bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single Amino Acid Substitutions Affect the Stability of the Dimer Interface of the E. coli β Clamp
Authors: Baez Joe 
Published: June 2021

The β sliding clamp is an E. coli DNA replication cofactor and a subunit of the DNA polymerase holoenzyme responsible for tethering the DNA polymerase to the DNA primer template to prevent polymerase dissociation, increase replication processivity, and mediate polymerase switching to enhance DNA damage tolerance. The circular protein is a three‐domain homodimer arranged in a head‐to‐tail orientation with an ability to exist in an open or closed conformation at the dimer interface found between domains I and III of each subunit. The clamp's ability to open and close controls associations with DNA and dictates the efficiency of bacterial replication. In this study we introduced mutations proposed to stabilize or destabilize the dimer interface of the clamp in order to probe the mechanism of clamp opening, and find potential target residues for improving or inhibiting clamp function and DNA replication. The ability to support replication in vivo, thermostability, clamp opening function, and effect on polymerase processivity have been studied for each mutant clamp. The results support the finding that specific mutations to the L82 residue found at the β clamp's dimer interface severely destabilize the clamp and diminish E. coli's ability to replicate.Support or Funding InformationSupported by NSF REU CHE‐1262734.
Llama Hemoglobin Binding to Heterologous Haptoglobins
Authors: Vanderpuye Oluseyi Adewale , Troutman Kicheryltus , Kellam Netanya , Dunn Christina 
Published: June 2021

The plasma protein haptoglobin binds hemoglobin released from lysed erythrocytes. The complex is internalized through the macrophage membrane protein CD163 thus preventing pathological oxidation of biological molecules and tissues. Hemoglobin primary structures can be as little as 50% homologous among species. Haptoglobin also shows structural variation among species and a genetic polymorphism is found in humans and not in other mammals. The goal of this study was to examine the extent to which hemoglobins can bind to haptoglobins from different species. The llama is adapted to high altitude and its hemoglobin might thus differ from those of many other mammals. By using non‐denaturing agarose gel electrophoresis, the charge properties and binding to human haptoglobin was compared for llama, human, dog, pig, horse and goat hemoglobins.Llama hemoglobin was found to be more positively charged than the other mammalian hemoglobins. All hemoglobins tested showed a band of increased anodal mobility when incubated with human plasma which suggested formation of hemoglobin‐haptoglobin complexes. For one plasma sample there was only one new band whereas for a second sample two new anodal hemoglobin bands were formed. This suggested the ability of the different species hemoglobins to bind to different genetic polymorphisms of human haptoglobin. When the different hemoglobins were incubated with purified haptoglobin variants Hp 1:1 and Hp 2:2, one new anodal hemoglobin‐containing band was seen. The new band in the Hp 1:1 samples had higher mobility than those in the Hp 2:2 samples. In all cases the llama hemoglobin‐haptoglobin samples had lesser mobility than those of the other mammals. Because incubation with one of the plasma samples produced two new bands rather than one as with the Hp 1:1 and Hp2:2 it is surmised that this plasma sample had the Hp 2:1 variant and that all animal hemoglobins tested can bind the three genetic variants of human haptoglobin although this variation is regarded to be absent in non‐human species. It is concluded that the binding sites on hemoglobin and haptoglobin for each other have been evolutionarily conserved despite differences in primary structure and marked difference in the charge of llama hemoglobin.Support or Funding InformationThe research was supported by Furman University Research Experience for Undergraduates 2016 session, NSF grant NSF CHE‐1460806 – awarded to K. Buchmueller and T. Hanks at Furman University and by Albany State University, Center for Undergraduate Research
Developing Monovalent Ion Parameters for the Optimal Point Charge (OPC) Water Model
Authors: Clark Daniel E , Dood John C , Krueger Brent P 
Published: June 2021

Molecular dynamics (MD) simulations are used to model the structure and movement of macromolecules. The gold standard for MD is to explicitly include water molecules using one of several standard models. Recently, a new water model, Optimal Point Charge (OPC), has been developed with simulation performance that compares better to experiment than existing models in its class (Izadi, Anandakrishnan, Onufriev, J. Phys. Chem. Lett., 2014, 5, 3863‐3871). For this new water model to be useful, Lennard‐Jones (LJ) parameters must be developed for at least a few monovalent ions. In this study MD simulations were used to develop these parameters. Preliminary results are presented including: extensive convergence testing of Hydration Free Energy, Lattice Constants (LC), and first peak position of radial distribution functions (RDF's); as well as surfaces showing the dependence of the RDF and LC on various LJ parameters within the OPC water model.Support or Funding InformationNational Science Foundation awards 1058981 and 1039925.
Specificity studies of the aromatic desulfinase, 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) from Nocardia asteroides A3H1
Authors: Hoang Dylan Minh , Smith Emily , Watkins Linette 
Published: June 2021

