bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of bacterial cytochrome P450's involved in gibberellin biosynthesis
Authors: Nagel Raimund , Peters Reuben J. , Carver Roy J
Published: November 2023

Gibberellins are essential plant hormones that regulate growth and development. Besides plants, phytopathogenic fungi and plant‐associated bacteria also produce gibberellins. The recently elucidated bacterial pathway uses four soluble cytochromes P450 (CYPs) for gibberellin biosynthesis. While the sequence of the reactions is exactly the same as the plant pathway, rather than two 2ODDs for the last two steps bacteria use two CYPs instead. We have successfully expressed the CYP117 responsible for the oxidation of ent‐kaurene to ent‐kaurenoic acid and with 18O2 labelling experiments could identify the sequence of catalysed reactions in this multistep transformation. The next biosynthetic step is the coupled formation of an aldehyde with ring contraction, here using UV/VIS‐spectroscopy we have determined the binding affinities of the relevant CYP114 for its substrate, ent‐kaurenoic acid, as well as close structural analogues. Notably the ring contraction reaction relies on a specific ferredoxin; other ferredoxins only enable production of the intermediate ent‐7a‐hydroxykaurenoic acid. Subsequently, CYP112 catalyses a series of reactions that lead to coupled demethylation‐lactone ring formation, transforming GA12 into GA9. Again we have used UV/VIS‐spectroscopy to determine the binding affinities of CYP112 for the substrate GA9 as well as both intermediates (GA15 and GA24), and multiple structural analogues. In addition, we also performed 18O2 labeling experiments with CYP112 that establish the sequence of catalysed reactions in this multistep transformation, and show that the carbon lost in the demethylation reaction leaves as CO2.
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 Information

Franklin 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)
Aspartate N‐acetyltransferase: a key enzyme in neurobiology and cancer
Authors: Wang Qinzhe , Zhao Mojun , Parungao Gwenn G. , Thangavelu Bharani , Mutthamsetty Vinay , Viola Ronald E.
Published: November 2023

N‐acetylaspartate (NAA) is the second most abundant amino acid in human brain. Several genetic mutations in newborns lead to a disruption of the N‐acetylaspartate metabolic pathway which causes a fatal, neurodegenerative disease named Canavan disease (CD). Also, several recent reports have discovered a positive correlation between the NAA concentration in at least two types of cancer cells (lung cancer and ovarian cancer) and tumor growth. Aspartate N‐acetyltransferase (ANAT) catalyzes the transacetylation reaction between acetyl‐Coenzyme A and L‐aspartic acid in neuron mitochondria as well as in cancer cells. Despite the importance of ANAT in brain development and cancer therapy, a full characterization has been lacking because ANAT is an integral membrane protein which is sensitive to detergents. Recently, we were able to express and purify a stable, soluble and active enzyme form as a fusion protein with maltose binding protein from an E. coli expression system. The fusion enzyme has been kinetically characterized, and several sub‐micromolar inhibitors have now been developed by library screening as well as by focused inhibitor development. A series of protein engineering studies have been conducted to obtain insights into the nature of membrane association. Crystallization trials of ANAT have been initiated in order to shed light on the catalytic mechanism of this key metabolic enzyme, as well as to provide structural guidance for further inhibitor development. Our success in production and characterization of human ANAT has set the stage for the development of selective inhibitors that can lower the abnormally elevated concentrations of NAA that are observed in CD and in certain cancer patients.
ATP synthase: a molecular drug target for olive oil constituent tyrosol and its analogs
Authors: Amini Amon , Ahmad Zulfiqar
Published: November 2023

Background

A 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.

Methods

Wild 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.

Results

Tyrosol 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.

Conclusions

Tyrosol 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 Information

This 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.
Effects of 8‐Arm Polyethylene Glycol (PEG8) Addition on Thermal Stability of Bovine Serum Albumin (BSA)
Authors: Parker Jacob S , Hill Jonathan , Olsen Kenneth W
Published: November 2023

