bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biophysical and Structural Studies of Chelator Fragment‐Library Candidates as Inhibitors of Metallo‐β‐lactamases
Authors: Williams Cameron , Tierney David L , Crowder Michael W , Cohen Seth , Fast Walter , Page Richard C 
Published: November 2023

Antibiotic resistance is a serious worldwide health problem. In the 2013 Antibiotic Resistance Threat Report, the CDC estimates that over 2 million people suffer from antibiotic‐resistant infections every year in the U.S. and 23,000 of these individuals die as a result. Metallo‐β‐lactamases are a class of enzymes that catalyze the hydrolysis of almost all β‐lactam antibiotics and, as a result, confer dangerous antibiotic resistance to the bacteria expressing them. Gram‐negative bacteria possessing metallo‐β‐lactamases are of utmost concern because these enzymes also break down carbapenems, a class of antibiotics commonly used as a last resort to treat antibiotic‐resistant bacterial infections. Enterobacteriaceae with carbapenemase activity are becoming increasingly widespread. This is a major problem because these gram‐negative bacteria of the intestinal flora are easily spread between humans and commonly found in clinical settings. Metallo‐β‐lactamase inhibitors are of particular interest because they could potentially restore the efficacy of current generation β‐lactam antibiotics. A library of chelator fragments has been developed to inhibit metallo‐β‐lactamases by taking advantage of the active site Zn2+ ions critical for proper enzymatic function. Our in vitro activity assays have identified several potent candidates from the library for further study. VIM‐2, a variant of Verona integron‐encoded metallo‐β‐lactamase (VIM), was overexpressed and purified for the purpose of studying these chelator fragments. We utilized differential scanning fluorimetry to demonstrate that high concentrations of chelators destabilize VIM‐2 by removing Zn2+ cofactors. Co‐crystallization and crystal soaks of VIM‐2 with several chelators was attempted in order to gain insight into the structure of the protein‐ligand complex but x‐ray diffraction data suggests that the chelators are not present in the crystal structure. Future work will involve binding affinity studies by isothermal titration calorimetry (ITC) and structural studies with advanced crystallographic techniques. Additional studies will be employed to examine the importance of a loop near the active site of all metallo‐β‐lactamases. Overall, these studies will contribute to the development of more effective chelators.Support or Funding InformationThis work was supported by NIH grant R01 GM111926.
Synthesis and application of small cationic hexamers as potential antibiotic agents
Authors: Sheffield John W , Kyle Charles T , Rockenbach Jacob D , Anderson C William , Wolyniak Michael J 
Published: November 2023

The development of antibiotic resistance among pathogenic bacteria is caused by mutations in bacterial DNA and represents a significant challenge to modern medicine. Among the several mechanisms of antibiotic action that have been documented, including inhibition of protein synthesis, inhibition of DNA synthesis, and cell membrane degradation, the delocalization of cytochrome facilitating ATP production shows great potential as a powerful emerging method in the quest to develop new therapies to fight bacterial infection. Wenzel et al. (2014) attempted to harness this technique through the synthesis of peptides consisting of arginine and tryptophan repeats (RWRWRW‐NH2) with a terminal amine group. His group found that this peptide chain interacts with the cell wall of Gram‐positive bacteria and successfully disrupts ATP synthesis and impedes osmoregulation, ultimately leading to cell death. Our project expands on this study as well as work done by John Brandt and Christopher Ferrante, former members of our lab group. We are working to find an optimal concentration of peptide hexamers that Brandt and Ferrante previously found to be effective as potential antibiotic agents using the mechanism proposed by Wenzel et al. We are using Merrifield synthesis for synthesizing the peptides KKKWWW‐NH2 and KWKWKW‐NH2, which Brandt and Ferrante have showed to be effective as antibiotic agents against a wide range of bacterial species. These peptides are being tested for antibiotic efficacy against eight Gram positive species, including Bacillus subtilis, Staphlococcus aureus, Staphlococcus epidermidis, Streptococcus pneumonia, Lactobacillus, Streptococcus pyogenes, Bacillus cereus, and Clostridium tetani. We are also further testing the specificity of these hexamers by expanding antibiotic analysis to cover a range of Gram negative bacteria, including Escherichia coli and Psuedomonas aeruginosa. Here, we will present a thorough characterization of these hexamers as potential simple yet effective antibiotic agents against a wide array of pathogenic bacterial species.Support or Funding InformationHampden‐Sydney College Honors Council
Mechanisms by which plant extracts delay aging in yeast by targeting certain signaling pathways and modulating lipid metabolism
Authors: Titorenko Vladimir , Lutchman Vicky , Medkour Younes , Leonov Anna , Arlia‐Ciommo Anthony , Svistkova Veronika , Dakik Pamela 
Published: November 2023

