bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Preliminary Study on Serum Proteomics in Yin‐Deficiency‐Heat Syndrome Rats using iTRAQ‐coupled 2DLC‐MS/MS
Authors: Liu Changming , Mao Liangen , Ping Zepeng , Jiang Tingting , Wang Chong , Chen Zhongliang , Xu Dandan , Li Ji‐Cheng 
Published: November 2023

This study aimed to observe the expression profiles of serum proteins and to discover potential biomarkers in Yin‐deficiency‐heat (YDH) syndrome rats induced by hot and pungent Chinese herbs. Totally 50 Sprague–Dawley rats were randomly divided into the normal group and the YDH model group. The concentrated decoction of three Chinese herbs such as aconite root, dried ginger, and cinnamon were applied to establish YDH model. The signs and symptoms of rats were observed dynamically. All rats were sacrificed at the end of the 4th week and serum was collected by using carotid artery retaining needle. Serum protein profiles in YDH rats and normal rats were investigated by iTRAQ‐coupled 2DLC‐MS/MS technique. Compared with the normal group, rats in the YDH model group showed obvious YDH symptoms, including dry hair, restlessness, and weight loss. Rats in the YDH model group gained weight more slowly (p<0.01) than did those of the normal group, and the thymic index decreased significantly (p<0.001) in the YDH model group. When setting quantification ratio of >1.25 as up‐regulated threshold and <0.8 as down‐regulated threshold, a total of 165 differentially expressed proteins (110 up‐regulated proteins and 55 down‐regulated proteins) were identified in the serum of YDH syndrome rats. Significant differences in protein Kininogen (KNG), Apolipoprotein C‐III (ApoC‐III) and Paraoxonase1 (PON1) were found between the YDH model group and the normal group by ELISA. Our work provided valuable insights into the changes of the protein expression in YDH syndrome rats, and identified 3 differentially expressed proteins (KNG, ApoC‐III and PON1) that can be used as potential biomarkers for diagnosing YDH syndrome.Support or Funding InformationThis work was supported by grants from National Basic Research Program of China (No.2014CB543002), National Special Sci‐Tech Projects (No. 2012ZX10005001‐006), and National Natural Science Foundation of China (No.81273882, NO. 81573709).
A Phosphoglycolate Phosphatase Virulence Factor From Staphylococcus aureus
Authors: Moreno Isreal , O'Handley Suzanne F 
Published: November 2023

Staphylococcus aureus is a major cause of hospital‐acquired infections. The multi‐drug resistant nature of certain S. aureus strains makes the discovery of new S. aureus drug targets vital. A newly discovered virulence factor from S. aureus was described as a homolog of NagD from E. coli, a member of the nitrophenyl phosphatase family of the HAD (Haloacid Dehalogenase) superfamily. We cloned the gene, expressed and purified the protein, and determined its activity. This virulence factor is not an ortholog of NagD UMPase from E. coli, but rather a phosphoglycolate phosphatase (PGPase). If phosphoglycolate accumulates in the cell, it will inhibit the glycolytic enzyme triose phosphate isomerase (TPI). In S. aureus, TPI also serves as an adhesion molecule that can bind to host cells via sugar‐side chains; phosphoglycolate would interfere with this adhesion process and thus make it harder for S. aureus to infect a new host cell. Thus, this S. aureus PGPase may act as a virulence factor by degrading the TPI inhibitor phosphoglycolate. We have subcloned this PGPase into a His‐tag vector, purified the protein using nickel affinity and size exclusion chromatography, and have characterized the enzyme's enzymatic activity, optimal conditions, and kinetics.Support or Funding InformationSupported by NIH AREA grant GM066715 and RIT Graduate Student Research Grant
Transition‐State Analogs as Effective Inhibitors Against Helicobacter pylori 5′‐Methylthioadenosine Nucleosidase
Authors: Payan Angel , Hirsch Brett , Schramm Vern 
Published: November 2023

Helicobacter pylori is a gram‐negative pathogenic bacteria responsible for ulcer progression and contributing to stomach cancer in humans. We targeted 5′‐methylthioadenosine nucleosidase (MTAN) in the futalosine pathway, a critical enzyme in menaquinone metabolism. A potent inhibitor, BTDIA, was previously designed from the MTAN transition state. It is a powerful inhibitor of bacterial growth. This study evaluates a small library of compounds stemming from this initial design. Through in vitro and culture inhibition assays, we compare this new series of potential inhibitors against the lead compound BTDIA. Future studies aim to test other analogs as potential therapeutic agents for H. pylori as well as to continue pre‐clinical trials with the current library of compounds for treatment of H. pylori infections.Support or Funding InformationAlbert Einstein SURP Program
Alpha‐glucosidase Inhibitory Activity of Mormodica charantia
Authors: OBIH PATIENCE OGECHI , NGUYEN ANTHONY , OBIH JOHN‐CLIFFORD AGBASI 
Published: November 2023

