bluuebunny/crossref_metadata_2025 huggingface dataset:
Macromolecular Crowding Slows Glutamate Dehydrogenase Kinetics
Authors: Slade Kristin , Desrochers Andrea , Rundlett Emily , Schleicher Maddie
Published: May 2022

Most of our understanding of enzyme kinetics is based on experiments conducted in dilute solution, which does not accurately represent crowded cellular environments. Cells contain high concentrations (300‐400g/L) of various macromolecules such as proteins, carbohydrates and ribosomes. This crowding reduces the volume of solvent available for other molecules in the solution and has previously been shown to impact the behavior of enzymes. To study these potential consequences, the Michaelis‐Menten kinetics of glutamate dehydrogenase (GDH) was monitored in the presence of high concentrations of “crowding agents” such as dextran, a glucose polymer. Given this enzyme’s central role at interface of nitrogen and carbohydrate metabolism, GDH is highly regulated. Our results suggest that crowding may influence this regulation. For example, leucine is a less effective activator of GDH in the presence of dextran. Furthermore, crowding enhances substrate inhibition of GDH in a pH‐dependent manner. The resulting effects from crowding were substrate dependent such that the presence of dextran decreased the Vmax more with the NADP+ cofactor than with NAD+. Both dextran and BSA decreased the Km of NADP+ but did not affect NAD+. The effects of crowding on the enthalpy (ΔH҂) and entropy (ΔS҂) of activations determined from Eyring plots also were also substrate and pH‐dependent. Overall, these results highlight the importance of studying enzymes especially heavily regulated ones like GDH under crowded conditions that more realistically mimic the crowded cellular environment.
Biochemical Characterization of a Human Ribonucleotide Reductase (HsRNR) Variant Lacking Activity Regulation
Authors: Rios Paulina , Perez Goncalves Gerardo , Miller Kelsey R. , Drennan Catherine
Published: May 2022

Ribonucleotide reductase (RNR) is responsible for the synthesis of deoxyribonucleotides used in DNA synthesis and repair. Class Ia RNRs are composed of two dimeric subunits: a catalytic subunit (α2) which contains the active site and two allosteric regulation sites, and radical generating (β2) subunit housing the diferric‐tyrosyl radical cofactor. The two subunits form an α2β2 heterotetrametric complex that allows the radical to travel from β to α subunit for catalysis. RNR activity is regulated by the binding of ATP/dATP to an N‐terminal cone domain. Human RNRs (HsRNRs) are class Ia enzymes that form a homohexameric α6in the presence of either dATP or ATP. Although, both α6 assemblies are morphologically identical, only the α6‐ATP can be disturbed by the addition of β to form the α2β2 complex necessary for catalysis. Recent studies have shown that α subunits contact each other via their cone domains in the α6 rings, but it is unknown if the cone domains contact β2 in the α2β2 active complex. Here, we aim to probe the role of the cone domains in catalysis through biochemical and biophysical studies of an α subunit variant lacking the cone domain. We used site‐directed mutagenesis to delete residues 2‐92 of α, and expressed and purified the variant a protein. The variant protein was used for HPLC‐MS based activity assays of HsRNR. Preliminary results show the cone domain is important for catalysis. Understanding more about RNR’s structure and activity regulation can facilitate development of new cancer therapies.
Pro‐survival lipid metabolism activates intracellular complement signaling to induce inflammasome‐mediated tumor metastasis
Authors: Janneh Alhaji H. , Ogretmen Besim
Published: May 2022

Tumor metastasis remains one of the major causes of cancer‐related deaths in patients with solid tumors. Mechanisms by which metabolic alterations regarding pro‐survival lipid signaling activation in inducing cancer cell migration and metastasis are largely unknown. Here, we demonstrate that sphingosine 1‐phosphate (S1P) metabolism activates endogenous complement signaling for inducing tumor metastasis via inducing inflammasome‐mediated pro‐inflammation. Our studies using molecular, pharmacologic, and genetic tools showed that activation of S1P and S1P receptor 1 (S1P/S1PR1) induces tumor metastasis via enhanced intracellular complement signaling by the regulation of the C3‐PPIL1 complex and subsequent inflammasome activation in cancer cells and in vivo xenograft‐derived tumors.
Triggering toxicity: How antibiotics can enhance the release of outer membrane vesicles from Escherichia coli during sepsis
Authors: Michel Lea V. , Konovalova Anna , Hellman Judith , Gaborski Thomas
Published: May 2022

OMVs are spherical buds derived from the outer membrane of Gram‐negative bacteria, containing lipopolysaccharide (LPS) and phospholipids, proteins, nucleic acids, peptidoglycan, and other cellular molecules. Some of those molecules, such as LPS and peptidoglycan associated lipoprotein (Pal), have been shown to be toxic and to contribute to sepsis‐related inflammation. While OMVs are released constitutively from Gram‐negative bacteria, environmental factors like temperature changes, stress, and antibiotics can enhance OMV release. We proposed that some antibiotics, such as beta lactams that target the peptidoglycan layer of the cell, enhance OMV release more than other types of antibiotics. We used ultracentrifugation, immunoblotting, and nanoparticle tracking analysis to purify, detect, and roughly quantify OMV release from Escherichia coli in the presence of different antibiotics. Preliminary results support our hypothesis, although data collection is ongoing. Finally, we propose a novel use of released OMVs as molecular biomarkers for bacterial sepsis.
Investigating Mitochondrial Dysfunction in Barth Syndrome
Authors: Sniezek Olivia L. , Anzmann Arianna , Senoo Nanami , Butschek Grant , Claypool Steven , Vernon Hilary
Published: May 2022