Bovine Serine Albumin (BSA) conjugated to 8‐arm Polyethylene Glycol (PEG‐‐‐‐8) via Strain‐Promoted Azide‐Alkyne Cycloaddition (SPAAC) has been proven to be a viable drug delivery system. In our experiments, BSA was de‐fatted and then activated using a short dibenzocyclooctyne (DIBO) as well as an extended DIBO activator (DIBO‐PEG4). The activated BSA was then conjugated to N3‐functionalized PEG8 and purified via size exclusion chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) of the product showed multiple species verifying the cycloaddition. BSA has two main drug binding sites, DS1 and DS2. It has been shown sulfamethoxazole (SMZ) preferentially binds to DS1 while phenytoin (PHT) preferentially binds to DS2. Fluorimetry was performed using SMZ and PHT to test the functionality of both drug binding sites of PEGylated BSA at room temperature proving both sites functional after PEGylation. The objective of this study is to explore the potential usage of this product by analyzing thermal stability of the PEG8‐BSAx compound. In this study, UV‐Visual Spectroscopy (UV‐Vis) was used to monitor aggregation of denatured PEG8‐BSAx over a range of temperatures. Initial assays via UV‐Vis showed less aggregation of PEGylated sample than unmodified BSA. Circular Dichroism (CD) was then performed to determine the temperature range conjugated BSA would remain functional as indicated by maintenance of its secondary structure. To verify the functionality of PEGylated BSA after exposure to high temperature followed by cooling, fluorimetry experiments were repeated to measure drug binding of SMZ and PHT.
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 Information

This 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 Information

This 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.
Crystal Structures of the Nuclear Receptor, Liver Receptor Homolog 1, Bound to Synthetic Agonists Reveal a Novel Mechanism of Activation
Authors: Okafor Denise , Mays Suzanne G. , Whitby Richard J. , Goswami Devrishi , Stec Jandoacutezef , Flynn Autumn R. , Dugan Michael C. , Jui Nathan , Griffin Patrick , Ortlund Eric
Published: November 2023

Liver receptor homolog 1 (NR5A2, LRH‐1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. While preclinical and cellular studies demonstrate that LRH‐1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH‐1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH‐1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH‐1 is activated by synthetic ligands are entirely unknown. Here, we use x‐ray crystallography and other structural methods to explore conformational changes and receptor‐ligand interactions associated with LRH‐1 activation by a set of related agonists. Unlike phospholipid (PL) LRH‐1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth, nor do they expand the pocket like PLs. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable, face‐to‐face π‐π‐stacking with LRH‐1 residue H390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand‐binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH‐1 agonist development and offers insights into rational design strategies.
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 Information

DePaul – RFUMS Collaborative Pilot Research Grants
Investigating Electrostatic Fields at Protein Interfaces for Drug Discovery
Authors: Strong Lisa , Novelli Elisa , Chang Benjamin , Webb Lauren
Published: November 2023

Non‐Covalent molecular interactions are becoming increasingly interesting to study especially for their pharmacological potential. Ras, a GTPase implicated in many cancers, is regulated by these interactions and can be used as an anti‐cancer target when non‐covalent interactions are disrupted. Despite years of intensive effort, no drug targeting Ras has been discovered. Instead of focusing our efforts on this protein, we are investigating the ADP Ribosylation Factor 1 (Arf)/Guanine Exchange Factor (Gea) model system, which has a natural inhibitor, Brefeldin A (BFA). We are quantifying the electrostatic field that determines non‐covalent interactions by using vibrational stark effect (VSE) spectroscopy with nitrile probes. Spectral shifts of a probe oscillator's energy are directly related to the probe's local electrostatic environment. Nitrile probes are strategically placed along the interface of the two proteins by mutating key amino acids to cysteine. This enables the two step SN2 reaction with 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (DNTB) and KCN to create a nitrile probe. These probes have a distinct signal in the infrared region which we can quantify using FT‐IR. After creating a library of these fields from various nitrile locations we can model the electrostatic field at the protein interface in solution with an inhibitor. In the future, this electrostatic field model can then be applied to the Ras system to identify potential inhibiters for Ras.
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 Information

Sponsor: Dr. Sandra Clement

Cal Poly State University

Biology Dept.

