bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosine phosphorylation of BipA and its effect on the binding affinity of BipA
Authors: Blaha Gregor , Conn Adam , Fan Haitian , Hahm Joseph , Diggs Stephen , Preston Williams , Wang Yinsheng
Published: November 2023

BipA is a translational GTPase with homology to translational elongation factor‐G. BipA changes its binding preference from ribosome to small ribosomal subunit depending on the intracellular ppGpp concentration. The cellular levels of ppGpp are correlated inversely to the availability of nutrients. BipA is also known to be responsible for interactions between enteropathogenic E. coli (EPEC) and human small‐intestinal epithelial cells in vitro. BipA knock out mutants of EPEC cannot attach and efface intestinal cells. BipA is tyrosine‐phosphorylated in vivo but only in the pathogenic strains of E. coli such as EPEC. We identified the specific residues that are in vitro phosphorylated and determined their effect on the BipA's binding affinity for ppGpp, ribosomes, and ribosomal subunits.
Development of Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Authors: Escamilla Yaritza , Hughes Casey , Hu Yanmei , Bullard James M
Published: November 2023

Introduction

Pseudomonas aeruginosa is a common cause of nosocomial infections and the leading cause of mortality in patents with cystic fibrosis. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis.

Results

P. aeruginosa glutaminyl‐tRNA synthetase (GlnRS) was overexpressed in E. coli cells, and purified to homogeneity. GlnRS from P. aeruginosa is a discriminating synthetase with respect to the requirement for the presence of tRNAGln to produce a stable glutaminyl‐AMP intermediate. When compared with GlnRS from E. coli the amino acid sequence of GlnRS from P. aeruginosa are only 60% conserved. The kinetic parameters for interaction with tRNA were determined and the KM and Vmax were 1.0 μM and 0.23 μM min−1, respectively. The observed kcat was calculated to be 0.15 s−1 resulting in a kcat/KM value of 0.15 s−1μM−1. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa GlnRS in a high throughput format. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with the ability to inhibit function of the enzyme. Three compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM02E04, BM04B05, and BM04H03 inhibit the activity of P. aeruginosa ArgRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. All three of these compounds exhibited promising MICs against Gram+ bacteria and moderate activity against Gram‐ bacteria causing respiratory infections.

Conclusion

GlnRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.

Support or Funding Information

Research was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017) and from the NIH UTPA RISE program grant # 1R25GM100866‐01.
Knockdown of axonemal inner arm dynein protein IC138 Trypanosoma brucei causes flagellar detachment and defects in motility
Authors: Marchese Anthony , Wilson Corinne , Springer Amy L.
Published: November 2023

Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Motility is critical for its life cycle progression and pathogenesis. However, the regulation of flagellar function is not well understood. T. brucei utilizes a flagellum that attaches along the length of the cell, beating from tip‐to‐base to move the cell forward. Studies have found that the assembly and regulation of proteins within the axoneme, the microtubule‐based cytoskeleton of the flagellum, are well conserved. Within the axoneme are outer and inner arm dyneins, structures known to be involved in microtubule sliding which drives cell motility. Inner arm dynein I1/f has been found to play an important role in regulation of the flagellar beating. We are interested in the characterization of T. brucei IC138, an intermediate chain of dynein I1/f. This protein has been has been studied in green alga Chlamydomonas reinhardtii and shown to be a phosphoprotein important for the regulation of dynein I1/f. A T. brucei strain was created to inducibly target IC138 by RNA interference. By 72‐hours post‐induction, quantification of the knockdown revealed that IC138 protein levels were reduced by 87%. The knockdown cells were monitored for phenotypic changes over 108 hours following induction, and exhibited reduced doubling times, slowed or abnormal motility and partially or completely detached flagellum. Abnormal motility phenotypes included slow twitching or irregular beating that became more severe over time, such that by 72 hours post‐induction approximately 80% of the cells were immotile. Flagellar detachment increased over time, although less dramatically than immotility, affecting less than 60% of the population. The motility defect is seen early following induction and is evident in both detached and intact flagella and thus is consistent with the role of IC138 as a regulator of motility. Depletion of IC138 may disrupt the assembly of dynein I1 or other interacting proteins, possibly contributing to the phenotypes observed. Classification of other proteins missing in IC138 depleted cells will help to identify C. reinhardtii orthologs or lead to the discovery of novel IC138 binding partners. This identification could reveal additional components of T. brucei motility regulation and a provide basis for future research. Further knowledge of the protein interactions within dyneinI1/f may lead to a better understanding of the phenotypic changes resulting from IC138 depletion.

Support or Funding Information

NIH 7R15AI101941‐02
Assessing Species‐Specific Determinants of DNA Binding in McrB Homologs
Authors: Hosford Christopher John , Johnson Virginia Mitchell , Chappie Joshua
Published: November 2023

Antibiotic resistance poses a significant threat to human health. The lagging development of new antibiotics and the rapid exchange of resistance genes have created a need for alternative methods to combat emerging ‘superbugs’. One promising strategy involves using lytic bacteriophages (phages) – bacterial‐specific viruses that lyse and kill their hosts while releasing their progeny. Modification‐dependent restriction systems (MDRS) are innate bacterial defense systems that recognize and degrade modified phage DNA, thus preventing mature phage production in the host. MDRS are highly conserved in antibiotic resistant bacteria and a number of phage‐encoded inhibitors have been identified. Understanding the molecular mechanisms of these systems can therefore provide a means to increase the efficacy and therapeutic potential of phages.