Barth syndrome (BTHS) is a rare, X‐linked disorder of mitochondrial phospholipid metabolism caused by variants in the gene TAFAZZIN.TAFAZZIN is a transacylase involved in the remodeling of cardiolipin (CL), a dimeric phospholipid localized to the inner mitochondrial membrane. Lack of TAFAZZIN‐based remodeling results in irregular cardiolipin content, characterized by increased unremodeled CL, increased monoloysocardiolipin (the CL remodeling intermediate), and a decrease in remodeled CL, which is enriched in polyunsaturated fatty acyl chains that are tissue‐specific in composition. BTHS is clinically characterized by cardiomyopathy, neutropenia, and myopathy, with a high morbidity and mortality. There are no approved disease‐specific therapies.

To investigate the cellular pathology and to identify new areas of potential therapeutic intervention, we developed two CRISPR‐edited cell lines: TAFAZZIN‐knockout (KO) HEK293 cells and iPSCs with which to perform broad‐based discovery experiments and to study tissue‐specific disease effects, respectively. A combined multi‐omics approach including proteomics, lipidomics, and metabolomics in TAZ‐KO HEK293 cells revealed diverse mitochondrial abnormalities, including defects in complex I of the respiratory chain, abnormal PDK2 expression, and dysregulation of proteins involved in mitochondrial quality control including PARL and PGAM5. Importantly, we discovered that molecules that bind to cardiolipin (SS‐31) or inhibit nascent cardiolipin deacylation (bromoenol lactone), partially remediate these mitochondrial defects.

We next explored cell‐type specific dysfunction in iPSC‐derived TAZ‐KO and wild‐type cardiomyocytes and neurons via lipidomics, RNA‐seq, and functional studies. We identified disturbances in cellular lipid content including an expected increase in the monolysocardiolipin content and a reduction that exhibited cell‐type specificity. RNAseq identified dysregulation in pathways regulated by PARL and PGAM5 including Wnt signaling, apoptosis, and autophagy in the undifferentiated state, with differentiated cell types highlighting pathways such as glucose metabolism and response to cellular stimuli. Oxygen consumption studies show impaired maximal respiratory capacity in TAZ‐KO cardiomyocytes and neurons. Ongoing investigations aim to address cell type specific mitochondrial dysfunction by characterizing PARL abundance, PGAM5 cleavage, and mitochondrial morphology in TAZ‐KO iPSC derived cell types. Additionally, we are currently targeting cardiolipin metabolism in differentiated TAZ‐deficient cells with the goal of remediating cellular lipids, mitochondrial gene expression, and oxygen consumption abnormalities.
Using autonomy to drive the development of students into budding scientists
Authors: Dries Daniel R. , Williams Ursula J.
Published: May 2022

How do we motivate students to become independent scientists? Self‐determination theory (SDT) posits an individual is maximally motivated when three basic psychological needs are met: competence, relatedness, and autonomy. Instructional laboratories intrinsically focus on competence and relatedness, as students complete experiments (competence) typically in groups (relatedness). Addressing autonomy in laboratories, however, is more challenging. Contemporary instructional labs aim to engage students in authentic scientific inquiry, with students demonstrating independence. Independence, however, is not equivalent to autonomy, which includes student choice. Here, we describe the conversion of a traditional analytical chemistry laboratory into a guided‐inquiry bioanalytical chemistry laboratory, using SDT as a theoretical framework. Throughout the laboratory, students move progressively through less structured experiments and conclude with a campus‐wide presentation on a project that they themselves have autonomously completed – from inception to design to implementation and analysis. A mixed‐methods analysis shows autonomy and relatedness as recurring themes in the ways students experience the lab. This project, then, provides a less resource‐intensive laboratory experience that helps transition students from participants in prescriptive laboratory coursework to fledgling inquisitive scientists.
Sex‐related differences in how hyperactive platelets exacerbate the cytokine storm in the peritoneal cavity during intra‐abdominal sepsis
Authors: Albayati Samara , Dorsam Glenn , Liverani Elisabetta
Published: May 2022

Intra‐abdominal sepsis, a complex clinical syndrome resulting from a serious infection, is a major healthcare problem associated with high morbidity and mortality. Inflammatory cytokine levels in the peritoneal cavity (PC) are much higher than in blood, and the enhanced release of these mediators is a key participant to the deleterious effects of sepsis. Platelets are well‐known for their role in modulating proinflammatory cytokine secretion. We have previously shown that in vivo blockade of P2Y12 signaling, an important regulator for platelet secretion, resulted in lower inflammatory cytokines in the blood. We hypothesize that proinflammatory cytokine levels in the peritoneal cavity will be elevated in a murine model of sepsis. To this end, male and female mice underwent a cecal ligation and puncture (CLP) procedure to induce sepsis and were treated with ticagrelor to block P2Y12 signaling. Peritoneal cavity cells and fluid (PCF) were collected 24 hrs post‐surgery to measure changes in proinflammatory cytokine levels and immune cell interactions. Our data show that during CLP‐induced sepsis, PCF exhibited very low platelet counts and platelet‐immune cell aggregates compared to sham‐treated mice irrespective of sex (*p £ 0.05, n=4). Surprisingly, despite low platelet numbers, soluble p‐selectin and PF‐4 levels were elevated, indicative of platelet secretion. Moreover, P2Y12 antagonist treatment significantly decreased platelet markers and immune cell aggregates in PCF from male but not female mice (*p £ 0.05, n=5). In conclusion, intercellular communication mediated through systemic platelet activation influences the peritoneal cavity during intra‐abdominal sepsis, even in the absence of platelets locally in a sex‐related manner.