The yeast Saccharomyces cerevisiae is a unicellular eukaryote that has been successfully used as a model organism for discovering signaling pathways and chemical compounds that modulate cellular aging, define organismal lifespan, and influence organismal fitness not only in yeast but also in various multicellular eukaryotes. Using a robust quantitative assay for measuring yeast chronological lifespan, we discovered six plant extracts (PE; i.e. PE4, PE5, PE6, PE8, PE12 and PE21) whose longevity‐extending efficiencies greatly exceed those of currently known natural anti‐aging compounds, such as resveratrol, rapamycin, spermidine, caffeine, and lithocholic acid. Our genetic, biochemical, and mass spectrometry‐based lipidomic analyses uncovered the following mechanisms by which these PE delay yeast chronological aging by targeting certain signaling pathways and modulating lipid metabolism: (1) PE4 attenuates the inhibiting effect of the pro‐aging TOR (target of rapamycin) signaling pathway on the anti‐aging AMP‐activated protein kinase (AMPK/Snf1), increases the intracellular concentration of phosphatidic acid (PA), and decreases the concentration of triacylglycerols (TAG; the major form of energy storage in yeast and other eukaryotes); (2) PE5 mitigates the pro‐aging cAMP/PKA (cAMP/protein kinase A) signaling pathway, greatly elevates the concentration of PA, reduces the concentration of TAG, and rises the concentration of cardiolipin (CL; a phospholipid synthesized only within mitochondria in yeast and other eukaryotes); (3) PE6 targets currently unknown pro‐ and/or anti‐aging pathways of longevity regulation, increases the concentration of PA and phospholipids other than CL, and decreases the concentration of TAG; (4) PE8 weakens the inhibiting effect of the pro‐aging cAMP/PKA signaling pathway on the anti‐aging AMPK/Snf1, enlarges the concentrations of PA, CL and all other phospholipids, and lessens the concentration of TAG; (5) PE12 activates the anti‐aging protein kinase Rim15 (on which the pro‐aging TORC1 and cAMP/PKA signaling pathways converge), and decreases the concentrations of CL and TAG; and (6) PE21 attenuates the pro‐aging protein kinase Sch9 (which is activated by the pro‐aging TOR and Pkh1/2 signaling pathways), elevates the concentrations of PA and phospholipids other than CL, and reduces the concentration of TAG.Support or Funding InformationSupported by NSERC of Canada.
Small Molecule Activation Affects Cellular Localization of NFκB and Fox03 During LPS‐induced TLR4 Signaling
Authors: Day Rachael Anne , Stoub Darren , Gupta Vineet 
Published: November 2023

Mac‐1, a β2 integrin, which is involved in cellular communication, has been implicated in leukocyte‐mediated inflammation. We recently discovered a class of small molecules, termed leukoadherins (LA), which are suggested to be Mac‐1 agonists, modulate cell adhesion, alter leukocyte‐induced phagocytosis and are, in general, anti‐inflammatory agents. In this study, we investigated how LA‐induced Mac‐1 activation modulates LPS‐induced TLR4 signaling pathways by affecting cellular localization of transcription factors Fox03 and NFκB. Using an optimized protocol for immunofluorescence, the cytoplasmic and nuclear levels of Fox03 and NFκB were analyzed both qualitatively and quantitatively, in the presence and absence of LA and LPS, in both RAW cells (murine leukocytes) and isolated PBMCs (human leukocytes). Our results show that LA causes NFκB to remain cytosolic and causes Fox03 to localize to the nucleus. Since Fox03 is a transcription factor that inhibits production of pro‐inflammatory chemokines, such as IRF‐7 & IFNα, and since NFκB is a transcription factor that promotes the production of pro‐inflammatory chemokines, such as TNFα and IL‐18, modification of cellular localization of either factor impacts inflammation. The results suggest that the anti‐inflammatory effects of LA may involve the Mac‐1 induced degradation of MyD88, which inhibits the IKKα/β pathway, which retains NFκB in the cytoplasm, and inhibits the AKT pathway, which allows for Fox03 to localize to the nucleus. Leukoadherins, therefore, represent a novel class of anti‐inflammatory medicines that have potential to treat disease such as glomerular nephritis, cancer metathesis and Systemic Lupus Erythematosus.Support or Funding InformationThis work was supported in part by grants from the NIH, Rush Medical School, and Dordt College.
Acetylating Polymyxin Antibiotics: Clues Toward Substrate Specificity of PA3944 Gcn5‐related N‐acetyltransferase of Unknown Function
Authors: Kuhn Misty L , Joe Layton , Amsler Brian , Zhang Brian , Majorek Karolina , Yen Robert , Wu Weiming , Gassner George , Baird Teaster , Minor Wladek 
Published: November 2023