2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) is a desulfinating enzyme that catalyzes the final carbon‐sulfur bond cleavage reaction in the biodesulfurization (BDS) of dibenzothiophene (DBT), a major organosulfur compound in fossil fuels. Homology studies suggest that Alanine 195 and Alanine 200 are important in determining the specificity of the active site. Point mutations were created to determine the effect of switching A200 to an Arginine (A200R), as well as switching A195 to an Arginine (A195R) and switching both A195 and A200 to Arginine (A195/200R). By altering amino acids in the active site of DszB, the effects of amino acid A195 and A200 on the active site specificity can be determined. Codon optimized DszB was cloned into pTAC‐MAT‐Tag‐2, overexpressed in E. coli, and purified using a Ni2+ column. Enzyme stored in aliquots containing 15% DMSO was stable at −80 °C for several weeks. The natural substrate 2‐(2′‐hydroxyphenyl)benzenesulfinate (HPBS) was tested, along with the smaller ring analogs 2‐(2′‐hydroxyphenyl)enthen‐1‐sulfinate (HPES) and benzenesulfinate (BS), in order to test whether the active site prefers one, two or three rings. The kinetic data of the codon optimized DszB showed a Km of 3.71 ± 0.60 μM and kcat of 52.8 ± 5.49 hr−1. The specificity of DszB‐A3H1‐A200R showed a change in specificity, preferring HPES over BS.Support or Funding InformationNational Science Foundation (CHE 1062629)
Factors that Influence Recombinant Lysine Deacetylase Activity
Authors: Imbraguglio Samantha A , Hylton Brandon J , Toro Tasha B , Watt Terry J 
Published: June 2021

Lysine deacetylases (KDACs) are the enzymes that reverse the post translational modification of acetylation, resulting in an unmodified lysine residue and acetate. KDACs play a critical role in the cell and changes to their activity have been linked to diseases. In order to characterize KDAC activity in a biologically relevant fashion, in vitro assay buffer should ideally mimic the in vivo environment. In this project, our research objective is to determine the components of buffer that influence KDAC activity while remaining biologically relevant and cost efficient. The starting buffer used consisted of phosphate, glycerol and potassium chloride. The buffer parameters varied included the viscosity, buffering agent, and metabolite concentrations. The effect of these alterations was measured through the use of fluorescence‐based activity assays. KDAC8 was the primary enzyme used to observe the effects of these variations. Although the variation of most of these parameters either did not affect KDAC8 activity or decreased it, the addition of a metabolite mixture to the reaction slightly increased KDAC8 activity. In order to determine which metabolite caused the activity increase, citrate was selected for further investigation due to its metal chelating properties. KDAC8 and other Class IIa KDACs require a metal cation at their active site to function. In excess, some of these metals can also bind to an inhibitory site on the enzyme. When citrate was added to the reaction in the presence of excess zinc, activity was partially restored. However, the addition of citrate alone had no effect on KDAC8 activity. In conclusion, a minimal phosphate buffer optimizes KDAC8 activity while remaining biologically relevant and cost efficient. Furthermore, the addition of citrate is a biologically relevant method to ensure that excess trace metals are not inhibiting KDAC8.Support or Funding InformationU.S. Army Research Laboratory and the U.S. Army Research Office DoD W911NF‐13‐1‐0129, Summer Undergraduate Research Fellowship and the Center for Undergraduate Research at Xavier University of Louisiana.
Expression, Purification, and Characterization of Codon Optimized and mutant variations of DszB from N. asteroides
Authors: Gumpf Jacob John , Idrizi Keid , Watkins Linette 
Published: June 2021