McrBC is a two‐component MDRS that restricts DNA containing 4‐, 5‐, or 5‐hydroxy‐methyl cytosines. The first component, McrB, contains an N‐terminal domain that recognizes and binds the modified site and a C‐terminal AAA+ motor domain that hydrolyzes GTP and mediates nucleotide‐dependent oligomerization. Recent crystallographic data shows that Escherichia coli (E.coli) McrB flips the methylated cytosine out of the duplex and stabilizes the resulting gap by inserting a tyrosine residue. However, this residue is not conserved suggesting that other mechanisms may exist for binding modified DNA. To examine the species‐specific determinants of McrB DNA‐binding, we have purified the putative DNA binding domains of different bacterial and archaeal McrB homologs and compared their ability to bind modified DNA with the prototypical E.coli McrB DNA‐binding domain. Thermococcus gammatolerans (T.gammatolerans) McrB binds the same DNA sequences as E. coli despite very little sequence homology. Bacillus cereus (B.cereus) McrB, in contrast, lacks a canonical N‐terminal domain and instead contains a novel domain that is inserted in the AAA+ motor core. This insert domain does not bind DNA containing methylated cytosines, suggesting it directs the conserved cleavage machinery to a different target. Furthermore, we have also crystallized the putative B.cereus and T.gammatolerans DNA binding domain and carried out preliminary X‐ray diffraction analysis. Together, these data provide insights into the substrate preferences of different McrB family members and have the potential to aid in the design of novel species‐specific therapeutics for treating multi‐drug resistant bacterial infections.
The Role of the C1q Domain of Zebrafish Otolin1a in Otolith Morphogenesis
Authors: Das Olipriya
Published: November 2023

Otolins are constituent proteins of vertebrate otoliths and otoconia, acellular biominerals that are essential for detecting linear accelerations in the inner ear. Based on their sequence similarity to the atypical collagens VIII and X, the otolin peptides are predicted to comprise an N‐terminal signal sequence, an internal collagen domain, and a C‐terminal C1q domain. C1q domains have been shown to trimerize on their own as well as direct the trimerization of larger proteins. In the case of collagen VIII and X, the protein trimers then form a three‐dimensional matrix, with the C1q and collagen domains as the hubs and spokes, respectively. By analogy, we hypothesized that the C1q domain of otolins directs the assembly of protein trimers that are required to form the extracellular matrix of otoliths and otoconia. We tested whether the C1q domain confers trimerization on a protein that normally exists as a monomers: we investigate whether a fusion of the Otolin1a C1q domain with thioredoxin (TrxC1q) also trimerizes. The zebrafish Otolin1a C1q domain was expressed in bacteria fused to thioredoxin. Multimerization of purified recombinant fusion proteins was assessed by size‐exclusion chromatography and gel electrophoresis. Under denaturing conditions, the recombinant fusion proteins formed monomers and trimers, but not dimers or higher‐ order complexes. Under native conditions, they formed only higher‐order complexes. In contrast, thioredoxin alone appeared as a monomer. Our results are consistent with the proposed ability of the Otolin C1q domain to form trimers that, in turn, assemble into higher‐order complexes. Experiments are underway to characterize the various complexes in more detail and to test whether the C1q domain is necessary to trimerize full‐length Otolin1a.

Support or Funding Information

Supported by NIH R01DC006962
Altering the Enantioselectivity of Tyrosyl‐tRNA Synthetase by Insertion of a Stereospecific Editing Domain
Authors: First Eric Allen , Richardson Charles
Published: November 2023

Translation of mRNA by the ribosome is stereospecific, with only L‐amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of D‐amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl‐tRNA synthetases, (2) binding of aminoacyl‐tRNAs to EF‐Tu, and (3) recognition of aminoacyl‐tRNAs by the ribosome. As a first step towards incorporating D‐amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl‐tRNA synthetase. This enzyme is unusual among aminoacyl‐tRNA synthetases, as it can aminoacylate tRNA with D‐tyrosine (albeit at a 30‐fold reduced rate compared to L‐tyrosine). To change the enantioselectivity of tyrosyl‐tRNA synthetase, we introduced the post‐transfer editing domain from phenylalanyl‐tRNA synthetase into the connective polypeptide 1 domain of tyrosyl‐tRNA synthetase. We show that the phenylalanyl‐tRNA synthetase editing domain is stereospecific, hydrolyzing L‐Tyr‐tRNA, but not D‐Tyr‐tRNA. We further show that inserting the editing domain into tyrosyl‐tRNA synthetase decreases the activity of the synthetic site in tyrosyl‐tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the L‐Tyr‐tRNA product. Overall, inserting the phenylalanyl‐tRNA synthetase editing domain, results in a two‐fold shift in the enantioselectivity of tyrosyl‐tRNA synthetase towards the D‐Tyr‐tRNA product. This effect is further enhanced by introducing hyperactivating mutations into the editing domain, with one variant (F145A/S211A) preferentially aminoacylating tRNA with D‐tyrosine when equimolar concentrations of L‐ and D‐tyrosine are present.