Diabetes mellitus is a metabolic endocrine disorder characterized by high blood glucose that has become global and is ever increasing in prevalence. One of the approaches for treating diabetes is by regulating the postprandial blood glucose via inhibition of alpha‐glucosidase. Alpha‐glucosidase is an enzyme that metabolizes complex sugars converting them to absorbable monosaccharide. Alpha‐glucosidase inhibitors like acarbose and miglitol are already in the market for the treatment of diabetes, but they have some draw backs because of their side effects. The aim of this study is to evaluate Mormodica charantia as an inhibitor of alpha‐glucosidase. To this effect, ethanol extract of Mormodica charantia was assayed in vitro by the method of Ramirez et al., 2012 for alpha‐glucosidase inhibitory activity in a 96‐well micro titer plate. This was read on a spectrophotometer at 400 nm using acarbose as a positive control. Our results indicated that Mormodica charantia possesses alpha‐glucosidase inhibitory action with an EC50 of 0.306 ±0.06113 mg/ml whereas acarbose produced an EC50 of 0.0.0000143 ± 6.32 mg/ml in its pure form. In this study, Mormodica charantia has demonstrated significant alpha‐glucosidase inhibitory activity suggesting that it has a potential for regulating postprandial blood glucose.Support or Funding InformationCenter of Excellence (COE)
Recombinant Production of a PLA2 Inhibitor: Implications for Production of Venom Inhibitors
Authors: Elmashhady Hady , Loh Chin Yi , Michael Chris , Ma Minh , Sahudin Sydney , Schutz Victoria 
Published: November 2023

Globally, bites from venomous snakes cause a wide variety of health issues, ranging from localized tissue damage to paralysis and death. However, such events are fully preventable with the timely use of anti‐venoms. Thus, increasing the availability of these anti‐venom treatments is essential to decreasing the mortality rates of those bitten by venomous snakes. Alternatives to anti‐venoms include venom inhibitors produced by the venomous snakes themselves. Many pit vipers, including the South American Lachesis muta, express the toxic enzyme, Phospholipase A2 (PLA2) in its venom complex. The snake prevents harmful effects of its own venom by expressing an inhibitor, beta‐phospholipase inhibitor (βPLI), to counteract the activity of PLA2 within the snake. Here we show the ability to clone both genes into pET expression vectors for recombinant expression in E. coli. An active fraction of the PLA2 was soluble and purified by nickel affinity chromatography. Measurements of βPLI inhibition of in vitro activity of PLA2 is underway.Support or Funding InformationThis research was conducted and funded by the University of Missouri‐Columbia student‐led organization, Mizzou Synthetic Biology Research Group (Mizzou Syn BRG).
Cloning and Purification of Human Prolyl Endopeptidase (hPEP) for Biochemical Comparison to Sunn Pest Prolyl Endoprotease (spPEP)
Authors: Moore Travis K 
Published: November 2023

Eurygaster integriceps Puton, common name sunn Pest, is one of the primary sources of wheat crop wastes in North Africa, Middle East, and Eastern Europe. It feeds by injecting the wheat grain with an enzyme characterized as prolyl endoprotease (spPEP) that breaks down Gluten, the wheat's main constitutive protein necessary for bread production (Darkoh et al., 2010). Previously, it has been shown that peptides isolated from Lactobacillus hydrolysates of caseins in bovine milk are able to inhibit mammalian PEP in colon cells, as well as bacterial PEP (Juillerat‐Jeanneret et al., 2010). While recombinant versions of these peptides are also potential inhibitors of the spPEP, their specificity must be tested against hPEP. The primary objective of this study was to clone hPEP into the same expression vector as spPEP in order to compare hPEP to spPEP with regards to substrate binding, recognition, and inhibition. Initially, hPEP was PCR amplified in order to incorporate the 5′ and 3′ ends necessary for ligation independent cloning (LIC) into the expression vector. This was then expressed in BL21(DE3)/pTFs and purified on a nickel column. Future studies will include comparing inhibition between hPEP and spPEP using 16 peptides of varying length that contain the non‐allergenic (as demonstrated by Ruiter, 2005) inhibitory sequence LNENLLRFFVAPFPEVFG, isolated from bovine αS1 casein.Support or Funding InformationFunding: Office of Research and Sponsored Projects, SFASU and Ed and Gwen Cole, Nacogdoches TX.
Mechanism and Catalytic Potential of a Cytochrome P450 (OleT) that Catalyzes C‐C Bond Scission
Authors: Hsieh Chun H , Huang Xiongyi , Keys Carson L , Groves John T , Austin Rachel N , Makris Thomas M 
Published: November 2023