1 Grand Av

San Luis Obispo, CA 93407
EFFECT OF AN ELECTRIC BIAS ON THE ANTIBACTERIAL PROPERTIES OF AG‐GQD NANOCOMPOSITES
Authors: Alvarez‐Colon Nelson Ivan , Marra Andre , Caro Giovanni , Avalos Javier , Morell Gerardo
Published: November 2023

Ag‐Graphene nanocomposite solution exhibits antibacterial activity against both Gram positive and Gram Negative bacteria. The functionalization of this Ag‐Graphene nanocomposite with polyethylene glycol provides stabilization and higher solubility in aqueous solution. The Ag‐Graphene nanocomposite was characterized by UV‐Vis spectroscopy, transimission electron microscopy (TEM), Raman spectroscopy, X‐ray photoelectron spectroscopy (XPS), field emission scanning electron microscope (FE‐SEM) coupled with energy dispersive spectrometry (EDS), and Fourier transform IR spectrocopy (FT‐IR). The antibacterial property of this nanocomposite was preoviously tested by Kirby‐Bauer assay disk diffusion and Minimun Inhbitory Concentration (MIC) assay using Pseudomonas aeruginosa and Staphylococus aureus as model strains for both types of bacteria. We hypothesize that by applying and external electric bias; a decrease in the bacterial inhibition time (less than 6 hrs.) can be attained. Gram positive and negative bacterias cell wall differ greatly in structure; therefore concentrations of the Ag‐GQD solution will vary for both types of bacteria. The success of this method allows us to propose a mechanism that describes the bactericide effect in terms of the ion transport between the solution and the time exposed to the bacterias cell wall. Preliminary data from this method suggests a significant difference in terms of decreased bacterial growth between control and experimental groups treated with the bias electrical field. As expected, the concentration of Ag‐GQD had to be different between strains due to their cell wall composition and different interactions between the Ag ions and the negatively charged proteins in ther cell wall. Taking advantage of the high biocompatibility of graphene and the antibacterial activity of silver, the Ag‐GQD nanocomposite becomes an effective nontoxic biomaterial suitable for numerous applications, such as self‐sterile textiles, biomedical devices, coatings, and cosmetics.
Tracking of epidermal growth factor receptor in cancer cells using nanodiamond
Authors: Lin Yu‐Wei
Published: November 2023

Nanodiamond (ND), a carbon‐based nanomaterial, has constituted a promising tool for bi‐imaging applications due to its excellent properties, including photostability, biocompatibility, and surface functionalization. Epidermal growth factor receptor (EGFR) is highly expressed in human cancer cells such as colorectal cancer (CRC). Cetuximab (Cet), a specific EGFR monoclonal antibody, has been approved for CRC therapy. Currently, nanoparticle‐antibody formulations display their ability to selectively bind on tumor cells through the ligand‐receptor interactions and increase the therapeutic efficacy. Here, we show the ND‐Cet complex that can be simply manipulated for tracking the EGFR in cancer cells. We demonstrated that ND‐Cet increased the uptake ability of drug complexes in the EGFR expressed cancer cells but not the EGFR absent cells. The fluorescence image of NDs can track the location of the binding of EGFR and Cet. We demonstrated a novel strategy using ND‐Cet to track the location of EGFR in cancer cells, which can be developed for the detection of specific cancer antigens.
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 Information

This 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 Information

DFG, 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.
Efficient Automatic Induction of Rules in Biological Systems
Authors: Whiting Mark E , Leduc Phil R , Cagan Jon
Published: November 2023

Rule based analysis of biological phenomena has rich potential but has been limited by our lack of methods for establishing systems of rules that discretely represent arbitrary structured data. Graph grammar is a formalism affording this kind of rule based representation of arbitrary information, and many tools have been developed to use graph grammars in engineering and design domains. However, inducting new graph grammar rules directly from a dataset is computationally complex and has not been automated; today it is done almost exclusively by hand, limiting the impact of the approach and mitigating its generalized use for analyzing biological phenomena.

This work introduces a method for improving the complexity bound of this problem by using chunking and probabilistic matching of new rules, and a system architecture for facilitating the quick evaluation of rules in new and unknown datasets. The new method has been evaluated with engineering, architectural and statistical datasets and offers insight into rules about the construction, function and fundamental dynamics of systems. This work provides novel insights to areas from multiscale modeling to representations of biological phenomena.
Determining the Effects of Intracellular pH on the Translational Response to Heat Shock
Authors: Triandafillou Catherine G , Dinner Aaron R. , Drummond D. Allan
Published: November 2023

Single‐celled organisms must be able to dynamically respond to changes in their environment to ensure survival and replication. The budding yeast S. cerevisiae grows best in acidic conditions, yet maintains cytosolic pH at or just above neutrality, expending a considerable portion of cellular ATP resources to do so. When cells experience stresses such as high temperatures or energy depletion, intracellular pH drops, equilibrating with the acidic environment. Given the strong buffering capacity of the cytosol, due mainly to an abundance of ionizable groups in biological molecules, this pH drop reflects a dramatic chemical change with wide‐ranging consequences for biological reactions in the cell. It has been shown that acidification drastically alters the material state of the cytoplasm, leading to solidification and decreased diffusion. Furthermore, acidification has been shown to be important for long‐term fitness following energy depletion.

