bluuebunny/crossref_metadata_2025_split huggingface dataset:
E3‐bglobin‐Dd mice: A new model to study hepatic zonal regulation of sex‐biased cytochrome p450 mRNA expression
Authors: Nail Alex N. , Jiang Jieyun , Peterson Martha L. , Spear Brett T. 
Published: November 2023

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and is the third leading cause of cancer death overall. In addition, HCC rates are much higher in males than in females. Our lab primarily focuses on liver genetic regulation and how dysregulated genes contribute to liver development and disease, including HCC.The cytochrome p450 (Cyp) family of enzymes are expressed mainly in the liver and have a critical role in the metabolism and biotransformation of endogenous and xenobiotic compounds. Many Cyps are expressed zonally in the livers of both mice and humans and, in addition, have been found to exhibit sex‐biased expression.Our lab has developed a novel model to study zonal hepatocyte populations within the liver. We have placed the H‐2Dd reporter gene under control of pericentrally active alpha‐fetoprotein E3 enhancer. Using antibodies against the Dd protein, which is expressed on the cell surface, we can separate pericentral and periportal hepatocytes using flow cytometry. This system enables us to analyze previously identified and novel zonally regulated genes. Using this system, we have identified Cyps that are both sex‐biased and zonally restricted within the mouse liver. Interestingly, the highly related Cyp2a4 and Cyp2a5 are female‐biased genes expressed periportally and pericentrally, respectively. These two genes have 97% genomic sequence identity over a 30 Kb region, providing a model to study sex‐biased and zonal regulation in the liver. Our central hypothesis is that sex‐biased zonally regulated Cyp enzymes contribute to sex‐biased HCC development. Understanding the roles of sex‐biased pericentral and periportal genes may lead to new insights into the basis for HCC predominance in males.
A Conserved Ser/Thr Kinase from Malaria Parasites is Inhibited by Tyrosine Kinase Inhibitors
Authors: Kirkman Lucy , Perez Astrid , Lin Benjamin , Harris Darcy , Schermerhorn Janse , Qian Yiwen , Brandt Gabriel 
Published: November 2023

Inhibitors were identified for a Ser/Thr kinase shared by all malaria parasites and shown to be essential. This kinase has a number of unusual sequence features, one of which is a gatekeeper residue much more commonly associated with eukaryotic tyrosine kinases than serine/threonine kinases. The recombinant kinase domain was expressed and purified from E. coli, then screened against a library of small molecules including known Ser/Thr and Tyr kinase inhibitors. The compounds emerging from the screen as potential inhibitors were all compounds with reported activity against Tyr kinases. An enzyme‐coupled assay was used to establish the affinity of each of these compounds. The most potent is a relatively high‐affinity inhibitor of the recombinant kinase domain (Ki = 1.2 uM). Comparison of a small family of selected analogs shows that the kinase discriminates among them, allowing the first view of the pharmacophore of this kinase. These are the first reported inhibitors of any member of this class of kinases and thus represent potential lead compounds for the development of new anti‐malarials.Support or Funding InformationFranklin Research Grant (American Philosophical Society)Hackman Fund (Franklin & Marshall College)Eyler Fund (Franklin & Marshall College)Marshall Scholarship (Franklin & Marshall College)Leser Scholarship (Franklin & Marshall College)
ATP synthase: a molecular drug target for olive oil constituent tyrosol and its analogs
Authors: Amini Amon , Ahmad Zulfiqar 
Published: November 2023