Moreover, P2Y12 antagonist treatment significantly decreased platelet markers and immune cell aggregates in PCF from male but not female mice. In conclusion, intercellular communication mediated through systemic platelet activation influences the peritoneal cavity during intra‐abdominal sepsis, even in the absence of platelets locally in a sex‐related manner.
The RNA Binding Protein AUF1 Regulates Phosphorylation of The mTORC2 Target Akt
Authors: Ragupathi Aparna , Li Mei‐ling , Patel Nikhil , Werlen Guy , Brewer Gary , Jacinto Estela
Published: May 2022

mTOR, which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA binding protein, AUF1 (ARE/poly(U)‐binding/degradation factor 1), modulates mTORC2/Akt signaling. AUF1 is required for Akt phosphorylation. It also mediates phosphorylation of the mTORC2‐modulated metabolic enzyme GFAT1 at Ser243. Reciprocally, mTORC2 could also modulate AUF1. Conditions that enhance mTORC2 signaling, such as serum restimulation or acute glutamine withdrawal augments AUF1 phosphorylation while mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in mTORC2/Akt signaling. Targeting AUF1 could serve as a novel treatment strategy for cancers with upregulated mTORC2/Akt signaling.
Exploring the Role of Msh2‐Msh6 in Processing Holliday Junction DNA
Authors: Du Amy , Lombardo Zane , Thayer Kelly M. , Mukerji Ishita
Published: May 2022

Msh2‐Msh6 is a eukaryotic ATP‐dependent mismatch repair (MMR) protein that plays a key role in maintaining the fidelity of the genetic code. The protein recognizes and initiates repair for single base pair mismatches and short insertion deletion loops (IDL). Msh2‐Msh6 functions via a molecular switch mechanism, whereby ATP hydrolysis and ADP/ATP exchange facilitates the binding and release of the protein from the mismatch. Previous research has revealed that the protein binds G:T mismatch DNA with the highest affinity. Interestingly, in‐solution binding assays have revealed that Msh2‐Msh6 binds Holliday Junction DNA, a key recombination intermediate, with 1:1 stoichiometry and a similarly high affinity. However, the functional importance and structural details of this interaction are unclear. Thus far, X‐ray crystallography and Molecular Dynamics (MD) simulations are limited to the protein’s interactions with mismatch DNA only. To address the lack of structural information, we modeled the Msh2‐Msh6‐HJ interaction using the human Msh2‐Msh6 crystal structure in which we have docked in junction DNA instead of the mismatched DNA. The junction DNA used in all experiments and simulations is J3, a non‐migrating junction with 17 base pair long arms. These MD simulations will help refine the structural model and assess significant interactions between the protein and DNA. Furthermore, ATPase assays and equilibrium binding assays are used to elucidate the protein’s ATP hydrolysis activity and binding affinity in the presence of G:T mismatch DNA, J3 junction DNA, and J3 junction DNA containing a G:T mismatch. Preliminary experiments show that relative to the apoprotein’s basal ATPase activity, ATP hydrolysis is weakly stimulated when Msh2‐Msh6 is bound to G:T mismatch and J3. Gel mobility shift assays (GMSA) reveal binding affinities in the range of 9.1±2.6 nM to 23.5±11.8 nM for G:T mismatch, J3 junction, and mismatched J3 junction. These data illustrate that Msh2‐Msh6 not only binds G:T mismatch and J3 with similarly high affinity, but also that introducing a mismatch in J3 does not modulate the protein’s affinity for the junction. In addition, the GMSA data suggest that Msh2‐Msh6‐HJ binding may take on multiple conformations, consistent with previous Förster resonance energy transfer (FRET) calculations that show Msh2‐Msh6 opening the junction after binding.
Identification of Novel Regulators for GPCR‐signaling via Genome‐wide Analysis
Authors: Baker James , Cranley Juliana , Pauer Emma , Rajasekhar Abhinav , Karthikeyan Swathy , Wang Yuqi
Published: May 2022

G protein‐coupled receptors (GPCRs) are a large family of proteins responsible for receiving and responding to many signals such as smell, light, and hormones. GPCRs are also the target for approximately thirty‐four percent of all FDA approved drugs. Therefore, gaining new insight about GPCR signaling regulation can be useful for improving our understanding of cellular physiology and enhancing the effectiveness of the drugs we use. GPCR signaling is highly conserved, enabling the use of model organisms, such as budding yeast Saccharomyces cerevisiae, to study its regulation. Yeast responds to pheromone using a well‐characterized GPCR pathway. In this project, we screened a collection of yeast mutants that all had one gene deleted from their genome, using a well‐established assay that measures pheromone response. From this screen, we have identified seven novel mutants that clearly and consistently alter the pheromone response. Further analysis of these mutants indicates that some of them affect the pheromone response at the level of or upstream of MAP kinase Fus3. We are in the process of cloning these genes and examining if the abnormal pheromone response and Fus3 activation of these mutants can be rescued. Our analysis suggests that unbiased genome‐wide genetic screening remains a viable approach in identifying novel regulators for GPCR‐mediated signaling.
A Quantitative Comparison of the Knockdown Efficiencies of CRISPR/Cas9 and CRISPR‐Cas12 in Caenorhabditis elegans
Authors: Patel Anya , Agrawal Nikki , Feldman Sydney , Hauser Paul , De Luke
Published: May 2022