2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase (DszB) catalyzes the final carbon‐sulfur bond cleavage reaction in the rate‐limiting step in the biodesulfurization of dibenzothiophene, a recalcitrant organosulfur compound found in fossil fuels. The dszb gene from Nocardia asteroides (A3H1) was codon‐optimized and overexpressed in E. coli. Expression was examined by FT‐IR and activity assays. His‐tagged DszB expressed from a codon‐optimized gene requires co‐expression with GroEL and GroES to fold properly. The DszB enzyme was purified using a Ni2+ column, and a kcat of 52.8 ± 5.49 hr−1 and a Km of 3.71 ± 0.60 μM was determined. Enzyme stored in aliquots containing 15% DMSO was stable at ‐80 °C for several weeks. DszB has optimal activity at 35 °C but is not stable for longer than 30 minutes at room temperature. Three mutated enzymes were expressed, purified and characterized. The A195R, A200R, and A195R/A200R DszB mutant altered kinetic parameters, increased temperature optimum, and increased temperature stability.Support or Funding InformationJames Madison University, National Science Foundation (CHE 1062629)
Regulation of the Arabidopsis thaliana Ca2+‐dependent protein kinase, CPK28, by autophosphorylation and Calmodulin‐binding
Authors: Bender Kyle W. , Zielinski Raymond E , Huber Steven C. 
Published: June 2021

Ca2+dependent protein kinases (CPKs in plants, CDPKs in protists) fulfill central roles as signal transducers downstream of myriad biotic and abiotic stress stimuli in plant systems. Related to Ca2+/CaM‐dependent protein kinases from the animal lineage, CPKs consist of a conserved Ser/Thr protein kinase domain tethered to a Ca2+‐binding regulatory domain known as the CDPK‐activation domain (CAD). CPK activation by Ca2+‐binding has been extensively studied, but the role of autophosphorylation and binding of interacting proteins in CPK regulation is less well understood. Using recombinant proteins, we tested the hypothesis that plant CPKs are regulated by Calmodulin (CaM) binding, and assessed the impact of autophosphorylation on Ca2+‐activation of a CPK involved in plant immunity. CaM specifically bound CPK28 and binding inhibited CPK28 peptide kinase activity. To test the effect of autophosphorylation on Ca2+‐activation of CPK28, we produced dephosphorylated and in situ autophosphorylated forms of the kinase in E. coli. Ca2+‐sensitivity of peptide kinase activity was tested at different Ca2+ concentrations spanning the physiological range (~0.1 to 1 μM in plants). Dephosphorylated and autophosphorylated CPK28 were both responsive to Ca2+ in the physiological range, however, the autophosphorylated kinase was activated more robustly and at lower levels of Ca2+ compared to the dephosphorylated protein. Thus autophosphorylation serves as a mechanism to ‘prime’ Ca2+‐sensitivity of CPK28. Collectively, our analyses reveal subfunctionalization of plant CPKs in terms of CaM‐binding and suggest the possibility for multiple modes of regulation of CPK28 in vivo by Ca2+, autophosphorylation, and CaM‐binding. Importantly, our study provides mechanistic support for the Ca2+‐sensitivity priming hypothesis for Ca2+‐signaling specificity in plant systems. Current work is focused on identifying the phosphosite(s) mediating Ca2+‐sensitivity priming and understanding the in vivo significance of both autophosphorylation and CaM‐binding.Support or Funding InformationFunding for this work was provided by the National Science Foundation (grant number IOS‐1354094 to REZ and SCH) and the USDA Agricultural Research Service (to SCH).
Identification of Novel Variants of the CB1 Cannabinoid Receptor in Cancer Cells
Authors: Yarbrough Azure , Pyrek Sebastian , Wood Lindsey , Urbaniak Alicja , Bush John , Prather Paul , Radominska‐Pandya Anna 
Published: June 2021