BackgroundA wide range of natural and synthetic compounds including dietary polyphenols are known to bind and inhibit ATP synthase. Dietary polyphenols can be found in a variety of fruits and vegetables such as olives, grapes, tea, coffee, etc. Polyphenols from olives and extra virgin olive oil are known to exhibit antimicrobial, anticancer, and antioxidant properties. Our lab is interested in understanding if the antimicrobial properties of polyphenols are linked to the selective inhibition of microbial ATP synthase. In this study, we examined the inhibitory profiles of Escherichia coli membrane‐bound ATP synthase and cell growth in the presence and absence of olive oil constituent tyrosol and its analogs; hydroxytyrosol, oleuropein, and dihydroxyphenylglycol.MethodsWild type E. coli pBWU13.4 and mutant strains were grown on minimal media to late log phase. Cells were harvested and then French Pressed to isolate and purify membrane‐bound F1FO ATP synthase. E. coli pUC118 with deleted ATPase gene was used as null control. Tyrosol and its analogs induced inhibitory profiles of wild‐type and mutant membrane‐bound enzymes were performed at 37 °C. Wild‐type, mutant, and null cell growth assays on limiting glucose were evaluated at OD595 using an AccuSkan Plate reader over a 24 hour period.ResultsTyrosol and its analogs induced a variable degree of ATP synthase inhibition. While tyrosol induced 100% inhibition of wild‐type ATP synthase, hydroxytyrosol, oleuropein, and dihydroxyphenylglycol induced between 40–60% inhibition. Degree of inhibition was also variable for mutant enzymes. Also, tyrosol and its analogs caused variable abrogation of wild‐type, mutant, and null cell growth.ConclusionsTyrosol and its analogs induced inhibitory profiles of membrane bound ATP synthase along with the variable effect on E. coli growth suggests that the antimicrobial properties of tyrosol and its analogs can be associated to the inhibition of ATP synthase. Thus, ATP synthase seems to be a potential molecular drug target to combat microbial infections.Support or Funding InformationThis work was supported by the National Institutes of Health Grant GM085771 to ZA and KCOM Biomedical Sciences Graduate Program Grant Award # 850‐611 to AA and ZA.
Engineering a Biocatalyst to Support a Novel Preparation of Blockbuster Statin Drugs
Authors: Belecki Katherine 
Published: November 2023

Rosuvastatin (Crestor®) and atorvastatin (Lipitor®) belong to a class of cholesterol‐lowering blockbuster drugs known as statins. In the US alone, approximately 25% of the population over 45 years old takes statins, and new prescription guidelines may lead to an increase in that number. Based on these projections, it is likely that statin therapy will continue to remain the standard of treatment for hyperlipidemia and that the market for these cholesterol‐lowering drugs will remain strong. Accordingly, the development of more efficient and “greener” strategies for the preparation of rosuvastatin in particular, and of statins in general, is a timely and relevant goal that has clear economic and environmental advantages.Nature's capability to introduce chirality into small molecules is unmatched, and scientists are beginning to harness the power of biocatalysis for enantioselective synthesis of pharmaceuticals and other important target molecules. All statins share a chiral 3,5‐dihydroxyacid side chain that is essential for their pharmacological activity. Construction of this pharmacophore has historically represented the most challenging and wasteful aspect of statin production. Although recent advances have improved stereoselective access to this chiral pharmacophore, its efficient incorporation into the final active ingredient has remained a challenge in some cases. In this work, an engineered biocatalyst based on a naturally occurring aldolase enzyme will be described. Using a structure‐guided rational mutagenesis approach, the substrate tolerance of a bacterial deoxyribose‐5‐phosphate aldolase (DERA) has been altered in order to generate the chiral statin pharmacophore with an additional unique functional handle. This biocatalytic approach enables the direct and efficient incorporation of the statin side chain, resulting in a novel synthetic route to rosuvastatin and other members of this widely‐prescribed class of drugs.Support or Funding InformationThis work was supported by a VCU Presidential Research Quest Fund Award.
Improving Personalized Medicine by Examining Structure‐Function Patterns in the Non‐Conserved Regions of Lactate Dehydrogenase (LDH)
Authors: Alao Esther Dami , Ayella Allan , Beck Moriah Rene 
Published: November 2023

