bluuebunny/crossref_metadata_2025 huggingface dataset:
In Vivo Assessment of a Tissue‐Engineered Vascular Graft in a Rat Model
Authors: MacIsaac Corina , Stringa Pablo , Lausada Natalia , Nieponice Alejandro , Geest Jonathan Vande
Published: November 2023

Heart disease is the leading cause of death in the United States, of which coronary artery disease (CAD) is one of the primary types. The current treatment of CAD involves bypassing the blocked coronary artery with an auxiliary blood vessel obtained from elsewhere in the patient's body. As an alternative to using this type of bypass graft, much research has been done on the fabrication of a biocompatible tissue engineered vascular graft (TEVG). The development of this TEVG is hypothesized to eliminate problems of bypass graft failure and compliance mismatch present in the current treatment. The basis of the TEVG is a scaffold which must provide (1) mechanical support of the TEVG and (2) a mesh for cells to proliferate and migrate through. This is sought to be achieved by electrospinning non‐synthetic biopolymers (gelatin, fibrinogen, collagen and tropoelastin) into a tubular construct. This procedure generates a thin fibrous mesh which allows for cell growth within the construct. The use of non‐synthetic biopolymers allows the scaffold to better compliance match a native coronary artery, as well as favor cell attachment. Preliminary experiments have been performed with different combinations and layering of the biopolymers, various speeds at which the construct was electrospun, and different amounts of glutaraldehyde cross‐linking time. Success has resulted from electrospinning constructs from a 10% by volume solution 80:20 gelatin/fibrinogen (w/w) in 1,1,1,3,3,3‐Hexafluoro‐2‐propanol (HFP). By analyzing the mechanical properties of these constructs with a microbiaxial optomechanical testing apparatus, the constructs have shown stress and strain relationships comparable to previously tested porcine left descending coronary arteries. Fluorescence imaging has also shown that porcine aortic smooth muscle cells will migrate and proliferate within this TEVG.

In addition to mechanical and in vitro cell culture testing, in vivo assessment of the acellular TEVGs has also been performed. This was achieved by performing infrarenal aortic interpositional graft placement surgery to implant the construct within a rat's aorta. This is a survival surgery and the rats remained alive for up to a month before sacrificing for analysis. Preliminary results of the in vivo assessments necessitated the use of synthetic polymer incorporated into the construct for added strength to be sutured at the anastomoses. However, results of these preliminary assessments show the effectiveness of using this surgery for analysis of graft function in vivo. Future research involves increasing the strength of the construct without the addition of a synthetic polymer, as well as seeding endothelial cells onto the constructs before implantation.

Support or Funding Information

Funding for this research is provided by: NIH 1R2HL111990 to JPVG (Co‐I Doetschman and Slepian) and HHMI 52006942 to the Biomedical Research Abroad: Vistas Open! program.
Varying Assay of Proteins Vital to Regulation of the Epithelial Sodium Channel (ENaC)
Authors: Wolfe Collin , Roach Brett E. , Booth Rachell E.
Published: November 2023

The epithelial sodium channel (ENaC) is a transmembrane heterotrimeric protein complex that transports sodium into epithelial cells. This channel plays a crucial role in sodium regulation, which is important for proper blood pressure and fluid balance. Located in the distal portion of nephrons within the kidney, the channel selectively regulates the reabsorption of sodium. Genetic mutations in ENaC have shown to cause disease states of hypertension in Liddle's syndrome and hypotension in pseudohypoaldosteronism type 1. Prior studies have focused on regulation of ENaC via protein‐protein interactions, such as an ubiquitin‐protein ligase Nedd4‐2, a ubiquitin transferase domain HECT, and serine/threonine‐protein kinases Sgk1, much is still unknown about ENaC. Our studies include expression of ENaC in yeast deletion strains to identify proteins necessary for ENaC function. A serial dilution pronging assay was performed for each separate deletion yeast strain. Results showed that although no tested strains had direct effect on ENaC functionality, the coated vesicle protein COPII, a Rubb1p (NEDD8) activator protein, and a p24 protein component involved in vesicle quality control all had negative effects on cell growth with and without salt present when particular ENaC subunits (β/γ) or all ENaC subunits (α/β/γ) were present. Further analysis of genes encoding proteins associated with ENaC will provide more insight into ENaC and its role in sodium homeostasis.
Determination of the Molecular Interactions of Bovine Gamma B Crystallins Using NMR Spectroscopy
Authors: Mathews Kaylee , Zanet John , Payan Angel , Michel Lea Vacca , Thurston George , Mills Jeffrey
Published: November 2023