Cannabinoid receptors (CBRs) are G‐protein coupled receptors (GPCRs) that are activated by the binding of cannabinoid ligands. It has recently been shown that CB1, found mostly in the brain, and CB2, found mostly in white blood cells and the spleen, have been found to be overexpressed in different cancer models. It has been found that besides the wild‐type CB1, different, shorter versions of the CB1 protein, called CB1 splice variants, are also found in the human brain. However, their function is unknown and requires further investigation. Currently, there is no information available on the expression of these CB1 variants in cancer. Preliminary results using semi‐quantitative reverse transcription polymerase chain reaction (RTPCR) utilizing primers corresponding to CB1 brain variants was used to demonstrate that in several cancer lines of brain, pancreatic, and breast tissue, there were CB1 transcripts of varying sizes were, in fact, being expressed. High, inter‐cancer variations were observed; some cancer lines did not possess any of the constructs while others expressed brain specific variants, and some lines even expressed other potential variants of differing lengths. Based upon our analysis of the results, we hypothesize that in these cancer cells, variants identical to those observed in the brain were being expressed, and that different, cancer‐specific CB1 variants were also being expressed. Currently, we are identifying numerous PCR products obtained by cloning and sequencing. Eventually, the isolated novel variant CBR1 genes will be overexpressed in mammalian cells, and functional analysis of each variant receptor conducted. If successful, the physiological significance of these splice variants and their use as potential novel targets for cannabinoid anti‐cancer drug developments will be determined.Support or Funding Information[DoD‐W81XWH1110795, UAMS TRI ABCRP to AR‐P, P20 RR‐16460 INBRE, NIH/NIDA DA039143 to ARP and PLP]
New Delhi Metallo‐Beta‐Lactamase Variants NDM‐4 and NDM‐12 from E. coli Clinical Isolates Exhibit Increased Activity and Stability
Authors: Williams Cameron , VanPelt Jamie , Poth Robert , Cottingim Kelsey , Stewart Alesha , Nix Jay C , Fast Walter , Tierney David L , Crowder Michael W , Page Richard C 
Published: June 2021

Metallo‐Beta‐Lactamases (MBLs) confer resistance to carbapenems, cephalosporins, and penicillins in several clinically relevant Gram‐negative bacteria including Acinetobacter, Pseudomonas aeruginosa, and many Enterobacteriaceae. New Delhi Metallo‐Beta‐Lactamases (NDMs) are amongst some of the most worrisome and prevalent MBLs. This family of MBLs is capable of hydrolyzing all generations of bicyclic beta‐lactams. Two new NDM variants, NDM‐4 and ‐12, have recently been identified in E. coli clinical isolates. NDM‐4 contains an M154L substitution while NDM‐12 bears both M154L and G222D mutations. Considering the clinical significance of Gram‐negative species possessing these enzymes, there is a need to characterize the structures and activities of these variants in order to provide a better basis for drug discovery efforts. We have found that NDM‐4 and NDM‐12 possess higher hydrolytic activity compared to NDM‐1. Our differential scanning fluorimetry studies suggest that the M154L mutation confers increased thermal stability and improved affinity for active site catalytic Zn metal ions. The additional G222D mutation of NDM‐12 further improves stability and Zn affinity. M154L, found in the secondary coordination sphere, accounts for changes in stability by acting as a buttress to the loop containing His122, a direct Zn coordinating residue. G222D, on the other hand, is a mutation located in a loop just above the active site approximately 10 Å from the active site zinc ions. Together, our crystal structures of these NDM variants, including the first known structure of NDM‐12, biochemical analyses, and biophysical analyses provide a basis for understanding the roles of key residues surrounding the active site within NDM variants. A better understanding of clinically relevant NDM variants is essential in the fight against MBL‐mediated antibiotic resistance. Our structural, biochemical, and biophysical studies will help to inform drug design efforts to develop new MBL inhibitors.Support or Funding InformationThe authors acknowledge financial support from the US National Institutes of Health (Award No. R01 GM111926 to RCP, DLT, MWC, and WF) and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.
The Role of Good Governance and an Enabling Environment in Delivering Good Nutrition
Authors: Kennedy Eileen Theresa 
Published: June 2021

It is generally agreed that good governance is essential for effective policy and program development and implementation. There is, however, no formulaic approach to governance that can be applied universally. Using data from Ethiopia this paper provides a analysis of factors contributing to effective governance in the design and implementation of a multi sector nutrition program in Ethiopia.Support or Funding InformationFunded by the United States Agency for International Development.
Male testicular development is affected by estrogens but not altered in neonatal piglets receiving soy infant formula
Authors: Mercer Kelly E , Chen Celine , Hennings Leah , Dawson Harry , Yeruva Laxmi , Ronis Martin , Badger Thomas 
Published: June 2021