Although Gcn5‐related N‐acetyltransferases (GNATs) have been studied for several decades in the context of aminoglycoside resistance and histone acetylation, the majority of their functions across all kingdoms are unknown. Members of this family typically catalyze the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to an acceptor substrate, which may include a protein, antibiotic, polyamine, or other metabolite. The goal of our laboratory is to determine the structure/function relationships of bacterial GNATs of unknown function. To achieve this, we use a broad‐substrate screening assay to identify initial substrates to kinetically characterize the enzymes and to provide ligands for crystallization trials. We selected the uncharacterized GNAT PA3944 enzyme from Pseudomonas aeruginosa, determined its three‐dimensional structure, and found that it acetylates polymyxin antibiotics (polymyxin B and E). Polymyxin antibiotics are cyclized antimicrobial peptides with multiple L‐2,4‐diaminobutyric acid residues that carry a positive charge and can disrupt the cell membrane. Since the rate of developing new antibiotics lags considerably behind the rise in bacterial antibiotic drug resistance, polymyxin antibiotics are being revisited as viable candidates for treating P. aeruginosa infections. It is not known if the PA3944 enzyme is important for polymyxin resistance and if it specifically acetylates the antibiotic at one position or can multiply acetylate the antibiotics. Here, we present the kinetic characterization of the PA3944 enzyme and provide insight into its substrate specificity toward polymyxin antibiotics, which will be important for understanding the necessary requirements for potential adaptive drug resistance mechanisms.Support or Funding InformationSupport for this project was provided to MLK through San Francisco State University in the form of an ORSP Small Grant and Startup funds.
Cell‐specific Glycomic and Signaling Alterations Associated with Human Ganglioside Deficiency
Authors: Dookwah Michelle , Aoki Kazuhiro , Kulik Michael , Dalton Stephen , Steet Richard , Tiemeyer Michael 
Published: November 2023

Salt & Pepper Syndrome (S&PS) is an autosomal recessive neurological disorder characterized by seizures, severe intellectual disability, choreoathetosis, dysmorphic facial features, and altered skin pigmentation. S&PS is caused by a mutation in the ST3GAL5 gene that encodes GM3 synthase, a glycosphingolipid‐specific sialyltransferase. ST3GAL5 synthesizes GM3 ganglioside by adding sialic acid to lactosylceramide. Both GM3 and ST3GAL5 are highly enriched in neural tissues. S&PS fibroblasts completely lack GM3 and comprehensive glycomic analysis revealed collateral alterations in N‐linked, O‐linked, and GSL glycans in comparison to normal fibroblasts. S&PS and normal fibroblasts were reverted to pluripotency and the resulting induced pluripotent stem cells (iPS cells) were subsequently differentiated to neural crest cells (NC cells) to investigate neural specific glycosylation and signaling alterations associated with loss of GM3. Complete loss of GM3 was recapitulated in the S&PS iPS and NC cells. N‐linked glycosylation was uniquely altered in S&PS NC cells, resulting in greatly increased abundance of high‐mannose glycans. Since glycosphingolipid abundance and glycoprotein glycosylation both modulate receptor signaling in numerous contexts, we assessed the activity of a spectrum of receptor tyrosine kinase pathways by antibody array. A decrease in ErbB3 phosphorylation was detected in the S&PS NC cells in comparison to normal cells. Western blot analysis also showed a sharp decrease in ErbB3 receptor expression as well as an increase in EGFR expression during the course of differentiation of S&PS iPS to NC cells. S&PS iPS cells provide a novel platform to investigate the influence of glycosphingolipids on cell signaling in specific cell types.Support or Funding InformationNIGMS/NIH
Lectin‐Like Properties of Human Antibodies Against the Red Meat‐derived Antigen N‐Glycolylneuraminic Acid: A Mitogenic Factor Promoting Carcinoma Growth
Authors: da Silva Frederico Alisson , Nguyen Nickolas , Varki Ajit 
Published: November 2023