Crystallins are proteins found in the eye and are responsible for regulating the transparency of the lens. Interactions between crystallins can cause phase separation and aggregation, which can decrease lens clarity and disrupt vision by causing cataracts. To study the orientation‐dependent interactions between crystallins, we utilized recombinant bovine γ‐B crystallin (CRYGB) proteins expressed in Escherichia coli. We acquired nuclear magnetic resonance (NMR) spectra on 15N/13C labeled CRYGB and completed the backbone residue assignments. Standard triple‐resonance spectra were acquired at NMR Facility at Madison (NMRFAM) on 600, 800, and 900 MHz spectrometers. HSQC experiments at variable concentrations revealed subtle chemical shift perturbations that, we propose, are the result of CRYGB interactions. Overall rotational correlation times (τ c) and translational diffusion rates demonstrated the concentration and temperature dependences on protein diffusion times. Preliminary results suggest that the sensitivity of NMR allows for a detailed look at how CRYGB proteins interact.
Calorimetic Study of Calcium Binding Proteins with their Interacting Peptides
Authors: Wei Chin‐Chuan , Schaffer Kyle , Jensen Drake , Ngo Rebecca
Published: November 2023

Several Ca2+‐binding proteins, including Calmodulin (CaM), NADPH Oxidase 5 (NOX5), and dual oxidase (Duox), were employed in calorimetry to understand their structural change upon ion and peptide ligand binding. A general mechanism for these proteins is that Ca2+ binding triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. Previously, we have characterized chimera proteins constructed by exchanging EF‐hands of CaM with those of Troponin C (TnC) to be used as an informative probe to better understand the functionality of each EF‐hand. Our results revealed that Ca2+ binding to chimeras impairs their hydrophobicity, and such conformational changes altered their interaction with a CaM‐binding domain (CMBD) peptide of Orai. 3TnC and 4TnC (3rd and 4th EF‐hand exchange position, respectively) exhibited a lower ANS binding and solvent accessible surface area change (ΔASA) based on structural homology modeling, but binding to Orai‐CMBD remained similar to CaM, suggesting that the exchanged EF‐hands in the C‐terminal lobe are capable of dynamically accommodating peptide binding. Our isothermal titration calorimetry (ITC) data indicates that approximately 0.3–0.7 protons are released to the buffer when CaM and chimeras are titrated with saturating Ca2+, suggesting that key residue(s) involved in Ca2+ binding are not altered significantly by the exchanged EF‐hands. The heat capacity change of Ca2+ binding (ΔCp) for CaM was determined to be ~ −0.66 kCal K−1 mol−1. The observed large negative value is probably due to 1) the increase in nonpolar ASA and decrease in polar ASA, which results in a positive ΔCp and a higher binding entropy change (ΔSb) and 2) the reorganization of hydrogen bonds including water molecules (i.e. solvation), contributing a relatively larger negative ΔCp. Interestingly, the ΔCp values of 1TnC and 2TnC are similar to CaM. On the other hand, 3TnC and 4TnC have very small ΔCp values ranging +0.004 to −0.011 kcal K−1 mol−1, possibly due the decrease of helical content determined by Circular Dichroism. The EF‐hand domain (EFD) of NOX5 has been suggested to be structurally similar to CaM. However, the proton release and ΔCP are ~0.07 and −0.0026 kCal mol−1K−1, respectively, indicating a fundamental structural change in CaM that is supported by modeling. We are currently investigating the heat capacity change upon peptide binding for these three Ca2+‐binding proteins, as well as their chimeras and mutants, to give insight about the binding modes and to rationalize apolar/polar surface changes to their ΔCp values. Differential scanning calorimetry (DSC) was also used to gather information about non‐covalent interactions in protein folding.

Support or Funding Information

This work is supported in part by National Science Foundation CCLI grant and Cottrell College Science Awards (CCSA) from Research Corporation.
Database for Storage and Rapid Retrieval of ProMOL Results
Authors: Baker Cameron D. , Mills Jeffrey L. , Bernstein Herbert J. , Craig Paul A.
Published: November 2023

ProMOL is an extension to PyMOL, a widely used molecular visualization program, developed by students at RIT and Dowling College to help determine the function of proteins. The process is computationally intensive. This is exacerbated by the fact that we have a large number of motifs to search against. A database was created to store the results for fast and easy retrieval. The database is stored on a standalone server and results are transferred by TCP/IP. The implementation itself is very simple and provides a base for future development. Results show a runtime improvement from over 30 minutes to just under a minute for alignment results for a single protein. The speed gain comes from the retrieval of prior results. This greatly improves search time for users looking for matches that have been encountered previously, either by themselves or by other users. Future improvements may include a NoSQL database on top of MySQL to increase the speed of data retrieval.