Personalized medicine relies on our ability to make predictions about disease susceptibility based on differences in protein or gene sequences. Recent research has suggested that the non‐conserved regions of proteins, those regions with the greatest variation across species, may act as rheostats in protein function by adjusting protein function gradually. Whereas the conserved regions, or those with high degree of sequence similarity, typically involve the active sites or are regions that have a critical function. Mutation at a conserved region, might result in a non‐viable life form because of a dramatic switch, however non‐conserved regions may be more flexible in accommodating changes. Hence the sole purpose of this research is to determine what happens when regions of the non–conserved domain of the enzyme lactate dehydrogenase are mutated and how this affects its enzymatic activity and thermal stability. Lactate Dehydrogenase (LDH) is a ubiquitous tetrameric oxidoreductase that catalyzes the conversions of pyruvic acid to lactate and vice versa. Previous research has revealed that the enzymatic rates for LDH from different Barracuda species can be linked to differences in the range of climates where these fishes live. We have focused on the non‐conserved regions of Barracuda LDH for this project and used bioinformatics to identify sites to mutate in order to find out what happens to the protein function, the thermal stability, the catalytic rate and the substrate affinity. Site‐directed PCR mutagenesis was carried out on the Barracuda DNA to generate point mutations. After purification of these mutant proteins, we carried out enzyme kinetic measurements to determine the enzyme affinity (Kcat) and the enzymatic rates (Vmax) of mutants. Circular dichroism was also used to compare the conformation and stability of the mutants to the wild type LDH. We will present our results generated thus far, which have focused on glycine 68, a non‐conserved residue of LDH that we have mutated to serine, alanine, aspartic acid, lysine and histidine.. to discover the effects of charge on protein stability and enzyme function. Our work has also explored how pH affects protein stability of these mutants. Here we will present our comparisons of enzyme function and protein stability for a series of LDH proteins that differ in nonconserved residues. Developing a better understanding of the effects of protein substitutions at nonconserved sites could be used to enhance our ability to distinguish between minor and major consequences of genetic alterations and thus impact our ability to diagnose disease.Support or Funding InformationThis project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418.
Inhibition of the Herpesvirus Fusion Protein Using Targeted Small Molecules
Authors: Gallagher Shannon L , Deokar Hemantkumar , Buolamwini John K , Connolly Sarah A 
Published: November 2023

Herpesviruses are DNA viruses that cause significant disease in humans and animals. Most adults are infected with one or more herpesviruses. During herpesvirus entry into cells, fusion of the viral membrane with the host cell membrane is mediated by glycoprotein B (gB), a class III viral fusion protein that is conserved across all herpesviruses. Fusion proteins are metastable proteins that mediate fusion by inserting into a target membrane and refolding from a prefusion to a postfusion conformation to bring the viral and cellular membranes together. Crystal structures of the postfusion gB conformation have been solved, however its prefusion conformation and the details of how it refolds to execute fusion are unclear. A previous mutagenesis study demonstrated that a region in herpes simplex virus (HSV) gB, termed the coil‐arm region, contributes to fusion. This gB coil‐arm region resembles the six‐helix bundle that is predicted to provide energy to drive fusion in class I fusion proteins. We have developed a panel of small molecule compounds that are designed to target the coil‐arm region of HSV gB. The compounds were tested for the ability to inhibit entry of HSV1 and HSV2 reporter viruses and two compounds significantly decreased virus entry, exhibiting 6–11 μM IC50. Compounds were not cytotoxic and they inhibited entry when added at a step after virus binding, but before fusion. Successful identification of fusion inhibitors could lead to drug candidates that inhibit this conserved fusion protein and clarify how this fusion protein functions, potentially providing new tools to study prefusion gB.Support or Funding InformationDePaul – RFUMS Collaborative Pilot Research Grants
Phage Templating of Nanoparticles, Proteins, and Biologically Relevant Molecules
Authors: Prabhu Siddharth , Smith‐Boeck Morgan , Santan Pranav , Martinez Nathaniel W 
Published: November 2023