Currently, many gene editing studies employ CRISPR/Cas9, however its low accuracy and knockdown efficiency is problematic. CRISPR‐Cas12, a newer and potentially more accurate variant, is hypothesized to have a higher knockdown efficiency than CRISPR/Cas9 in Saccharomyces cerevisiae and various plants. However this has not been quantitatively verified in mammalian cells. Our study compares the loss of gene function efficiencies of CRISPR/Cas9 and CRISPR‐Cas12 through decreasing green fluorescent protein (GFP) expression in Caenorhabditis elegans via CRISPR‐mediated deletion. We used the LP162 strain of C. elegans which endogenously expresses GFP. We used C. elegans due to the similarity of molecular pathways to those of humans, their simple anatomy, and their fast reproductive cycle. Through bacterial transformation, we expressed our modified plasmid (containing the gRNA sequence, Cas endonuclease sequence, and C. elegans‐specific promoters) in Escherichia coli. The C. elegans subsequently ingested the transformed bacteria. Fluorescence and protein expression were used as assays to determine the efficacy of CRISPR‐mediated GFP deletion. We hope to show a quantitative assessment of the difference in loss of function efficiencies between CRISPR/Cas9 and CRISPR‐Cas12 when used to delete GFP in C. elegans. Our goal is for these findings to be valuable for researchers seeking to use the most efficient Cas endonuclease for gene knockdown experiments.
RNA Therapeutics for Brain Tumors: Targeting Pre‐mRNA Splicing Motifs to Generate Therapeutic Gene Isoforms
Authors: Hintelmann Thomas , Sine Laura C. , DeMarco Victoria , Reardon Sean , Hicks Martin J.
Published: May 2022

Individuals diagnosed with glioblastoma multiforme (GBM) have a short life expectancy of 12‐15 months. This project is to develop therapies for effective and continuous drug delivery to the brain, targeting cancer‐driving genes. Tumor cell proliferation in GBM is often stimulated by epidermal growth factor receptor (EGFR) and is important for tumor cell survival. In our lab, we are developing RNA therapies to alter the splicing mechanism of EGFR to block its activation, thus stop tumor cell growth. Our approach uses an adeno‐associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre‐mRNA transcript. In this project, we cloned therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. We have found that our therapies have led to a shift in Intron 10A retention increasing alternative intronic polyadenylation generating a short soluble therapeutic decoy. Our antisense therapies targeted the EGFR pre‐mRNA transcript, specifically, regions of exon 10 and intron 10. Therapies were either designed to enhance recognition of the alternative intronic polyadenylation signal or block recognition of canonical exon 10/exon 11 splicing. We targeted the Exonic Splicing Enhancer (ESE) wild‐type 5’ Splice Site (wt‐5’SS) of exon 10, and the regions surrounding the alternative intronic polyadenylation signal. Each target showed a significant increase in intron retention. Targeting the ESE showed a 5‐fold increase in intron retention (p < 0.04), the wt‐5’SS was 15‐fold (p < 0.001), and the region surrounding the alternative intronic polyadenylation signal showed the strongest effect, with greater than 60‐fold increase in intron retention (p < 0.002). Currently, we are examining additional antisense therapies, as well as generation of the therapeutic soluble decoy protein using ELISA.
Providencia alcalifaciens is a Highly Antimicrobial Resistant Bacteria Found in a Suburban Creek
Authors: Carlson Clayton D. , Senti Evan , Oostema Julia
Published: May 2022

Antibiotic resistant bacteria have been found in diverse environments all over the world with greater rates of resistance in those locations most closely in contact with human populations. The Navajo Creek is a small, suburban waterway 15 miles southwest of Chicago, IL that flows through neighborhoods and forest preserves. Among bacteria isolated from the creek, resistance to five different antibiotics has been discovered, and metagenomic analysis of these populations shows diversity among the resistant microbes. Strikingly, one bacterial species, Providencia alcalifaciens,shows resistance to all five antibiotics simultaneously. Full genome analysis reveals that this species displays a diverse set of resistance genes to overcome these drugs. The interplay between these genes and the drugs they defeat is still being determined. Additionally, the full range of P. alcalifaciensin the local environment is being measured by identifying the microbe in regional water, soil, and animal waste. This project offers careful analysis of a highly resistant microbe and potential pathogen that resides in a suburban waterway.
Tight junction membrane protein's role in neural tube defects
Authors: Mizrachi Dario , Allen Wesley
Published: May 2022

More than 300,000 infants worldwide are born with neural tube defects (NTD), making it the second most common birth defect in the world. Many genetic and environmental factors have been identified as drivers of NTDs, some of which have been tied to damage of tight junctions (TJ), which are critical for the success of neural tube folding. Claudins (CLDN), occludin (OCLN) and junctional adhesion molecules (JAMs) are essential components of the TJ and are key players in its function. In the chicken embryo, clostridium perfringens enterotoxin (CPE) induces NTDs, by interacting broadly with CLDN3, ‐4, and ‐8. These interactions increase TJ permeability to an influx of calcium ions, causing cell death. There is no information for the role of OCLN or JAMs in NTDs. Our laboratory has created Synthetic Biology approaches to produce soluble CLDNs, OCLN and JAMs. These are detergent independent proteins that result in molecules that retain structure and adhesive properties of the native membrane protein. We present evidence that specific interactions, rather than the broad specificity of CPE, can be used to identify specific mechanisms leading to NTD formation. Our approach expands to the role of OCLN and JAMs in the formation of NTDs. These new tools may be used in developmental research as an alternative for genetic manipulation with equal value.
Understanding the Lipid‐dependent function of SARS‐CoV‐2 E Protein
Authors: David Emily , Stahelin Rob
Published: May 2022