Support or Funding Information

This work was supported in part by NIH GM078077.
Identification of the Recognition Sequence in Gluten by the Prolyl Endoprotease of Eurygaster integriceps
Authors: Melton Stephanie
Published: November 2023

Eurygaster integriceps Puton, Sunn pest, is the most globally destructive insect pest of bread wheat and durum wheat infecting 15 million hectares (HA) in Western and Central Asia and in Eastern Europe. To discontinue the harmful effects that current pesticide treatments have, environmentally safe biopesticides, inhibitors of the Sunn pest's digestive enzyme, are being sought. The objective of this study is to advance knowledge and understanding of the recognition sequences of the Sunn pest prolyl endoprotease (spPEP) within its natural substrate, gluten, as well as provide information towards the development of peptide inhibitors. The first method to the study is to separate gluten into glutenins (high molecular weight) and gliadins (low molecular weight). Gliadins were separated according to pI with BioRad's Rotofor Cell system and then by molecular weight with 12% SDS gels into their α/β, γ, and ω isoforms, Current results from the SDS gels indicate several gliadins have similar pI's between 7.22 pH and 8.72 pH consistent with the theoretical pI and molecular weight generated using the ExPASy Bioinformatics Resource Portal. The information from the NCBI database for the subdivisions of gliadins allowed identification of the proteins for spPEP in‐gel digestion. To date, γ gliadin has been identified with the predicted characteristics. Once digested, the fragments will be analyzed using MALDI‐Mass spectrometry to determine the mass and amino acid sequence of each fragment. Future experiments will include sending fragments to the Molecular Structure Facility at UC Davis, Davis CA for N‐terminal sequencing. When all the analysis is gathered, the data will be compared with the complete collection of gliadin sequences from NCBI to identify the cut and noncutting sites of the spPep for future design of inhibitors.

Support or Funding Information

Funded by Stephen F. Austin State University Graduate Research Assistant (GRA) Program
A Novel Lipolytic Enzyme from an Uncultured Bacterium Genome
Authors: Rangel Margarita Victoria , Palmieri Gabriel , Zoubeidi Nabil , Lee Becky , Ouverney Cleber
Published: November 2023

Lipases are extracellular enzymes with a wide range of cellular functions and technological applications. Some are critical in bacterial pathogenicity, whereas others are exploited by the food or pharmaceutical industries. The majority of prokaryotes are uncultured and harbor promising untapped resources, such as novel lipases with medical and biotechnological relevance. Our goal was to retrieve new lipases from uncultured bacteria and test them for novel activity. We used a culture‐independent metagenomic approach to obtain full sequences of uncharacterized lipases from an environmental complex bacterial community. We constructed a fosmid library from the environmental source and sequenced over 4,000 fosmids using Illumina MiSeq. Over 100,000 bacterial genes were annotated, most of unknown function. Clones were screened using a tributyrin agar assay. Over 136 novel lipases were identified and we pursued to characterize lip2465, which contained a distinct GDSL esterase/lipase motif, indicating it belongs to the GDSL family. A top hit on NCBI's BLAST and the UniProt database for lip2465 revealed 53% amino acid similarity to a GDSL esterase of an uncultured bacterium from soil. Although GDSL esterases are known for their broad substrate specificity, five lipids were tested and lip2465 was only active on tributyrin (C4) and tricaproin (C6), suggesting a preference for short carbon chains, and not the longer C8, C10, or C16 lipids tested. After subcloning lip2465 and assaying for activity, the enzyme retained its robust lipolytic activity on tributyrin and significantly surpassed a Staphylococcus aureus control. Sequence‐based studies are leading to predictive proteomic models for uncultured bacteria and while these provide valuable information about uncultured organisms, it is important to pair predictive models with validation in vivo. The approach described here will allow us to continue producing experimental access to the genes of uncultured bacteria, which we can align with deposited genome sequences and begin validating their function. Complex mixed microbial communities exist everywhere and this research is the next step towards building knowledge about organisms we currently know little about.
Noninvasive Biomarker Candidates for Cadmium‐induced Nephrotoxicity by 2DE/MALDI‐TOF MS and SILAC/LC‐MS Proteomic Analyses
Authors: Moon Aree , Kim Sun Young , Lee Hye Min , Ko Eun‐yi , Kim Kyeong Seok , Kim Hyung Sik
Published: November 2023