Support or Funding Information

This research was supported by funds from Louisiana State University Health Sciences Center in Shreveport and the Biomedical Research Foundation of Northwest Louisiana.
APOBEC3F‐Vif Binding Interface Elucidated by Multiple Experimental Approaches
Authors: Harris Reuben , Richards Christopher , Shaban Nadine , Anderson Brett , Demir Ozlem , Amaro Rommie
Published: November 2023

Background

APOBEC3 family DNA cytosine deaminases provide overlapping defenses against pathogen infections. However, most viruses have elaborate evasion mechanisms such as the HIV‐1 Vif protein, which subverts cellular CBF‐beta and scaffolds the assembly of a polyubiquitin ligase complex to degrade these enzymes. Despite advances in APOBEC3 and Vif biology, a full understanding of this direct host‐pathogen conflict has been elusive.

Methods

We combine virus adaptation, structural, and computational studies to interrogate the APOBEC3F‐Vif interface and build a robust model for this vital host‐pathogen interaction.

Results

First, new structures of the Vif binding domain of APOBEC3F were solved by x‐ray crystallography. Second, a recurring compensatory amino acid substitution in HIV‐1 Vif from virus adaptation experiments provided an initial docking constraint, and microsecond molecular dynamics simulations were used to optimize interface contacts. Third, single‐cycle and spreading virus infectivity experiments were used to validate a long‐lasting electrostatic interaction predicted by molecular dynamics simulations to occur between APOBEC3F E289 and HIV‐1 Vif R15.

Conclusions

Our studies have yielded a comprehensive structural model for the APOBEC3F‐Vif interaction, which explains most prior genetic data. Taken together with the dynamic nature of this host‐pathogen interaction, our data suggest a “wobble model” to explain how HIV‐1 Vif has evolved to bind different APOBEC3 enzymes and, more generally, explain how pathogens may evolve to escape innate host defenses (i.e., a molecular explanation for the Red Queen hypothesis). Our studies also suggest that the APOBEC‐Vif interface may be a less favorable target for new antiviral drug development in comparison to other heterologous surfaces of the Vif‐CBF‐beta ubiquitin ligase complex.
The HECT Domain and RCC1‐like Domain 5/Herc5 Protein Exhibits Intrinsic Ubiquitin Ligase Activity
Authors: Ronchi Virginia P , Klein Jennifer , Streich Frederick , Haas Arthur
Published: November 2023

HERC5 is a Type1 interferon‐induced Hect ligase having an essential role in the cellular innate immune response to viral and inflammatory signals. HERC5 is thought to be the principle ligase for conjugation of the interferon‐induced ISG15 ubiquitin‐like protein to a broad subset of cellular targets; however, HERC5 also is reported to ubiquitinate NM23B and the Hect domain forms a ubiquitin thioester intermediate with its Cys994 active site residue. The specificity of HERC5 for components of both ISG15 and ubiquitin conjugation has not been previously explored. In the current studies, HERC5‐limiting kinetic studies of 125I‐ISG15 chain formation showed cooperative sigmoidal kinetics with respect to UbcH8~ISG15, as evidenced by a linear Hill plot (nH ≈2) and a biphasic curve consistent with substrate inhibition, requiring two functionally‐ distinctive UbcH8~ISG15 binding sites (K0.5= 12 ±1 nM and kcat‐ISG15t = 1.8 ± 0.02 10−5 s−1). By exploiting the intrinsic ability of Hect ligases to form polyubiquitin chains in the absence of target proteins, we now show that recombinant HERC5 exhibits intrinsic ubiquitin ligase activity. A biochemically‐defined semiquantitative E2 screen showed that HERC5 assembled 125I‐polyubiquitin chains and displayed a broad E2 specificity. Similar to ISG15, HERC5 displayed cooperative sigmoidal kinetics (nH ≈ 2) for polyubiquitin chain assembly and showed substrate inhibition. The UbcH5 family was the cognate E2 for HERC5 with a value of K0.5 ≈ 400 nM and kcat‐ub range of 0.01 s−1. The inactive UbcH5BC85S‐ubiquitin oxyester substrate analog and UbcH5BC85A product analog were inhibitors of the ligase function. Inactive HERC5C994A and the blocked derivative Ac‐PheNH2 inhibited the ligase activity by possible affecting HERC5 oligomerization. Isotope dilution assays of 125I‐wild type polyubiquitin chain formation using unlabeled K>R point mutants at each of the seven ubiquitin lysines showed that HERC5 forms K48‐linked chains capable for targeting proteins for 26S proteasome‐dependent degradation. Together with early observations that HERC5 binds polysomes and target nascent polypeptides for ISG15 conjugation, the present studies suggest interferon induction of HERC5 might upregulate ubiquitin‐dependent antigen presentation; additionally, HERC5 is a potential bifunctional ligase functioning in both ubiquitin and ISG15 ligation pathways.