We examine the utility of a bio‐panning technique to isolate unharnessed and potentially yet‐unidentified marine bacteriophage that may be able to play a role in the development of useful nanoparticle architectures for applications as diverse as nanoparticle‐based solar cells, nanosensors, to their use as antimicrobials. The most pressing bottleneck in the field is the tediousness of assembling nanoparticles at scales as small as a millionth or a billionth of a meter, however phage templating for the patterning of nanoparticles is proving to be a powerful way to overcome this problem. Marine and freshwater ecosystems hold the highest concentration of unidentified bacteriophage on the planet and we predict that tapping in to this unharnessed pool of phage will result in the discovery of a variety of novel phage‐nanoparticle interactions. To date, marine and freshwater phage have been successfully pooled and concentrated by polyethylene glycol (PEG) and iron chloride (FeCl3) precipitation, followed by low speed centrifugation in our laboratory. These pooled phage concentrates are currently under evaluation with ‘smart bio‐panning & propagation’ to identify varying phage‐nanoparticle interaction strengths. Isolated phage are genotyped via shotgun sequencing and categorized by nanoparticle interaction with the intention of developing a first of its kind library of unique phage‐nanoparticle interactions that may hold promise in advancing nanoscience research requirements of the future.Support or Funding InformationSponsor: Dr. Sandra ClementCal Poly State UniversityBiology Dept.1 Grand AvSan Luis Obispo, CA 93407
Probing the Global Kinome and Phosphoproteome in Chlamydomonas reinhardtii via Sequential Enrichment and Quantitative Proteomics
Authors: Werth Emily Grace , McConnell Evan W. , Karim Gilbert T. S. , Lianez Inmaculada Couso , Perez Carlos W. , Manley Cherrel , Graves Lee M , Umen James G. , Hicks Leslie M. 
Published: November 2023

Identifying dynamic protein phosphorylation events is critical for understanding kinase/phosphatase regulated signaling pathways. Research aimed at dissecting the roles of kinases on a global “‐omics” scale has grown within the past decade with the potential to become more comprehensive for discovery‐based, untargeted kinome analyses. Utilization of immobilized kinase inhibitors [e.g., multiplexed inhibitor beads (MIBs) or kinobeads] broadly captures protein kinases through binding to the ATP‐binding domain of intact native kinases. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Presented here is a method to study the global kinome and phosphoproteome in tandem in the model photosynthetic organism, the alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry‐based label‐free proteomics. A dual enrichment strategy targets intact protein kinases via capture on MIBs columns with subsequent proteolytic digestion of unbound proteins and peptide‐based phosphorylation enrichment. To increase depth of coverage, both data‐dependent and data‐independent (via SWATH) mass spectrometric acquisitions were performed to obtain a >50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative kinome and phosphoproteomic datasets yield 115 protein kinases and 2,250 phosphopeptides with 1,314 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events. For example, through the induction of stress in Chlamydomonas and the use of differential proteomic studies of the kinome and phosphoproteome, future studies using this tandem enrichment have the potential to target specific pathways and investigate global kinome and phosphoproteome changes in a quantitative fashion.Support or Funding InformationThis research was supported by a National Science Foundation CAREER award (MCB‐1552522) to L.M.H. Authors would like to acknowledge Dr. Gary Johnson for providing the MIBs beads.
Proximity labeling and interactomic study of primary cilia
Authors: Rinschen Markus , Kohli Priyanka , Benzing Thomas , Schermer Bernhard 
Published: November 2023

Cilia have recently emerged as sensory organelles present in many mammalian tissues involved in a wide variety of cellular processes such as fluid flow, cell movement as well as multiple signaling pathways. Over the last years an increasing number of human diseases have been linked to defects in cilia with autosomal‐dominant polycystic kidney disease (ADPKD) perhaps being the most prominent example. However, very little is known about the protein composition as well as the time‐resolved dynamic processes within this important organelle.To characterize the ciliary membrane‐associated proteome, we used the genetically‐engineered enzyme ascorbate peroxidase (APEX) and targeted it to the ciliary membrane (cmAPEX). With STED microscopy, we could confirm the subciliary localization of the tag specifically at the membrane of the cilium. Upon sequential addition of biotin phenol and H2O2 to the cells, APEX generates free biotin radicals that label proteins within the closest proximity, in particular at the ciliary membrane. Subsequently, cells are lysed, and biotinylated proteins are enriched with streptavidin beads and identified by mass spectrometry.We identified numerous known ciliary proteins, in addition to novel ciliary membrane proteins and several actin modifying proteins. Inhibiting these actin‐modulating proteins regulated ciliary length, reaffirming the importance of actin dynamics in ciliogenesis. The results also identified ciliary proteins that are differentially regulated upon inhibition of actin polymerization and functional assays characterized their effect on ciliogenesis.cmAPEX is a versatile tool to study perturbations in this small and dynamic organelle. This approach provides quantitative analysis of the proteomic composition of the cilium and at the same time eliminates the need to isolate ciliary factions. We believe that this work will provide further insights into the cilium biology and suggest potential therapeutic targets for cilia‐associated genetic disorders.Support or Funding InformationDFG, CECAD Graduate School of Ageing.
Temporal Analysis of Coagulopathy in Burn Sepsis using a Systems Biology Approach
Authors: Srinivasan Seshamalini , Donohue Duncan , Gautam Aarti , Detwiler Leanne , McLawhorn Melissa M. , Tejiram Shawn , Moffatt Lauren T. , Jett Marti , Shupp Jeffrey W. , Hammamieh Rasha 
Published: November 2023