OleT (CYP152L1) is a recently discovered cytochrome P450 that is structurally similar to peroxide dependent P450s that catalyze fatty acid hydroxylations. OleT has been shown to catalyze the cleavage of n chain length fatty acids to form n‐1 alkenes through C‐C bond scission of the terminal carboxylate. Using headspace GC‐MS and FTIR with isotopically labeled substrates, we have confirmed that OleT metabolizes eicosanoic acid to 1‐nonadecene, forming carbon dioxide as a co‐product of the reaction. No oxygen deriving from peroxide is observed in either product. In order to probe the mechanistic deviation of OleT from P450 peroxygenases, we have employed a “decoy” approach where a short chain length fatty acid provides the acidic group necessary for peroxide heterolysis, and also allows access of small molecule probe substrates. Using this approach, we confirm that OleT also catalyzes epoxidation and hydroxylation reactions. Products derived from the metabolism of radical clock substrates directly show that OleT is capable of efficient oxygen rebound with radical lifetimes that are similar to other P450 monooxygenases. A site‐directed mutant, in which the acidic group is instead provided by the protein framework, circumvents the need for a substrate carboxylate, and shows efficient metabolism of a wide range of hydrocarbon substrates.Support or Funding InformationThis work is supported by startup funds from the University of South Carolina, and funding from the U.S.C. Office of Research through the Aspire program to T.M.M.
Peptides Mimicking the Reactive Center Loop (RCL) as Potential Inhibitors of Serpin‐3 in Manduca sexta
Authors: Li Miao , Kanost Michael R. 
Published: November 2023

Insects employ innate immune responses to eliminate or kill invading pathogens and parasites. Manduca sexta, the tobacco hornworm, is widely used as a model insect for investigating innate immunity, including defenses mediated by serine protease cascade pathways. These pathways are regulated by serpins (serine protease inhibitors). Through forming inactive irreversible complexes with targeted proteases, serpins efficiently block activity of serine proteases in M. sexta plasma after the cascades are triggered by exposure to microorganisms. The hydrolysis of the scissile P1‐P1’ bond in a serpin reactive center loop (RCL) by a protease leads to a large conformational rearrangement, in which the RCL inserts into β‐sheet A in the serpin, resulting in the formation of an inactive complex between the serpin and protease. Previous studies on human antithrombin, α1‐protease inhibitor, and plasminogen activator inhibitor‐1 showed that short peptides mimicking N‐terminal residues of the RCL can insert into β‐sheet A and block the formation of covalent complexes, resulting in loss of serpin inhibitory activity. We are examining the use of serpin RCL peptides in M. sexta to block the function of hemolymph serpins, as a strategy for investigating biological roles of individual serpins. As a model for these studies, we are using M. sexta serpin‐3, which has been well studied and confirmed to effectively inhibit prophenoloxidase‐activating proteinase 3 (PAP3). We designed four peptides, varying from five to eight amino acids in length, whose sequences correspond to the N‐terminal region of M. sexta serpin‐3 RCL (P14 to P7), and synthesized them via solid phase peptide synthesis. The size and purity of these peptides was tested by MALDI and HPLC. Recombinant proPAP3 was expressed in Sf9 cells using a baculovirus system, followed by purification via several chromatography steps. Purified proPAP3 was activated by incubation with its activating protease, HP21. Recombinant serpin‐3 expressed in E. coli was purified by nickel‐affinity chromatography. After incubating serpin‐3 with the RCL peptides, inhibition of PAP3 by serpin‐3 was reduced, with more than 90% loss of inhibitory activity after treatment with the P14‐P9, P14‐P8, or P14‐P7 peptides. This result suggests that the RCL peptides are acting in an insect serpin as demonstrated previously with human serpins to disrupt serpin inhibitory function. Future experiments will focus on optimizing the use of RCL peptides for investigating the functions of serpin‐3 and other hemolymph serpins as regulators of proteases in immune cascades of M. sexta.Support or Funding InformationSupported by NIH grant GM041247
N‐terminal Extracellular Beta‐1 Adrenergic Receptor Cleavage by Matrix Metalloproteinase 2 (MMP‐2)
Authors: Gonçalves Andrezza Neves , Ramos Junia , Gerlach Raquel Fernanda 
Published: November 2023