Support or Funding Information

Funded by GM34009, GM47426 to A.L.H.
The Role of Dictyostelium UbpA in Nutrient Sensing and the Growth‐to‐Development Transition
Authors: Behrens Michael R. , Burghart Victoria J. , Lindsey David F.
Published: November 2023

UbpA is a ubiquitin processing protease required for the growth‐to‐development transition during development of the social amoeba Dictyostelium. UbpA, a homolog of yeast Ubp14 and human IsoT/USP5, functions in the disassembly of free polyubiquitin chains. ubpA− cells misregulate genes at the growth‐to‐development transition, do not show EDTA‐resistant cell‐cell adhesion, and fail to aggregate. To identify genes that operate in UbpA‐dependent pathways, we carried out REMI mutagenesis of ubpA− cells and screened for second‐site suppressors of the developmental defects. One suppressor mutant, SU35, developed finger‐like structures and showed EDTA‐resistant cell‐cell adhesion. The SU35 suppressor mutation was located in an open reading frame of 419 codons that could encode a 47.0 kDa protein not similar to any other sequence in the database. Northern analysis indicated that the SU35 gene was expressed in growing wild‐type cells and downregulated upon starvation; in ubpA− cells, it is expressed but not downregulated. In other experiments, we have shown that UbpA is required for the growth‐stage expression of lmcB, which is involved in sensing nutrient levels and signaling cells to develop upon starvation. To elucidate LmcB‐dependent pathways, pulldown experiments utilizing His‐tagged LmcB were used to identify proteins that interact with LmcB. These included UduA3 and the cytidine deaminase, CdaA. Direct binding experiments confirmed the interaction between LmcB and CdaA. cdaA is upregulated at the growth‐to‐development transition. These results will lead to a better understanding of the mechanisms cells use to sense starvation and respond by making the transition from growth to development.
Assessment of CCN2 Independent Modules Regenerative Capacity on Osteoarthritis and Further Selecting the Most Suitable Among them as a Potential Therapeutic Drug
Authors: Abdelkader Tarek , Aoyama Eriko , Nishida Takashi , Hattori Takako , Janune Danilo , Hara Emilio S. , Ono Mitsuaki , Tabata Yasuhiko , Kuboki Takuo , Kubota Satoshi , Takigawa Masaharu
Published: November 2023

CCN family proteins (Connective tissue growth factor (CTGF)/CCN2, Cystein rich protein (Cyr61)/CCN1, and Nephroblastoma overexpressed gene (nov)/CCN3) play important roles in differentiation and regeneration of cartilaginous tissues. CCN2/CTGF is known to promote the regeneration of articular cartilage. It's comprised of 4 highly interactive independent modules, each of which was suggested to have its own biological activity. This study aims to assess the effects of these modules independently and their combinations on chondrocytic cells in vitro and damaged cartilage in vivo, especially in relation to full length CCN2.

Methods

Effect of a single module, or their different combination, was evaluated with human chondrocytic HCS‐2/8 cells. The cellular phenotype was estimated by the gene expression of chondrocytic markers. Proteoglycan synthesis and proliferation were evaluated by [35S]‐sulfate and [3H]‐thymidine incorporation assays, respectively. Physical interaction of 2 modules was kinetically examined by a surface plasmon resonance (SPR) methodology. Cartilage regeneration in vivo was evaluated by using 2 rat models simulating osteoarthritis via surgical or chemical intervention. Angiogenic affinity of independent modules was assessed in vitro by tube formation assay using matrigel with human umbilical endothelial cells (HUVEC) and ex ovo by CAM (chorioallantoic membrane) assay with chick embryo.

Results

Functional analysis in vitro revealed a biological activity even stronger than the full length CCN2 in a particular module. Interestingly, mixed application of all 4 modules almost reconstructed the bioactivity to the level of the full length. SPR analysis uncovered significant interaction of 2 modules and full‐length CCN2. The result of cartilage regeneration experiments in vivo was also consistent with the findings obtained in vitro. The results of the matrigel assay indicate that the same module has tube formation potential under special circumstances and a similar effect could be observed in the CAM assay ex ovo.

Conclusion

These results indicate independent bioactivity of each module for cartilage regeneration and suggest unknown means of binding to construct a possible tetramodular complex that is functionally comparable to the full length CCN2. Also the effect of the proposed independent module as a regenerative drug on angiogenesis must be kept in mind and further analyzed as this characteristic might be beneficial for vascular regeneration in spite the fact that it's considered a side effect for a proposed OA treatment.
Aplip1 regulates Dynein‐dependent but not Kinesin‐dependent nuclear movement in muscle
Authors: Camuglia Jaclyn , Auld Alexander , Murphy Ciaran , Folker Eric
Published: November 2023

Muscle cells are composed of multinucleated cells called myofibers. The nuclei in myofibers undergo a series of dynamic movements to maximize internuclear distance. The importance of these movements is illustrated by the high correlation between mispositioned nuclei and muscle disease. Though the positioning of nuclei is a distinctive feature of the myofiber, the mechanisms for nuclear movement and positioning are only now emerging. It is known that two separate pathways contribute to nuclear movement in muscle. In the cortical pathway, Kinesin moves Dynein to the cell cortex where it is anchored. From that position, Dynein applies a pulling force on nuclei. Additionally, in the proximal pathway, Dynein and Kinesin apply force directly to the nuclei. Although Dynein and Kinesin are required for both pathways, the two pathways are spatially segregated suggesting that separate pools of Dynein and Kinesin must be specified for each pathway. To understand how the two pools of Dynein and Kinesin are differentially regulated, we have tested factors known to regulate motor activity in other contexts. One candidate, Aplip1 has been demonstrated to coordinate Kinesin and Dynein during axonal transport and was therefore a strong candidate to regulate Dynein and Kinesin in muscle. We found that Aplip1‐eGFP localizes at the cortex of the muscle cell, suggesting a role for Aplip1 in the cortical pathway. Consistent with this, embryos that were heterozygous for null mutations in both Aplip1 and Dynein displayed mispositioned nuclei. However, embryos that were heterozygous for null mutations in both Aplip1 and Kinesin were unaffected, suggesting that Aplip1 does not contribute to Kinesin‐dependent trafficking of Dynein. Together, these data suggest that Aplip1 specifically regulates Dynein activity at the cell cortex by a Kinesin‐independent mechanism.
Visualization explorer for classification and functional enrichment of fecal microbiome to metabolites
Authors: Kumar Raina , Yang Ruoting , Gautam Aarti , Hammamieh Rasha , Mudunuri Uma , Jett‐Tilton Marti
Published: November 2023