In the case of heat shock, very little is known about the interplay between changes in pH and other, more well‐studied cellular changes such as the transcription and translation of the heat shock genes, reduction in cellular energy production, and the formation of mRNA‐protein granules. In order to understand how changes in intracellular pH are interpreted by the cell, and to what degree they affect the heat shock response and cellular fitness, we use a pH‐sensitive, ratiometric GFP derivative to measure intracellular pH during acute heat shock and recovery while simultaneously measuring the induction of heat shock response proteins using quantitative, multi‐color flow cytometry.

We find that the degree of cytosolic acidification alters the expression dynamics of molecular chaperones, independent of heat shock temperature and duration. We also find that cells which fail to acidify their interior suffer long‐term defects in pH regulation following stress, potentially indicating a loss of fitness.

Support or Funding Information

Research reported in this publication was supported by the National Institute of Biomedical Imaging And Bioengineering of the National Institutes of Health under Award Number T32EB009412 and the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE‐1144082.
Effect of thermal manipulation during Broiler chicken embryogenesis on the differential expression of Heat shock protein and Factor during heat stress on post‐hatch chicken
Authors: Alzghoul Mohammad Borhan
Published: November 2023

The objective of this study was to determine the effect of thermal manipulation during broiler chicken embryogenesis on the differential expression of heat shock proteins (HSPs) and heat shock factors (HSFs) during thermal stress at post hatch days 14 and 28 of age. Fertile chicken eggs (n=1500) were divided into five incubation treatment groups (300 each): control group was maintained at 37.8 °C 56% relative humidity (RH) throughout the incubation period; TM1 was subjected to TM at 38.5°C for 24 h and 65% RH daily during ED12–18; TM2 was subjected to TM at 39°C for 24 h and 65% RH daily during ED12–18; TM3 was subjected to TM at 39.5°C for 24 h and 65% RH daily during ED12–18 and TM4 was subjected to TM at 40°C for 24 h and 65% RH daily during ED12–18. At post‐hatch days 14 and 28 of age, chicks in TC groups were subjected to thermal challenge (TC) at 41.0°C for 6 h while naïve chicks were kept under regular conditions. Chick's response to TC was evaluated by determination of body temperature (Tb) and differential muscle mRNA expression of Hsp108, Hsp90, Hsp70 and Hsp60. During TC at days14 and 28 of age, the Tb of TM chicks was significantly lower than those of the controls. When compared to the control, TM resulted in a rapid significant increase in mRNA levels of Hsps and HSFs in the muscle during TC at post hatch days 14 and 28 of age.

Support or Funding Information

Deanship of Research, Jordan University of Science and Technology (Project ID# 66/2016).
ACTIVITY OF HEPATIC EXPRESSION OF GLUTATHIONE S‐TRANSFERASE ISOTYPES (ALPHA, PI, AND MU) OF MUS MUSCULUS EXPOSED TO ATRAZINE IN UTERO
Authors: Finley Lauren
Published: November 2023

Atrazine (ATR) is a commonly utilized herbicide that remains in the soil, where it can seep into ground water. The Environmental Protection Agency (EPA) has expressed concern, and has set a safe concentration to 3 ppb. While there have been numerous studies conducted to determine the detrimental effects of chronic exposure to ATR, very few studies have focused on in utero effects. Of interest is the family of liver enzymes specific to detoxification of ATR: glutathione S‐transferases (GST). Previous research has identified that chronic exposure to ATR in fish shows a decrease of enzymatic activity for the Mu isotype of GST, but has not looked at the other two isotypes, Alpha and Pi. The current study utilized CD‐1 mice (Mus musculus) newborn pups of mothers exposed to ATR (0, 3, 30 ppb) during gestation. Analysis of birth weight and GST enzymes activities were performed to determine the relationship between in utero exposure to ATR and the enzymatic activity of the three isotypes of GST.
Identification of a Novel Pathomechanism Underlying a Congenital Disorder of Glycosylation: Crosstalk between Hexosamine Biosynthetic Pathway, PI3K/AKT Signaling, and Protein Glycosylation
Authors: Wong Sunnie Yan‐Wai , Perez Stefanie , Huang Xiaoping , Stiers Kyle , Beamer Lesa , Foulquier Francois , Berry Gerard , Kozicz Tamas , Morava Eva
Published: November 2023