Noradrenalin, a primary neurotransmitter of sympathetic nervous system, signals via its interaction with α‐ and β‐adrenergic receptors (ARs), a family of G protein‐coupled receptors (GPCRs) expressed in cells of the cardiovascular system. Matrix metalloproteinase 2 (MMP‐2) has been described to be found in higher levels in aging vessels and heart, and in many cardiovascular diseases, such as hypertension and atherosclerosis. The presence of higher levels of MMP‐2 in heart and arteries in disease conditions has led many groups to propose the involvement of MMP‐2 in the worsening of many cardiovascular conditions. It has been suggested that MMP‐2 may cleave α‐ and β‐adrenergic receptors, and that this proteolytic events might link conditions such as diabetes and metabolic syndrome with increased proteolytic activity in plasma. Nonetheless, so far the cleavage of α‐ and β‐adrenergic receptors by MMP‐2 have not been shown in classical biochemical experiments, which is a fundamental piece of knowledge to further study the possible involvement of MMP‐2 with altered adrenergic responses in cardiovascular diseases. Thus, the aim of this study was to design peptides of the N‐terminal domain of the β1‐adrenergic receptor that had sequences that were likely to be cleaved by MMP‐2 (using in silico analyses) and are physically available on the outside of cells, and to synthetize such peptides for in vitro cleavage assays with rhMMP‐2. The analyses revealed a few cleavage sites on the N‐terminal extracellular domain of the β1‐adrenergic receptor, and based on this 3 synthetic peptides (ASEGSAPLSQWG; PPASEGSAPLSQ; ASPPASLLPPAS) were designed and synthetized. Incubation of these peptides with rhMMP‐2 and appropriate controls under standard conditions for MMP‐2 assays was followed by Mass Spectrometry analyses of the cleavages. Two of the three peptides were cleaved by MMP‐2, with 2 of the selected peptides showing cleavages. This initial study suggests that cleavage of the beta1‐adrenoceptor N‐terminal domain by MMP‐2 possible under in vitro conditions ideal for MMP‐2 activity, and further studies involving precipitation of the receptor in cells that are exposed to different concentration of MMP‐2 need to be carried out, in order to fully understand the ability of MMP‐2 to cleave this receptor in the cellular context.Support or Funding InformationThis study was supported by CAPES
Insights into rate‐limiting substrate dynamics during proteolysis of Kunitz‐BPTI family canonical serine protease inhibitors
Authors: Kayode Olumide , Caulfield Thomas R. , Soares Alexei , Radisky Evette S. 
Published: November 2023

Proteases are ubiquitous enzymes that catalyze the hydrolysis of peptide bonds within protein substrates; they have served as key model enzymes for studying the molecular basis for catalytic power and specificity. Protease substrate specificity is most often defined in terms of linear sequence motifs that flank the cleavage site; however, the natural substrates of proteases are proteins with 3‐dimensional shapes and complex conformational dynamics that are not well represented by 1‐dimensional sequence alone. These structural and dynamical properties can impact recognition and binding of substrates by proteases, as well as the efficiency of catalysis itself. In this study, we explore the importance of substrate structure and dynamics for proteolysis using as our model the cleavage of the Kunitz‐BPTI family of canonical serine protease inhibitors by mesotrypsin. Bovine pancreatic trypsin inhibitor (BPTI), an archetypal serine protease inhibitor of the Kunitz family, has a high affinity substrate‐like interaction with trypsin, yet is cleaved many orders of magnitude slower than other peptide substrates. Mesotrypsin, a trypsin variant, has been shown to hydrolyze Kunitz family inhibitors at accelerated rates; this is especially true of human Kunitz domain inhibitors. Amyloid precursor protein inhibitor (APPI) and amyloid precursor like protein‐2 Kunitz domain (APLP2‐KD), two human Kunitz domain family members, are hydrolyzed by mesotrypsin several hundred times faster than BPTI. Here, we present a new crystal structure of a cleavage intermediate of APLP2‐KD bound to mesotrypsin, refined to 1.4 Å resolution. This newly solved structure reveals a dramatic substrate conformational change that we hypothesize is required during Kunitz domain cleavage. Using this structure along with published APPI and BPTI complexes, we have modeled acyl‐enzyme intermediates of mesotrypsin and we have carried out molecular dynamic simulations to explore the conformational transformation that allows the progression of the hydrolysis reaction. We identify dynamic features of the inhibitor scaffolds, located far from the enzyme binding site, that correlate with observed inhibitor cleavage rates and plausibly explain their differential propensity to undergo both conformational transformation and cleavage. Our findings implicate global scaffold flexibility as a determinant of proteolytic rate, as rigid substrates possessing stabilizing features that render them highly resistant to conformational change are proteolyzed more slowly than more flexible substrates of similar structure.Support or Funding InformationNIH R01 CA154387Mayo Graduate school
Conjugation of BSA onto PEG8 via Strain‐Promoted Azide‐Alkyne Cycloaddition (SPAAC) as a Drug Delivery System
Authors: Hill Jonathan 
Published: November 2023