Advanced high throughput technologies such as Next Generation Sequencing have created opportunities for scientists to explore the human microbiome more intensively and effectively. This enables us to understand the impact of imbalances in normal microbial flora through determining changes in the microbial composition and investigating how these correspond with changes occurring at the molecular level and the physiological level in the human body. Here, we developed the visualization explorer component for our in‐house automated microbiome analysis pipeline. The explorer takes enriched microbial composition per sample as input and provides a unique way to classify and visualize the microbiome data, organized under tabs. Under the relative abundance tab is visualization of the microbial composition across different samples with taxonomic annotation using different charts such as bar and radial charts. The PCA/Diversity index tab allows one to perform and visualize the principal component analysis and microbial diversity index comparison between treatment and control categories. Under the functional enrichment tab, the metabolite enrichment for the present microbial composition per sample is conducted and provides the predicted significant pathways between two categories in a network view. This visualization explorer was built using R open source language and Shiny server API. PICRUST performs the metabolite prediction and pathway enrichment. For the network visualization, explorer uses Graphviz open source software. The other features of this explorer include several visualization options such as heatmaps, histograms and line charts to compare differentially abundant bacterial species between disease and control samples.

Support or Funding Information

DHPRDTEGDF 374000GJ
Purification and Expression of Rhodobacter sphaeroides: A Predicted Dioxygenase
Authors: Neumann Stephanie Michelle , Nichols Caytlin , Selinsky Barry
Published: November 2023

Prostaglandin H2 synthase is a dual function cyclooxygenase/dioxygenase whose structure has been studied extensively. Bacterial genes that generate proteins structurally resembling mammalian cyclooxygenases have been identified through bioinformatics techniques. One understudied protein in this peroxidase‐cyclooxygenase superfamily is predicted from the Rhodobacter sphaeroides genome. In this study, we subclone and express the putative dioxygenase from Rhodobacter sphaeroides both as a SUMO fusion protein and with a C‐terminal histidine tag. The expression and purification conditions for the Rhodobacter protein were optimized, yielding soluble protein suitable for enzymatic analysis. The substrate specificity and function of the protein, determined through GC/MS analysis of lipid utilization and product formation, will be reported. In future research we aim to conduct structural studies on the putative dioxygenase enzyme from Rhodobacter, which could have future implications regarding the evolutionary characteristics of this bacterial superfamily of enzymes.
Characterization of bacterial homologs of prostaglandin H2 synthase using GC/MS
Authors: Nichols Caytlin , Butchy Margaret , Selinsky Barry
Published: November 2023

Prostaglandins are formed by the enzyme prostaglandin H2 synthase (PGHS), which has been found in all vertebrates whose genomic sequence is known. In the peroxidase‐cyclooxygenase superfamily, several archaeal, bacterial, fungal, and plant open reading frames (ORFs) were predicted to have structures similar to mammalian PGHS. Our laboratory has subcloned and expressed several bacterial ORFs encoding for proteins resembling mammalian PGHS. This report describes the development of a GC/MS assay to follow substrate utilization in these bacterial enzymes. The method was tested using ovine prostaglandin H2 synthase (which uses arachidonic acid), and the linoleate 10S dioxygenase, expressed from an ORF copied from Nostoc punctiforme. The lipid preference of both enzymes was confirmed by our GC/MS results. Using site directed mutagenesis, active site variants will be made in attempt to convert the Nostoc 10S‐dioxygenase into an enzyme that will use arachidonic acid as a substrate, as seen in mammalian PGHS. The success of our mutagenesis and enzymatic analysis of the mutants will be discussed.
Gene Expression Patterns in Trauma Patients: A Systems Biology Approach
Authors: Srinivasan Seshamalini , Donohue Duncan E. , Gautam Aarti , Conroy Amanda S. , Nelson Mary F. , Cohen Mitchell J. , Hammamieh Rasha , Jett Marti
Published: November 2023