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the pathogen responsible for COVID‐19, a disease which has resulted in the death of millions worldwide. COVID‐19 ranges in severity of symptoms which are related to the inflammatory response. Although the field has progressed significantly, there is still a critical gap in understanding virus‐host lipid interactions that contribute to viral assembly and budding of this lipid‐enveloped virus. There are four structural proteins encoded in the SARS‐CoV‐2 genome: membrane, envelope (E), nucleocapsid, and spike. These proteins give shape to the bilayer lipid coat that encapsulate the genomic core necessary for virus infection and replication. These proteins are essential for viral reproduction but how they interact with each other and how lipid species regulate their assembly is not well understood. It is established that inflammation is common during infection. One enzyme that participates in this process is ceramide kinase (CERK), which synthesizes the pro‐inflammatory lipid, ceramide‐1‐phosphate (C1P). CERK is in fact a therapeutic target for a variety of inflammatory disorders. My primary goal is to define the relationship between CERK activity and SARS‐CoV‐2 assembly. We aim to understand the impact that CERK activity has on the localization and assembly of SARS‐CoV‐2 structural proteins. I am interested in how CERK regulates E protein localization and formation of virus‐like particles and the process by which E‐lipid interactions contributes to membrane curvature changes necessary for formation of new viral particles. Overall, I anticipate that this study will help define the relationships between a host enzyme and host membranes and the assembly of SARS‐CoV‐2.
The role of substrate deformation in context‐dependent non‐CG DNA methylation
Authors: Song Jikui
Published: May 2022

Cytosine‐5 DNA methylation is a major epigenetic mechanism that is important for gene regulation and genome stability. Across evolution, the DNA methylation pathways have diversified between different kingdoms. For instance, DNA methylation in mammals predominantly occurs in the context of CG dinucleotides, established by de novo DNA methyltransferases DNMT3A and DNMT3B and maintained by DNMT1. In contrast, DNA methylation in plants occurs in all three sequence contexts in plants, namely CG, CHG and CHH (H = A, C, or T), with CHG and CHH methylation maintained by CHROMOMETHYLASES (CMTs) and Domains Rearranged Methyltransferase 2 (DRM2). Emerging evidence reveals high context‐dependence of non‐CG methylation in plants: both CHG and CHH methylation is enriched with an A or T nucleotide at the +1‐flanking site, providing a mechanism in reinforcing DNA methylation at specific chromatin domains, such as AT‐rich TEs. To understand the context‐dependency of plant non‐CG methylation, we recently characterized the structural basis for the DRM2‐ and CMT3‐mediated non‐CG methylation. Remarkably, DRM2‐ and CMT‐mediated DNA methylation both involve intercalation‐induced base distortion at the +1 target‐flanking site. The conformational energy penalty associated with this DNA deformation is mostly stabilized by van der Waals contacts, rather than base‐specific hydrogen‐bonding interactions that were previously observed for mammalian DNMTs, allowing the potential influence of nucleotide composition at the +1 site on DRM2‐ and CMT‐mediated methylation. These findings establish a link between DNA deformation and context‐dependent non‐CG DNA methylation in plants, providing a new paradigm for the evolutionarily diversified DNA methylation mechanisms.
Regulation of Extracellular Matrix Gene Expression by Desmosomal Cadherins
Authors: Mateo Beatriz , Johnson Brandon , Thomas Sarah M. , Rudman Lara , Conway Daniel , Dubash Adi D.
Published: May 2022

Desmosomes are protein complexes crucial for maintaining cell‐cell adhesion and integrity of tissues. These complexes are made up of proteins from three families: transmembrane cadherins (Desmoglein and Desmocollin) link adjacent cells in the extracellular space, armadillo proteins (Plakophilin and Plakoglobin) stabilize the intracellular plaque, and the cytolinker Desmoplakin (DP) connects the plaque to the intermediate filament network. Desmosomal proteins have also been shown to coordinate gene expression pathways required for processes such as proliferation, differentiation and cell migration. In particular, several lines of evidence have linked desmosomal proteins to gene expression of extracellular matrix (ECM) proteins. Loss of Plakophilin‐2 or Desmoplakin causes increases in expression of fibronectin and collagen, while in contrast, loss of plakoglobin results in a significant decrease in expression of fibronectin. These data indicate that individual components of the desmosome control ECM gene expression via distinct cellular signaling networks. In our study, we sought to investigate the role of desmosomal cadherins in ECM gene expression, via use of A431 cells lacking Desmoglein‐2, generated via CRISPR‐mediated knock‐out. Compared to control cells, Dsg2 KO cells demonstrated a dramatic increase in expression of Fibronectin (Fn1) mRNA, and relatively minor changes in expression of Collagen 1 (Col1a1) and Collagen 2 (Col2a1) mRNA. An increase in expression of Fibronectin protein levels in Dsg2‐deficient cells was also observed via western blot. We show that loss of Dsg2 KO also caused a significant increase in mRNA expression of the pro‐fibrotic signaling molecule transforming growth factor beta 2 (Tgfb2), but not Tgfb1 or Tgfb3. Increased expression of Fn1, Col1a1, Col2a1 and Tgfb2 was also observed upon siRNA‐mediated knockdown of Dsg2 in both A431 cells and HaCaT keratinocytes, verifying that these changes are not clone‐specific or due to off‐target CRISPR effects. siRNA‐mediated knockdown of other desmosomal proteins could not rescue Fn1 or Tgfb2 increases in Dsg2 KO cells, indicating that changes are not due to mislocalization of other desmosomal components. An extensive analysis of signaling pathways known to be involved in regulation of ECM gene expression uncovered an important role for PI3‐Kinase and Akt signaling, as inhibition of either signaling protein resulted in a rescue of Dg2KO‐mediated increases in Fn1 gene expression. Moreover, we have documented a significant increase in phosphorylation of Akt in Dsg2‐deficient cells. Taken together, our study highlights a novel role for Dsg2 in mediating ECM gene expression via PI3‐K/Akt signaling, adding significant insight into the mechanisms by which desmosomal cadherins control the adhesive behavior of cancer cells.
Elucidating the Physiological Role of Guanidinium (Gdm+) in Bacteria
Authors: Lucero Rachael M. , Stockbridge Randy B.
Published: May 2022