Cadmium (Cd2+) is a major environmental pollutant that induces cytotoxicity by heavy‐metal accumulation. Prolonged Cd2+ exposure leads to cell damage by oxidative stress mainly in the kidneys, a critical organ for detoxification. In order to identify reliable on invasive protein biomarkers for Cd2+‐induced nephrotoxicity, we performed 2DE/MALDI‐TOF MS and SILAC/LC‐MS analyses using conditioned media (CM) of HK‐2 human kidney epithelial cells treated with CdCl2. Here, we identified heat shock cognate 71 kDa protein isoform1 (HSPA8) and α‐enolase (ENO1) as potential biomarker candidates for the evaluation of Cd2+‐induced nephrotoxicity. Treatment with CdCl2 increased the protein level of HSPA8 in CM and lysates of HK‐2 cells. The mRNA level of HSPA8 was also increased by CdCl2 treatment, indicating transcriptional regulation. The level of ENO1 was increased in CM, but not in lysates of CdCl2‐treated HK‐2 cells. CdCl2 did not affect the mRNA level of ENO1. We provide evidence that the increases of HSPA8 and ENO1 in CM were due to Cd2+‐induced cell death through oxidative stress. The increases of HSPA8 and ENO1 levels were also detected in CM of HK‐2 cells treated with other nephrotoxic agents such as HgCl2, NaAsO2, cisplatin, amphotericin B, and cyclosporine A. Urine and kidney tissues of CdCl2‐treated rats showed increased levels of HSPA8. Taken together, this study identified HSPA8 and ENO1as non‐invasive biomarker candidates by two comparative proteomic analyses. These new biomarker candidates may have potential as alternatives to traditional biomarkers for the efficient and sensitive assessment of nephrotoxicity.

Support or Funding Information

This work was supported by a grant from Korea Food & Drug Administration [10182KFDA992‐3202] and by the National Research Foundation of Korea (NRF) grants [2013R1A2A2A04013379].
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 Information

This 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).
An Investigation into the Extended Coordination Network of Catalytic Zinc Ions in a Class B β‐lactamase
Authors: Caruso Alessio , Eberhardt Eric
Published: November 2023

Antibiotics have a long history as essential tools in fighting bacterial infections. The β‐lactam subgroup of antibiotics has been widely and successfully used since the identification of penicillin in the early 20th century. In recent years, β‐lactamases have been responsible for an alarming increase of bacterial resistance to many β‐lactam antibiotics. Our study gains insight into the mechanism of action of New Delhi Metallo β‐lactamase (NDM‐1 EC: 3.5.2.6) to facilitate the development of mechanism‐based inhibitors to metallo β‐lactamases. NDM1 is a dinuclear zinc‐containing enzyme that demonstrates higher catalytic efficiency than its mono‐zinc counterparts. High‐resolution crystal structures of NDM‐1 by Zhang and Hao reveal insight into the role zinc ions play in the formation of a catalytic hydroxide as well as additional stabilization of the leaving group. Our inquiry explores the role of a key residue of NDM‐1: in particular, Asp‐223 that may play a role in the extended coordination network of the catalytic metals. Asparagine and alanine mutants have been generated, and the steady‐state kinetic parameters are being evaluated.
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 Information

Supported 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 Information

Albert 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 Information

Center of Excellence (COE)
Metallothionein Induction, Antioxidant Defence Systems and Lipid Peroxidation: An Amalgamation of Biomarkers of Heavy Metals Pollution in Mus musculus
Authors: Osuala Fidelia Ijeoma , Otitoloju Adebayo Akeem , Igwo‐Ezikpe Miriam
Published: November 2023