Coagulopathy in trauma patients is associated with a threefold to fivefold increase in mortality. An initial abnormal coagulation profile, as measured by routine clinical tests such as PT and PTT, increases the odds of dying by 35%–326%. (MacLeod et al. 2003). Yet, the current understanding of the mechanisms underlying the development of coagulopathy following severe injury is extremely limited making its prevention and treatment limited. The aim of this study is to use systems biology approaches to integrate multiple data types including clinical findings, various “‐omics” approaches from the onset of the disease to develop a universal picture of the progression of this hemostatic dysfunction. Adult trauma patients were recruited based on previously published inclusion/exclusion criteria. Blood collection was performed every 2–6 hours for the first 24 hours and daily up to 4 days. Comprehensive demographics, injury, treatment and outcome data was prospectively collected. A temporal change in gene expression was studied using whole genome microarrays which probe 50,000 biological features (Agilent Technologies, CA). 79 samples representing 25 patients were processed and analyzed. A comparison of samples from patients with ISS (Injury Severity Score) below the median of 19 (38 samples) vs. those at or above 19 (n=41) showed significantly higher expression (FDR adjusted moderated t‐test p<.05) for DHX58, which is involved in interferon signaling, and lower expression for MIAT, a known biomarker of myocardial infarction, in samples from high ISS patients. Work is ongoing to fully characterize mRNA expression changes in these patients to better understand the contribution of demographic, clinical, and treatment confounds towards identifying molecular targets with potential utility in informing treatment decisions.

DISCLAIMER

Research was conducted in compliance with IRB approved human subjects protocol #H7462‐25307‐07A. The views, opinions, and/or findings contained in this report are those of the author(s) and should not be construed as official Department of the Army position, policy, or decision, unless so designated by other official documentation.
Mapping Tyrosine Phosphorylation Networks by Chemical Proteomics
Authors: Rogers William Eric , Ghassemian Majid
Published: November 2023

Chemical Proteomics is an unbiased approach to cellular signaling, but it relies on the ability to chemically react a specific post‐translational modification for analysis. Previously, a Beta‐elimination/Michael's addition reaction has been developed to chemically probe phosphorylated Serine and Threonine residues. The stability provided to the phosphate in by attachment to the ring structure of Tyrosine makes this process impossible. Herein, I present a procedure for chemically labeling phospho‐Tyrosine (pY) through a modified Suzuki coupling on tryptic peptides. This is followed by a biotin‐streptavidin affinity based enrichment and mass spectrometric (MS) analysis. This procedure solves the two main problems of pY analysis. First, the “flyability” of the peptide is increased drastically by removing the negative charge that is present on the phosphate (pKa=1.76) at the standard pH (~2.0) used in MS. Second, and as importantly, the problem of rarity of phosphorylated peptides in a cellular system is also solved. With the addition of SILAC, ICAT, or other isotopic labeling techniques it is straightforward to map the propagation of signaling along pathways and possibly uncover novel effects by that signaling that can not be determined via conventional methods.

Support or Funding Information

W.E.R is a recipient of an Arne N. Wick pre‐doctoral research fellowship from the California Metabolic Research Foundation.
Phosphorylation of Neuronal Proteins in Drosophila melanogaster Changes With Age
Authors: Thomas Alexis , Haberman Adam
Published: November 2023

Cells, such as epithetical and red blood cells, have the capacity to regenerate throughout an organism's lifetime. However, neurons are unique because they do not regenerate; once most neurons are formed, they last the lifetime of an organism. This lack of regeneration is the reason that neurodegenerative diseases, including Alzheimer's Disease and Huntington's Disease, are so detrimental; the diseases target cells that can't be replaced if they're damaged. These diseases occur later in life, which indicates a problem with the normal aging process of neurons; these diseases are associated with abnormal protein accumulation. There are housekeeping genes that are associated with normal cell upkeep; this upkeep can happen by protein degradation by either proteosomal digestion or autophagy. The two degradation pathways are quality control mechanisms that allow for degradation of abnormal or old proteins without destroying the undamaged proteins, which allows for longevity in structures such as neurons. As neurons age, they acquire intracellular aggregates despite these degradation pathways, and some of these aggregates are key features of neurodegenerative diseases. We are interested in understanding how well degradation pathways work in neurons of different ages, and what changes in older cells lead to intracellular aggregates. One key way to understand how neurons age is to investigate how neuronal proteins change over time. Changes in protein abundance or post‐translational modification of existing proteins could severely affect how cells deal with the stresses of aging. To determine how proteins change with age, protein levels and post‐translational modifications are compared between young and old neurons to determine how the same protein could differ over time. The changes are then analyzed in order to identify post‐translational modifications that are important for healthy neuronal aging and investigate the functional significance of the modifications. We have focused on identifying changes in protein phosphorylation between young and old brains in wild type Drosophila melanogaster by two dimensional gel electrophoresis and liquid chromatography mass spectroscopy. Preliminary results have found increased levels of protein phosphorylation in old Drosophila melanogaster heads. Once we analyze these findings through mass spectroscopy, we will be able to identify the exact proteins that differ in order to study their function in Drosophila.
Using Polymerase Chain Reaction Inhibition to Study Small Molecule Binding to DNA
Authors: Ghannam Ryan , Smith Sheila R , Benore Marilee Ann
Published: November 2023

Detection of DNA‐molecule interaction can be challenging. Knowing that Polymerase Chain Reaction (PCR) can be inhibited by interfering agents, this interaction was exploited as a potential assay. Using whole plasmid and DNA fragments, the ability of small molecules to inhibit the PCR reaction was studied under diverse conditions and testing several potential inhibitors.

Support or Funding Information

University of MI Dearborn campus grants
Purification and Characterization of DNA from Fresh Versus Frozen Chicken Embryos
Authors: Byk Jennifer , Smith Sheila R , Benore Marilee Ann
Published: November 2023

Purification of DNA from tissues can be carried out by several methods. The ease of purification, yield and functionality of the DNA isolated varies depending on the method used. Using both frozen and fresh chick embryos, we tested several kits and traditional methods. Comparative analysis of the yields, purity, and integrity of fresh tissue versus frozen tissue were measured.