Until recently, guanidinium (Gdm+) was not known to be physiologically relevant in bacteria. Gdm+ is most commonly known for industrials purposes in production of explosives, rubber, plastics and as a chaotropic agent for protein denaturation. However, it has recently been shown that diverse bacteria have mechanisms to sense Gdm+, use Gdm+ as a nitrogen source, and export Gdm+ using guanidinium exporters (Gdx), establishing Gdm+ homeostasis as important to bacterial physiology. Here, we examine the effects of exogenous guanidinium ion on bacteria in context of planktonic growth and biofilm formation. We further investigate how and why bacteria endogenously produce Gdm+. Using flow cytometry, we test the competitive fitness of bacteria that are deficient in guanidinium exporter under conditions of varying carbon and nitrogen. These experiments begin to reveal the biological role of a metabolite that was once overlooked.
pH Effects on the Stability and Folding of Monomeric Caspases
Authors: Joglekar Isha
Published: May 2022

Caspases are an ancient class of cysteinyl proteases that play an integral part in cell development and apoptosis as an evolutionarily conserved function. During apoptosis, the intracellular pH decreases from 7.4 to ~ 6.8 and cytosolic acidification affects the activation of caspase‐3 and pH‐dependent conformational changes affect the rate of auto maturation in the dimeric procaspase‐3, which in turn influences the ability of this enzyme to execute the cell. Proteins are required to fold correctly and attain their native conformation which is vital for protein function and the survival of the organism. The protein folding landscape is sculpted through evolution and there exists an evolutionary selection for functional residues by selecting residues that stabilize the interactions to attain the native conformation while repressing the selection of non‐native interactions that take place during folding. Through the course of evolution, how these proteins fold to the native conformation to contribute to the fitness of the organism while maintaining or altering the energy landscape that is guided by trade‐offs between the stability and the activity of the protein remains elusive. To understand how the energy landscapes changed over time, we resurrected an ancestral caspase (existed about 650 milllion years ago) and we examined the folding pathways of caspases from Cnidarians, specifically corals. We studied the equilibrium unfolding of caspases from O.faveolata (species susceptible to disease) and P.astreoides (species resistant to disease) as a function of pH (between pH 3 and pH 10.5) to examine the changes in the context of stability and folding. The data shows that these monomeric proteins have a high secondary structure at the native state and unfold via a three‐state process closer to the physiological pH (pH 6.5~pH8.5), via an intermediate state which is aggregation prone. However, outside of this pH range, the native protein loses its secondary structure and exists in an intermediate state which is stable. Due to this, these proteins undergo a two‐state unfolding mechanism wherein, the protein transitions from an intermediate state to the unfolded state, reversibly. Overall, the free energy change for these primitive coral caspases is ~12.5kcal/mol, which is higher than what is observed for human caspases. This higher stability might be due to the decrease in the activity and the presence of certain stabilizing residues that are not observed in the human caspases.
Unique DNA Polymerase kappa Interactome Suggests Novel Cellular Functions
Authors: Paul Shilpi , Cifani Paolo , Pappin Darryl , Spratt Thomas
Published: May 2022

Translesion DNA synthesis (TLS) polymerases evolved to tolerate DNA damage that bypasses DNA lesions, thus preventing genomic instability. Multiple TLS polymerases exist with different damage tolerance capabilities, since they have low fidelity their access to the replication fork must be regulated to minimize mutations. The current paradigm is that a combination of kinetic partitioning and protein‐protein interactions are used to regulate TLS polymerase activity. A major knowledge‐gap in elucidating the roles of these polymerases is that it is difficult to identify which polymerase is active in a specific situation. We designed and synthesized a novel nucleotide analog N2‐benzyl‐ 2′‐deoxyguanosine (EBndG) that is highly selective toward DNA polymerase kappa (Pol κ), a Y family TLS polymerase. Pol k can bypass bulky lesions in the minor groove generated by a metabolite benzo[a]pyrene diolepoxide (BPDE), an environmental carcinogen. Although Pol κ has been identified to have multiple cellular roles, the mechanisms regulating its different cellular activities are unknown.