Soy infant formula contains many phytochemicals, including phytoestrogens, which are structurally similar to estradiol (E2). As a result of their potentially estrogenic properties, soy infant formulas have been the subject of concern with regards to reproductive toxicity. To address these concerns regarding male reproductive development, we fed male piglets a soy‐based infant formula, a dairy milk‐based infant formula, milk formula supplemented with E2, or milk formula supplemented with putative estrogenic soy phytonutrient genistein (GEN), in comparison with piglets receiving sow's breast milk from postnatal day 2–21. In the E2 supplemented group, serum testosterone and testicular weight were significantly reduced compared to the milk formula‐fed and sow's milk controls. Testis tubule area, Sertoli cell and germ cell numbers were also increased in the E2 supplemented group compared to milk formula controls, P<0.05. When compared to the sow's milk control, RNAseq analysis in testes showed that E2 supplemented milk formula altered expression of 2012 testicular genes (±1.5 fold, P<0.100). Functional annotation analysis indicated up‐regulation of several pathways in the E2 supplemented group, including canonical Wnt signaling, cell differentiation and migration, angiogenesis and spermatogenesis. E2 also down‐regulated cholesterol and androgen synthesis pathways. Real‐time RT‐PCR confirmed the decrease in steroidogenic acute regulatory protein (Star) mRNA in the E2 supplemented group, suggesting a potential mechanism for the observed decrease in serum testosterone in this diet group. In contrast, GEN supplementation of milk formula altered 104 genes, soy formula 49 genes, and the pathways impacted had no clear links to testes function or sex organ development. These data show that early infant diet exposure to phytoestrogens in soy‐based infant formula are not estrogenic in the male reproductive system.Support or Funding InformationThis work was funded by USDA‐ARS Project 6026‐5100‐010‐05S.
DUSP5 Functions in a Feedback Loop to Suppress TNFα‐Induced ERK1/2 Phosphorylation and Inflammation in Adipocytes
Authors: Habibian Justine S , Jefic Mitra , Schepps Abbie , Bagchi Rushita A , McKnight Robert A , McKinsey Timothy A , Morrsion Ron F , Ferguson Bradley S 
Published: June 2021

Adipose tissue inflammation is a central pathogenic element that links obesity to insulin resistance and type II diabetes. Evidence demonstrates that extracellular signal‐regulated kinase 1/2 (ERK1/2) acts as a key signaling molecule that can inhibit insulin signaling in the cytosol and promote pro‐inflammatory gene expression in the nucleus. Recent evidence demonstrates that dual specificity phosphatases (DUSPs) dephosphorylate (i.e. inactivate) ERK1/2, thus inhibiting inflammation and insulin resistance. In this report, we observed that the ERK1/2‐specific phosphatase, DUSP5, was induced in response to high fat diet‐induced obesity. Moreover, increased DUSP5 gene expression correlated with increased tumor necrosis factor α (TNFα) gene expression in response to high fat diet‐induced obesity over time. Based on these findings, we examined a role for DUSP5 on ERK1/2 dephosphorylation and pro‐inflammatory gene expression in 3T3‐L1 adipocytes exposed to TNFα. We demonstrate that DUSP5 functions as a negative regulator of TNFα‐mediated ERK1/2 phosphorylation leading to inflammation. We report a rapid phosphorylation and subsequent dephosphorylation of ERK1/2 in 3T3‐L1 adipocytes in response to TNFα stimulation; concomitant to ERK1/2 dephosphorylation we further observed increased DUSP5 expression in 3T3‐L1 adipocytes stimulated with TNFα, suggesting a role for DUSP5 in the regulation of ERK1/2 signaling. Consistent with this hypothesis, genetic knockdown of DUSP5 led to sustained ERK1/2 activation and enhanced inflammatory gene expression in response to TNFα. Mitogen‐activated protein kinases (MAPKs) have been reported to regulate DUSPs, thus creating a feedback inhibitory loop. Indeed, we report that inhibition of both ERK1/2 and c‐Jun N terminal kinase (JNK) attenuated TNFα‐induced DUSP5 expression. Further loss‐of‐function studies are underway to examine a role for DUSP5 on adipose tissue inflammation in vivo using DUSP5 knockout mice in response inflammatory stimuli and/or diet‐induced obesity. Collectively, these data support the postulate that DUSP5 functions in the feedback inhibition of ERK1/2 signaling in response to inflammation and that loss of DUSP5 function exacerbates the inflammatory response in adipocytes.Support or Funding InformationThis work is supported by the USDA National Institute of Food and Agriculture (Hatch‐NEV00727). Core facilities used for Research reported in this publication was supported by National Institute of General Medical Sciences of the National Institutes of Health (P20 GM103554).