Support or Funding Information

University of Michigan Dearborn Campus Grants
Evaluating the Extraction Efficiency of Food Borne Pathogens on Automated Homogenizer Platforms
Authors: Garrett Shari , Atwood James , Easparro Brandon
Published: November 2023

There is an ever increasing demand for food and beverage manufacturers to adhere to rigorous food safety regulations. As part of the global need for safe food, food and beverage manufacturers test their products for the presence of food borne pathogens. While safety regulations describe what pathogens should be controlled through testing there is no predefined method of sample preparation or detection. Typically, the first step in a food borne pathogen detection study is the disaggregation of the food product or the washing of the sample to release bacteria from the food surface using a Stomacher. While the later method is great for large masses of food stuffs, some researchers may require small amounts of product for analysis. Bead mill homogenizers, as well as rotor‐stator homogenizers, can be used to aid in the sample preparation process. Processing can be completed in less than one minute while maintaining bacterial cell and analyte integrity. Depending on the pathogen of interest and the food matrix, the downstream results, including sensitivity can vary greatly based on the sample preparation method chosen.

With this is mind, we analyzed two sample preparation techniques on commonly contaminated foods. Meat and spinach samples were inoculated with known levels of recombinant E.coli expressing Green Fluorescent Protein. The inoculated samples were processed on a bead mill homogenizer and an automated rotor‐stator homogenizer. The homogenates were grown on nutrient agar and the proportion of viable cell recovery was quantified. Limit of detections were determined by lysis of recovered homogenates, DNA purification and PCR amplification.

It was determined that both homogenization techniques were able to process food samples for the extraction of live cells and plasmid DNA. The processing efficiency can deliver a 76% cell recovery with a PCR detection limit to 1×10−5 CFUs/ml. A linear relationship was demonstrated between the number of CFUs to intensity of DNA amplified via PCR. A detection limit of 10−5 CFUs is comparable to some food testing methods; however the vigorous processing of bead mill and rotor stator technology has the potential to eliminate the standard pre‐enrichment step needed for the detection of food pathogens.
Development of an easy, inexpensive, and precise method to identify Hemoglobin C for use in underdeveloped countries
Authors: Ohiri Chiemeziem D , Randolph Tim R
Published: November 2023

Hemoglobin C is one of the most common structural hemoglobin variants in the human population, second only to HbS (sickle cell). Like HbS, the HbC mutation exists in places which have inadequate resources to detect the presence of Hemoglobin C. This study aims to develop an easy, inexpensive, yet precise method to detect the presence of Hemoglobin C and determine zygosity. The hypothesis is that Hemoglobin C crystal formation can be induced intracellularly when incubated in a hypertonic salt solution and visualized microscopically when stained with new methylene blue. Crystal formation was achieved by incubating de‐identified patient blood samples bearing the Hemoglobin SC mutation in sodium chloride solutions ranging in concentration from 2%–6% and in concentrated Dulbecco's phosphate buffered saline. Sodium chloride concentrations between 3% and 5% showed the highest crystal formation. With further study, we hope to optimize the method to detect Hemoglobin C and establish guidelines to determine zygosity of blood samples.

Support or Funding Information

CO is supported by the DeNardo Education and Research Foundation Fellowship.
Comparison of Enzymatic Activities of Pokeweed Antiviral Protein (PAP) Isoforms toward RNA from the Tobacco Etch Virus
Authors: Aitbakieva Valentina R , Domashevskiy Artem V
Published: November 2023

Plants produce ribosome‐inactivating proteins (RIPs) phytotoxins that serve an important role in plants’ defense mechanisms against pathogenic invaders. RIPs are RNA N‐glycosidases that inhibit protein synthesis of a host cell through depurination of highly conserved sarcin/ricin loop of large ribosomal RNA. After synthesized inside the cells, RIPs are exocytosed and stored within extracellular spaces. Once a pathogenic attack occurs, RIPs gain access into the cell, alongside with a pathogen. Lesions within depurinated ribosomal RNA, produced by enzymatic activity of RIPs, avert the translocation step of protein synthesis. As a result, ribosome stalls.. The common pokeweed plant, Phytolacca americana, produces pokeweed antiviral protein (PAP), a type 1 RIP. PAP had been interacts directly with viral RNAs, depurinates them directly, and does so without substantial damage to the host ribosoms. PAP lowers the infectivity of many plant and animal viruses, including HIV‐1, poliovirus, and influenza. Nine known isoforms of PAP are produced in different parts of the plant; these vary during seasonal development. The mechanism of PAP‐viral RNA selectivity is not well known, and antiviral activity among different PAP isoforms varies. The focus of this study is to gain a deeper understanding into PAP‐RNA selectivity mechanism, and examine which isoforms of PAP exhibit the greatest activity toward tobacco etch virus (TEV) RNA. This knowledge will further be used in the development of efficient antiviral treatments.