To interrogate the identity of proteins surrounding the Pol κ active sites, we performed an extensive study using modified iPOND (isolation of proteins on nascent DNA), called iPoKD (isolation of proteins on Pol kappa synthesized DNA). Human cell lines were treated with BPDE, subsequently 5‐ethynyl‐2′‐deoxyuridine (EdU) or EBndG was added and proteins bound to the DNA containing EdU and EBndG were analyzed by mass spectrometry. In addition, we performed quantitative analysis of the Pol κ active sites interactome using iPoKD followed by iTRAQ (isobaric tags for relative and absolute quantitation). Our data identified DNA replication and repair proteins previously identified with EdU pull‐downs; and interestingly enrichment of RNA binding, ribosome biogenesis, nucleolar proteins and transcriptional repressive complex(es) associated with EBndG pull‐downs. Chromatin modifiers, histone chaperones and histone variants are identified suggesting changes in chromatin structure that facilitates Pol κ‐mediated DNA lesion bypass, repair and restorative process. identification of unique proteins associated with EBndG‐containing DNA, suggests novel roles for Pol κ’s activity in the cell.

Using super‐resolution confocal microscopy, Pol κ activity is identified in the nucleolus after BPDE damage. EBndG is observed in the nucleolar DNA and Pol κ 's activity regulated by the canonical polycomb‐complex recruited by the PARylation of PARP1. BPDE lead to transcriptional stress and repression that is gradually recovered. We are investigating whether Pol κ maintains ribosomal DNA integrity after BPDE damage and is required for TLS DNA synthesis or repair. Pol κ active site associated candidate proteins are being validated using CRISPR‐Cas9 knockout or siRNA knockdown strategies. These data will provide first insight into Pol κ’s core interactome, it's regulation, chromatin surrounding Pol κ active sites and its novel cellular roles.
The Effect of Atrazine on Fear Behaviors in Rats
Authors: Rivera‐Lopez Melissa , Martines‐Guzman Osmarie , Caceres‐Chacon Mauricio , Hernandez‐Busot Gabriela N. , Rodriguez‐Rosado Sian , Figueroa‐Perez Alexdiel , Sierra‐Mercado Demetrio
Published: May 2022

Atrazine (ATR) is a widely used herbicide in agriculture. Unfortunately, excessive use of ATR has led to the contamination of food and drinking sources (Mosquin et al., 2012; Whitmore and Chen, 2013). Subsequently, this has led to humans becoming exposed to ATR in their daily life. Animal studies have shown that ATR can influence emotionally relevant behaviors such as anxiety in rodents (Ma et al., 2018; Chavez‐Pichardo et al., 2020). However, other emotional behaviors such as fear remain unexplored. To address this gap, rats were exposed to ATR in their drinking water at a dose considered safe for ingestion (0.035mg/kg; Environmental Protection Agency), whereas control animals received filtered water. Both groups had ad libitum access to water for four months. Following exposure, fear was assessed using Pavlovian fear conditioning where rats learn that a tone predicts a shock by exhibiting freezing behavior to the tone. We hypothesized that rats exposed to ATR would show increased fear expression compared to control rats. Anxiety‐like behavior was also examined using the open field test. During fear conditioning no differences were observed in freezing behavior between groups (ATR: 58%, Controls: 59%; n=10; p=0.3714). In the open field test, ATR rats trended towards having more center entries (ATR: 23.8, Controls: 14.8; n=10, p=0.0570) and decreased time spent in corners (ATR: 70.5 s, Controls: 114.2 s; n=10, p=0.0248) compared to controls. These results suggest that ATR does not affect fear behaviors, but it can decrease anxiety‐like behaviors. Future directions aim at examining brain regions key to emotional behaviors, such as the amygdala, using immunohistochemistry. Our findings may help elucidate how prolonged ingestion of atrazine at a dose considered safe for humans may influence emotionally relevant behaviors.
Purine biosynthesis and related enzymes play the oncogenic function in FGFR3 mutant urothelial carcinoma
Authors: Chang Yu‐Chan
Published: May 2022

Urothelial carcinoma (UC), as one of the malignant tumors, has a high incidence, mortality, and poor survival rate. FGFR3 is one of the frequency mutation genetic events of UC. Recent studies have reported that FGFR3 mutations are associated with lower aggressiveness and better prognosis of UC, and small molecule compounds with FGFR3 mutations have been developed and treated. However, they are still limited, especially for wild‐type cells. We used Ingenuity Pathways Analysis (IPA) to analyze the significantly expression of 351 genes (>2‐fold change cutoff value) between FGFR3 wild‐type and mutation/fusion UC cells by FGFR3 inhibitors treatment. Through the predicted canonical pathways, several metabolism‐related events have been regulated, including purine biosynthesis. Focusing on de novo purine biosynthesis, we found that several involved enzymes are upregulated in UC. In addition, we have also observed that several enzymes are inhibited by FGFR3 inhibitors in mutant cells, but not in wild type. We then hypothesized that purine biosynthetic events might confer drug resistance and promote UC tumorigenicity. To verify our observations, we determined the expression level of purine biosynthesis after FGFR3 classification. The results indicate a similar trend in clinical cohorts that overexpression of purine biosynthesis genes in FGFR3 wild‐type. Moreover, combining these genes can be used as an independent prognostic factor (HR=1.41, p=0.026). We further established the overexpression of purine biosynthesis genes in some FGFR3 mutant UC cells. We determined that purine biosynthesis genes cause FGFR3 mutations models to be more prone to mesenchymal and metastatic than controls. Our studies also observed that FGFR3 wild‐type cells promote UC cell radioresistance via purine biosynthesis and further activate the homologous recombination repair mechanism. Combining all evidence, we claim that purine biosynthesis should be focused and regarded as a novel strategy against UC.
Integrated Quantum Mechanical and Machine Learning Methods Enable Scalable and General pKa Prediction for Pharmacokinetic Modeling
Authors: Walden Daniel M. , Bundey Yogesh , Brunk Nicholas E. , Khotimchenko Maksim , Hou Hypatia , Chakravarty Kaushik , Hixon Mark S. , Varshney Jyotika
Published: May 2022

