests an alternative and cheaper media for this microbial culture. Thus this inexpensive media from locally sourced materials would reduce the cost of operations in industries requiring lipase in developing countries.Support or Funding InformationThe grant for this research was awarded by the Central Research Committee, University of Lagos, Nigeria (CRC, 2017/01)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization Chemical Compounds that Inhibit Lysyl‐tRNA Synthetase from Pseudomonas aeruginosa
Authors: Balboa Samantha , Hu Yanmei , Bullard James M 
Published: June 2021

IntroductionPseudomonas aeruginosa is an opportunistic pathogen and a major cause of nosocomial infections. The enhanced ability of P. aeruginosa to acquire resistance to most antibiotics places it in a special class of pathogens, the so‐called “superbugs”. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis and are validated targets for development of antibacterial agents. The gene encoding P. aeruginosa lysyl‐tRNA synthetase (LysRS) was cloned and the resulting overexpressed protein was purified to 98% homogeneity.Methods and ResultsAminoacylation assays were used to measure kinetic parameters for interactions of LysRS with tRNA and ATP:PPi assays were used to determine the kinetic parameters for interactions with lysine and ATP. The KM values for interaction with lysine, ATP and tRNALys were determined to be 45.3, 627, and 3.3 μM, respectively. The kcatobs values were calculated to be 13.3, 22.8, and 0.35 sec−1. This resulted in kcatobs/KM values of 0.29, 0.036, 0.11 s−1μM−1. P. aeruginosa LysRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen natural product (800) and synthetic (890) compound libraries to identify compounds with the ability to inhibit the function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified and inhibitory activity was confirmed. These compounds inhibited the ability of LysRS to aminoacylate tRNALys with IC50 values of 35.3, 66.9, and 36.4 μM, respectively. The minimum inhibitory concentration (MIC) was determined for each compound against a panel of ten pathogenic bacteria. Each compound exhibited broad spectrum activity. Time‐kill studies indicated a bacteriostatic mode of inhibition for each compound against cultures of S. aureus, however in cultures of M. catarrhalis, BT06F11 and BT08F04 was observed to be bactericidal. When tested in cultures of human embryonic kidney 293 (HEK‐293) cell lines using MTT cell proliferation assays, BT06F11 was not toxic at any concentration tested. BM01D09 was observed to only inhibit human cell growth at high concentrations (CC50 = 370 μM). However, BT08F04 was toxic at much lower concentrations (CC50 = 61 μM).ConclusionLysRS from P. aeruginosa was characterized and developed into a screening platform to identify potential anti‐bacterial compounds. Three compounds were identified as having inhibitory activity against the aminoacylation of LysRS. The compounds were then characterized for inhibition of enzymatic activity, bacterial growth and toxic effects in human cell cultures.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.