Support or Funding Information

Title V, HSI‐STEM and MSEIP programs within the U.S. Department of Education; the PAESMEM program through the National Science Foundation; and New York State's Graduate Research and Technology Initiative. Professional Stuff Congress of the City University of New York (PSC‐CUNY), Cycle 44 Award.
Spectroscopic and Electrochemical Characterization of a Unique Radical S‐Adenosylmethionine Methylase
Authors: Blaszczyk Anthony J. , Silakov Alexey , Zhang Bo , Maiocco Stephanie J. , Lanz Nicholas D. , Kelly Wendy L. , Elliott Sean J. , Krebs Carsten , Booker Squire J.
Published: November 2023

TsrM catalyzes the methylation of carbon 2 of the indole ring of L‐tryptophan. Its reaction is the first step in the biosynthesis of the unique quinaldic acid moiety of thiostrepton A, a thiopeptide antibiotic. The appended methyl group derives from S‐adenosylmethionine (SAM); however, the enzyme also requires cobalamin and iron‐sulfur cluster cofactors for turnover. In this work we demonstrate the ability to overproduce and purify TsrM and characterize its metallocofactors by UV‐visible, electron paramagnetic resonance, hyperfine sublevel correlation (HYSCORE), and Mössbauer spectroscopies, in addition to protein film electrochemistry. The enzyme contains one equivalent of its cobalamin cofactor in its as‐isolated state, and can be reconstituted with iron and sulfide to contain one [4Fe–4S] cluster with a site‐differentiated Fe2+/Fe3+ pair. Our spectroscopic studies suggest that TsrM binds cobalamin in an uncharacteristic five‐coordinate base‐off/His‐off conformation, whereby the dimethylbenzimidazole is replaced by a ligand lacking nuclear spin, likely a water molecule. Electrochemical analysis of the protein by protein‐film electrochemistry indicates a one‐electron redox wave that exhibits a midpoint potential of ‐550 mV, assigned to a [4Fe–4S]2+/[4Fe–4S]+ redox couple. Analysis of TsrM by Mössbauer and HYSCORE spectroscopies suggests that SAM does not bind to the unique iron ion of the cluster in the same manner as other radical SAM enzymes, yet still disturbs the electronic configuration of the Fe/S cluster. These biophysical studies suggest that TsrM is an atypical radical SAM enzyme, consistent with its reported inability to catalyze formation of a 5′‐deoxyadenosyl 5′‐radical.

Support or Funding Information

National Institutes of Health
Comparison of Stability and Kinetic Properties of DszB from N. asteroides A3H1 and R. erythropolis IGTS8
Authors: Hutchinson‐Lundy Alanna , Crithary Austin , Schmitz Jonathan Michael , Watkins Linette
Published: November 2023

Dibenzothiopehene (DBT) and its derivatives comprise up to 60% of the organosulfur contamination of crude oil. The enzyme 2‐(2′‐hydroxyphenyl) benzenesulfinate desulfinase (DszB) catalyzes the carbon‐sulfur bond cleavage in the final, and rate‐limiting step in the biodesulfurization of DBT. The DszB enzyme from Nocardia asteroides A3H1 and Rhodococcus erythropolis IGTS8 was overexpressed in E. coli, purified and characterized kinetically. Kinetic assays revealed a sigmoidal response when the velocity was plotted against [S], calling into question the monomeric structure of the enzyme. Size exclusion chromatography and formaldehyde cross‐linking was conducted to further investigate the oligomerization of HPBS desulfinase. The stability of the enzyme was measured under various storage conditions and increased stability was observed upon immobilization of the enzyme to CNBr‐activated Sepharose beads. These studies aid in the understanding of the factors that control the rate and stability of the desulfinase enzyme to learn how to make it more economically feasible.
Induction of Apoptosis in DLD‐1 Colorectal Cancer Cells using Water Soluble Compounds from Rumex Crispus
Authors: Bhandari Mukund , Lockwood Hannah , Clack Beatrice
Published: November 2023

Colorectal cancer is the third leading cause of cancer death in the United States (American Cancer Society, 2015). Colorectal cancer cells can become resistant to current chemotherapy agents which prompts the need for new anticancer compounds (Crawford, 2014). In this study, water soluble compounds from R. crispus root and leaf were extracted, purified and separated using reversed phase High Performance Liquid Chromatography (HPLC). Cell viability assays were performed on human colorectal adenocarcinoma (DLD‐1 ATCC® CCL‐221TM) cells for each fraction and were further screened for caspase 3/7 activity to show that cell death was through apoptosis. R‐26, L‐26 and L‐19 fractions from HPLC exhibited cell viability reduced to 8.7 %, 10 % and 9.8%, respectively. The caspase‐3/7 fold change was observed to be 12.3, 3.5 and 26.9 fold increase for R‐26, L‐26 and L‐19 respectively, compared to untreated cells. mRNA from L‐19 treated cells was used to determine caspase 3,7,8, 9 and 10 levels of gene expression by qRT‐PCR. The ΔΔCt values for each caspase was determined using GAPDH for normalization. The ΔΔCt values for initiator caspases 8 and 10 were found to be 3.0 and 6.5 fold higher relative to untreated cells respectively. The initiator caspase‐9 showed no change. These results suggest the extrinsic pathway of apoptosis to be involved. Gas chromatography‐Mass Spectrometry (GC‐MS) of the HPLC fractions provided possible candidate compounds.

Support or Funding Information

Office of Research and Sponsored Projects, SFASU and Ed and Gwen Cole, Nacogdoches, TX