We present the development of the BIOiSIM™ pKa model for predicting aqueous molecular ionization. Assessment and optimization of drug pKa occurs early in drug discovery; thus, models for high‐throughput pKa prediction must balance computational cost, scalability, accuracy, and applicability to diverse chemical structures. Key to our pKa prediction model is a set of low computational cost, quantum mechanical charge descriptors that incorporate not only ionizable group charges, but the additional atoms and bonds likely to modulate pKa. The finalized machine learning models produced good accuracy on the test holdout set (R2 = 0.9, mean absolute error = 0.6−0.7, root mean square error = 1.0) and show good generalizability predicting the most acidic and basic ionization centers of the SAMPL6 external benchmark validation set (mean absolute error = 0.8, root mean square error = 1.0). Performance of the model is comparable to commercial pKa predictors but is trained on less data and exhibits higher scalability needed for pharmacokinetic modeling.
Accessory Head(s) of the Palmaris Longus Muscle in an 86 Year‐Old White Female Cadaver and an 81 Year‐Old White Male Cadaver
Authors: Granite Guinevere , Sheridan Ariana , Jones Shiloh , Wind Gary
Published: May 2022

It is known that the palmaris longus muscle is one of the most variable muscles in the human body. The palmaris longus muscle has a high morphological diversity and variations are frequently reported in the literature. Recognizing anatomical variation in the palmaris longus muscle and tendon is important for clinical associations such as neurovascular entrapment and compression, as well as tendon graft and transfer for reconstruction. While neglecting to identify palmaris longus variations rarely leads to an increase in mortality, it is essential to identify for clinical relevance. During anatomical dissection of fifty cadavers in the 2020 undergraduate first‐year anatomy course at the Uniformed Services University of Health Sciences (USUHS), we found two separate cases of accessory heads of the palmaris longus muscle. The first case involved a preserved 86‐year‐old White Female who had one unilateral accessory head of the left palmaris longus muscle. The second case involved a preserved 81‐year‐old White Male who had a unilateral pair of accessory heads of the right palmaris longus muscle.
Electroacupuncture Decreases Chronic Inflammation, Sympathetic Activities, and Blood Pressure in Dahl Salt‐Sensitive Hypertensive Rats
Authors: Gong Yiwei , Fu Liang‐Wu , Malik Shaista , Guo Zhi‐Ling , Tjen‐A‐Looi Stephanie C.
Published: May 2022

Low grade chronic inflammation is detected in hypertensive patients. The dahl salt‐sensitive hypertensive (DSS HTNive) rat model has been investigated on different pathways how excessive salt intake induces elevation in blood pressure (BP). Previous studies have demonstrated low grade chronic inflammation and elevated sympathetic outflow in DSS HTNive rats. We have shown in series of studies that sympathoinhibitory electroacupuncture (SI‐EA) at acupoints ST36‐37 suppresses sympathoexcitatory cardiovascular responses and reduces BP in HTNive rats and patients. Other researchers reported that EA at acupoints SP6‐7 as well as ST 36‐37 decreases inflammation. Therefore, we hypothesized that combined EA (cEA) at ST36‐37 and SP6‐7 induces greater reduction in BP than SI‐EA through the modification of chronic inflammation and elevated sympathetic activity in DSS HTNive rats. DSS rats (n=36) were fed with 4% salt diet and treated with SI‐EA, cEA or Sham‐EA (without electrical stimulation) twice weekly for five weeks once the rats developed moderate HTN (SBP 160±5 mmHg). Systolic/diastolic BP (SBP/DBP) and heart rate (HR) were measured by weekly tail‐cuff non‐invasively. Upon completion of different treatments, cardiac perfusion was performed under anesthesia, and plasma samples were collected to measure the concentration of high‐sensitivity c‐reactive protein (hs‐CRP), an indicator of low‐grade chronic inflammation, and norepinephrine (NE) by ELISA. We observed that DSS rats fed with 4% salt diet developed moderate HTN (SBP 163±3.8 mmHg) in five weeks compared to those fed with standard rat chow (SBP 129±5.4 mmHg). After five weeks of treatments, the EA treated rats displayed significantly lowered SBP (138±4.7 mmHg for cEA group, 147±4.2 mmHg for SI‐EA group) compared to the Sham‐EA treated rats (SBP 191±6.8 mmHg). The cEA group showed greater reduction in SBP by 28% than the SI‐EA group (23%) compared to Sham‐EA group. The DBP but not HR among the groups exhibited similar trends. Furthermore, we observed that plasma NE concentrations showed greater reduction in the cEA group by 24.5% than the SI‐EA group (11.4% decrease) compared to the Sham‐EA group. In addition, the rats treated with cEA displayed lowest concentration of plasma hs‐CRP (225 ug/mL) in the three hypertensive groups. As such, our findings suggest that cEA at ST36‐37 and SP6‐7 induces larger BP‐lowering effect than SI‐EA in DSS HTNive rats and was associated with reduction of biomarkers of chronic inflammation and sympathetic outflow.