Phosphoglucomutase 1 (PGM1) deficiency is a recently identified subtype of congenital disorder of glycosylation that can be treated by dietary modification. PGM1 is required for the interconversion of glucose 1‐phosphate and glucose 6‐phosphate, and is a key enzyme in glycogen metabolism. Although the exact mechanism remains unclear, PGM1 deficiency has been associated with abnormal intracellular glucose and galactose metabolites, as well as reduced N‐linked glycosylation in the endoplasmic reticulum and Golgi. These metabolic disturbances are thought to be responsible for both missing and truncated glycans that have been detected in patient serum transferrin. A recent landmark study demonstrated that oral D‐galactose supplementation improved cellular glycosylation in six patients and resolved hypogonadotropic hypogonadism in two patients. While the clinical benefits of galactose supplementation in PGM1 deficiency are clear, its biological effect on the biochemical and cellular level has not been characterized in detail. It was assumed that dietary galactose supplementation improves galactosylation by increasing intracellular UDP‐galactose relative to UDP‐glucose; however, this has not been confirmed experimentally, nor has an alternative mechanism of action been explored. Here, we report no difference in the ratio of intracellular UDP‐glucose to UDP‐galactose between patient and control cells, despite significant improvement in N‐linked and O‐linked glycosylation in patient cells following galactose supplementation. We found no evidence to support galactose acting as a pharmacological chaperone for PGM1 missense variants. Surprisingly, we discovered disruptions in the hexosamine biosynthetic pathway (HBP) at the metabolic and transcriptional level, as well as downregulation of PI3K/AKT signaling in PGM1 deficient patient cells. Interestingly, galactose supplementation appears to rescue HBP and PI3K/AKT signaling. Collectively, our preliminary studies suggest a hitherto unexplored crosstalk between HBP, PI3K/AKT signaling, and protein glycosylation.

Support or Funding Information

This study is supported in part by the Hayward Foundation and by 1 U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the Louisiana Clinical and Translational Science Center.
From Bookworm to Team Player: Problem Based Learning
Authors: Rao Sandy
Published: November 2023

Medical education faces a daunting task: ensure medical students are equipped with a comprehensive knowledge base while simultaneously developing future clinical physicians. Often this is achieved through an educational dichotomy: academic learning during the first two years and clinical learning during the final two years of medical school. But wouldn't it be more effective to introduce medical students to clinical skills like critical thinking and working with groups before they start seeing patients? Wouldn't they be more prepared to translate their knowledge base into practice if they were taught to do just that from the start of their education? This is where Problem Based Learning (PBL) offers a means of transitioning medical students from bookworms to team players.

Problem based learning is a method of teaching where students work through a clinical case in small groups. It requires students to problem solve, guide their own learning, and work together with other students. Students must formulate questions, research solutions, and present their findings to the group (Gijselaears, 1996). These tasks require critical thinking and strong communication skills, both of which are vital for a career as a physician. A facilitator is present to help students strengthen these skills by offering guidance in formulating appropriate questions, conveying vital information, and potentially offering real‐life experiences (Gallagher, 1997).

Several studies have found improved anatomy test scores in students participating in PBL learning over didactic learning, suggesting that PBL is a more effective teaching style (Williams, 2014). However, some studies showed no difference between these scores and that PBL students rated themselves as possessing less medical knowledge (Koh, 2008). Further aspects of PBL learning were addressed through a questionnaire focused on student's experiences with an anatomy PBL. Some responses discussed the practicality of a PBL session; how each group has different experiences based on the effectiveness of their facilitator and how learning is difficult since learning objectives are not answered as clearly as in a didactic model. This lecture will focus on the advantages and disadvantages of PBL in anatomy education, as well as the personal experiences of medical students with this style of learning.