The hydrophobicity of many drugs poses a major problem for delivery to target tissues. One current technique for hydrophobic drug delivery utilizes Human Serum Albumin (HSA). Hydrophobic binding pockets on HSA protect the molecule from degradation due to redox reactions, hydrolysis and immunogenic response. Bovine Serum Albumin (BSA) provides a cheaper, more readily available alternative to HSA. Structural similarities between binding pockets of both species suggest similar binding capabilities. Conjugation of multiple BSA molecules onto an 8‐arm polyethylene glycol backbone (PEG8‐BSAx) addresses concerns about the effect on osmotic pressure, competition for binding sites, and immunogenicity. Utilizing SPAAC, multiple dibenzocyclooctyl‐functionalized de‐fatted BSA were attached to an azido‐functionalized PEG8. CD demonstrated no loss of secondary structure after PEGylation. Four high molecular weight PEG8‐BSAx species were separated from the reaction mixture via SDS‐PAGE and reversed phase HPLC. Ligand‐binding capabilities and dissociation constants were determined using the fluorescent probe ANS. Multiple BSA molecules were successfully conjugated onto a PEG8 framework while maintaining ligand‐binding capabilities. These results suggest that PEG8‐BSAx is a viable delivery system for water‐insoluble pharmaceuticals.Support or Funding InformationLoyola University Chicago
Chemical and Chemoenzymatic Synthesis of UDP‐N‐Acetyl Glucosamine Probes
Authors: Heiss Tyler Kelley , DeMeester Kristen E. , Liang Hai , Barbero Borja , Grimes Catherine Leimkuhler 
Published: November 2023

Uridine‐diphosphate‐N‐acetylglucosamine (UDP‐GlcNAc) is a ubiquitously used nucleotide sugar involved in bacterial cell wall biosynthesis and the posttranslational modification GlcNAcylation. If functionalized versions of UDP‐GlcNAc were more easily accessible, these chemical probes could be used to rapidly investigate biological systems. However, current chemical strategies for synthesizing UDP‐GlcNAc derivatives are difficult and lengthy. Herein we describe the chemoenzymatic synthesis of strategically functionalized UDP‐GlcNAc chemical probes from Glucosamine‐1‐Phosphate (GlcN‐1‐P) and synthetic acetyl donors known as N‐acetylcysteamine thioesters (SNAcs) using the bacterial cell wall biosynthesis enzyme GlmU. Hypothesis: If GlmU is shown to use GlcN‐1‐P and derivatized SNAcs to produce modified UDP‐GlcNAc's, then these altered building blocks will be used to investigate bacterial cell wall biosynthesis and O‐GlcNAc transferase (OGT) activity.Support or Funding InformationWe would like to thank the Koh Lab and the rest of the Grimes Group for their continuing help and support. We also thank the Howard Hughes Medical Institue (HHMI), the University of Delaware's Summer Scholars program, and the Pew Foundation for funding my work.
A Lipid Fraction from Catfish (Arius bilineatus, Val.) Potently Inhibits Collagen‐induced Aggregation in Vitro
Authors: Pace‐Asciak Cecil , Al‐Hassan Jassim M. , Afzal Mohammad , Paul Bincy Maniyalil , Oommen Sosamma , Liu Yuan Fang 
Published: November 2023

The catfish (Arius bilineatus, Val.) secretes a gelatinous substance from their skin upon threat or injury. The gel is composed of lipids and proteins. Preparations from the skin have previously been shown to possess a number of biochemical and pharmacological activities including blood clotting, healing of non‐healing diabetic foot ulcers and relief from neuropathic pain in man. The crude lipid preparation was subjected to chromatography on silica gel. The chloroform fraction was most potent in inhibiting collagen‐induced aggregation of washed human platelets in the presence of added calcium (IC50 = 5 ug/250e6 cells/0.5 ml). Purification of the active compounds is being pursued.Support or Funding InformationSupported by a grant from Kuwait Foundation for the Advancement of Science No. KFAS 201320701A‐D, and Kuwait University Research Grant No. SL03/14.