The exploration and exploitation of natural resources inadvertently results in the release of persist and toxic heavy metals into the environment. This warrants the need to formulate a combination of biomarkers as a biomonitoring tool to evaluate health effects of heavy metals as an early warning signal in environmental exposure. Male albino mice (M. musculus) were divided into six groups (n = 12 per group) treated for 28days to salts of Pb(NO3)2 = 0.24 mM, FeSO4 = 0.45 mM, CdSO4 = 0.05 mM, ZnSO4 = 0.24 mM MnSO4 = 0.57 mM and control administered distilled water. The effect of heavy metals exposure was evaluated in terms of Methallothionein (MT) in the serum by evaluating the sulphyhydryl residue, Superoxide dismutase (SOD) activity was determined as the difference between superoxide anion decomposition and production, catalase (CAT) was determined using the method of Beers and Sizer, Glutathione‐s‐transferase (GST) by monitoring the formation of a conjugate between 1 M GSH and 1 M 1‐chloro‐2, 4‐dinitrobenzene and Lipid peroxidation (LPO) by estimation of thiobarbituric acid reactive substances in the liver, kidney, heart and brain. The results showed that Iron (Fe) induced the highest concentration of MT followed by Cd > Mn > Pb > Zn. SOD and CAT significantly (P < 0.05) decreased. GST activity fluctuated with Cd inducing the highest activity in the brain and liver, Fe and Pb induced the highest activity in the kidney and heart respectively. Cd induced significantly (P < 0.05) higher levels of MDA in the brain. This indicates that Fe is an indirect MT inducer which is mediated primarily by stress response while the decrease of SOD and CAT activities could be attributed to competitive inhibition and generated internal free radicals. The initial significant increase of GST activity observed in this study, suggested an adaptive mechanism of mice in response to exposure to cd. The investigated combinations of biomarkers were found to have good potentials for early detection and diagnosis of heavy metals pollution in the mammalian model. This will also minimize misinterpretation in cases of complex pollution situations especially in highly industrialized areas as the Lagos metropolis, Nigeria.
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 Information

This 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 Information

Funding: 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 Information

This 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 Information

Supported 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 Information

This 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 Information

NIH R01 CA154387

Mayo 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 Information

Loyola 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 Information

We 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 Information

Supported by a grant from Kuwait Foundation for the Advancement of Science No. KFAS 201320701A‐D, and Kuwait University Research Grant No. SL03/14.
Antibacterial Activity of Organic Ligands and their Cu(II) Complexes
Authors: Hamaway Stefan , Fox Kristin , Tanski Joseph , Tyler Laurie
Published: November 2023

Cu(II) complexes with biologically relevant thiazole or imidazole ligand frames have been previously shown to exhibit nuclease activity. To further probe the biological activity of these species, antibacterial studies have been carried out. Zone of inhibition and minimum inhibitory concentrations (MIC50) were assessed for both free ligand and complex for each derivative. An ampicillin positive control was used as a reference for antibacterial activity while the solvent, DMSO, served as the negative control to ensure that the observed activity was attributed to either of the reactive species. The assays were performed using E. coli and S. aureus, gram‐negative and gram‐positive bacteria respectively. It was determined that ligand structure affected the antibacterial properties of the Cu(II) complexes while the pure ligand displayed little to no activity. Complexes containing imidazole groups displayed greater antibacterial activity compared to complexes with ligated thiazole ligands. Both complexes exhibited marginally greater activity against E. coli compared to S. aureus. While the ligands may be structurally similar to each other, the ring substitutions were found to greatly impact the antibacterial properties of these complexes.
Cell Membrane Permeabilization by Double, Bi‐monopolar and Bi‐polar, Electrical Pulses and Polycationic Peptides
Authors: Lemeshko Victor V. , Ortiz Diego A.
Published: November 2023

Electrical permeabilization of cell membranes is commonly used to deliver drugs, DNA plasmids and other molecules into living cells. We demonstrated that the cell permeabilization by polycationic peptides with antibacterial and anticancer activities may be strongly increased by a membrane diffusion potential, generated in the presence of valinomycin, or by high‐voltage electrical pulses, showing the synergistic effects. In this work, to provide insight into the mechanism of the plasma membrane electrical permeabilization, excluding possible thermal effects, we used double, bi‐monopolar and bi‐polar, square 1 Hz electrical pulses of 2850 V/cm with pulse widths of 10–200 microseconds. The pulses were applied to the red blood cell suspension in the physiological salt solution at room temperature. The application of 10 double 100 microseconds pulses, bi‐monopolar or bi‐polar, caused an equal, 4.5 °C increase in the suspension temperature and very different cell permeabilization effects. With this respect, the cell permeabilizing effect after 2 minutes of application of bi‐polar pulses with the separation time of 4 microseconds between mono‐pulses was almost five times lower than that caused by bi‐monopolar pulses, showing that each second mono‐pulse of bi‐polar pulses essentially reversed the effect caused by the first mono‐pulse. At the separation time of 65 milliseconds, the permeabilizing effect of bi‐polar pulses was only 2 times lower than that of bi‐monopolar pulses. The described experimental approach seems to be useful to estimate relaxation times of the electrical permeabilization of biomembranes under various experimental conditions (Research grants HERMES #29207, Universidad Nacional de Colombia).