Coagulopathy is described in the trauma patient population, but to date is studied less in burn patients. The pattern of changes in coagulation that have been described in burn patients are unclear, yet the changes resemble those found in patients with sepsis. The aim of this study was to use a systems biology approach to integrate multiple data types, including clinical findings and transcriptomics, to develop a universal picture of the progression of these physiological conditions. As part of a larger study, seventeen adult burn patients were selected and deemed ‘septic’ based on a positive blood culture and having two or more of the SIRS (systemic inflammatory response syndrome) criteria as abstracted from charts. Blood samples were collected every 2–4 h for the first 12 h, then every 12 h up to day 7, and on a weekly basis until 21 days post admission for a total of 20 samples. Comprehensive demographics, injury, treatment and outcome data were collected and tested for association with coagulopathy at baseline and continually throughout the study. This clinical data was integrated with time series analysis of gene transcript microarray data, which probes over 50,000 biological features, completed on all samples. Further characterization of coagulopathy seen in burn patients with sepsis may add important information to identify biomarkers of disease progression and associate these with appropriate therapeutic strategies as the illness proceeds.DISCLAIMER:The views, opinions, and/or findings contained in this report are those of the authors and should not be construed as official Department of the Army position, policy, or decision, unless so designated by other official documentation.
Altered Fecal Microbiota and Urine Metabolites Following Soman Exposure in a Rat Model
Authors: Gautam Aarti , Getnet Derese , Kumar Raina , Hoke Allison , Cheema Amrita K. , Rossetti Franco , Schultz Caroline , Hammamieh Rasha , Lumley Lucille A. , Jett Marti 
Published: November 2023

The clinical and experimental pathobiology of organophosophate nerve agent exposure has been extensively described. Here, were port for the first time an altered fecal microbiome and urine metabolome post‐exposure with soman, a highly lipophilic G‐class nerve agent. Non‐anaesthetized Sprague‐Dawley rats were administered soman via sub‐cutaneous injections versus a saline control group and feces, urine, and relevant organs were harvested at72 hrs and 3 months post‐exposure. To evaluate the direct effect of soman exposure on the bacterial biota, 16S rRNA‐based analyses were performed on feces and the ileum on the Illumina MiSeq platform. High resolution mass spectrometry was used to analyze and compare the metobolomic and lipidomic urine composition between exposure groups and the control group at both time points. Soman insult reshaped fecal bacterial biota by significantly decreasing the TM7phylum abundance and increasing the prevalence of the Cyanobacteria and Proteobacteria phyla in the exposed rats with increasing dose and seizure status. However, analogous changes were not observed in the bacterial biota of the ileum tissue, which remained the same irrespective of dose or seizure status after insult. Interestingly, the urine metabolome was markedly altered based on only seizure status of the host, where guanosine diphosphate, leukotriene C4, kynurenic acid, aldosterone, and neuraminic acids were excreted at a much higher rate after the first 72 hrs in seizure rat groups, suggesting a multi‐organ impact of soman exposure. These findings demonstrate the feasibility of using microbiota and urine metabolome‐based surveillance and monitoring for nerve agent exposure. These experiments were conducted under approved protocols (IACUC U‐908) at the U.S. Army Medical Research Institute of Chemical Defense.DISCLAIMERS: Research was conducted in compliance with the Animal Welfare Act and all other Federal Requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.