Considerable epidemiological data has shown that human susceptibility to various carcinomas (particularly colorectal cancer) is strongly impacted by diet, especially by the consumption of red meat (foods of mammalian origin). There are many proposed mechanisms for the cancer‐promoting effects of red meat including generation of mutagens by grilling or addition of nitrates, and free‐radical generation by heme iron. However, the grilling and nitrate addition factors also apply to poultry and fish (which are not associated with cancer risk), and the heme iron theory is not conclusively proven. Also unexplained is the fact that other carnivores do not seem to share this red meat related risk. Our group has recently discovered a novel human‐specific mechanism involving the consumption of a non‐human sialic acid N‐glycolylneuraminic acid (Neu5Gc), which is enriched in red meats. We have reported that despite the fact that humans are genetically unable to produce Neu5Gc, this molecule can be metabolically incorporated and expressed on the cell surface glycans of human carcinomas after dietary intake. Such Neu5Gc containing glycans interact with circulating anti‐Neu5Gc antibodies, leading to an inflammatory response (xenosialitis), which can increase tumor incidence in human‐like Neu5Gc‐deficient mice that have circulating anti‐Neu5Gc antibodies. These antibodies naturally exist at variable concentrations in all humans. Since anti‐Neu5Gc antibodies can interact with any cell surface glycans containing Neu5Gc, we considered the possibility that these antibodies may also have lectin‐like activity by cross‐linking multiple surface molecules, with pleiotropic effects on cell signaling during interaction with tumor cells expressing Neu5Gc. To test this hypothesis, human colorectal cell lines were fed with Neu5Gc and incubated with human serum samples containing high levels of anti‐Neu5Gc antibodies. We observed that incubation of colorectal cells with the serum samples led to surface deposition of anti‐Neu5Gc IgG and IgM antibodies and complement in Neu5Gc‐fed but not in unfed cells. The antibody and complement deposition induced cross‐linking of cell surface glycoproteins like Muc‐1 and increased phosphorylation of S6 ribosomal protein. Corroborating these findings, our preliminary data suggests that anti‐Neu5Gc antibodies have mitogenic properties that can promote proliferation of colorectal cancer cells expressing Neu5Gc, via a mechanism similar to that of growth factors. Our results show for the first time the lectin‐like activity of human anti‐Neu5Gc antibodies as a mechanism for the promotion of carcinoma growth associated with red meat consumption.Support or Funding InformationProgram Science Without Borders ‐ Conselho Nacional de Aperfeicoamento de Pessoal de Nivel Superior (Capes) BEX‐9254‐13‐7‐ Brazil to FAS and NIH R01GM32373 to AV.
Investigation of Enzymatic Mechanism and Cellular Expression of DPY‐19L1 and DPY‐19L4
Authors: Kastigar Alexis , Murdock Brandon , Rakus John 
Published: November 2023

Throughout the process of protein synthesis, many proteins require a co‐ or post‐translational carbohydrate molecule addition to properly achieve fundamental structural and functional roles. This process, known as glycosylation, is enzyme‐directed, site‐specific, and usually achieved by an N‐ or O‐linkage. However, there exists a specific class of glycosyltransferases that forms a glycosidic bonds with sugar moieties through a C‐linked carbon‐carbon bond. The mammalian enzyme DPY‐19 a C‐glycosyltransferase that attaches a mannose sugar to the second carbon of the indole ring of tryptophan residues in a tryptophan‐x‐x‐tryptophan consensus sequence (‘x’ is any amino acid). DPY‐19 was first identified in Caenorhabditis elegans, where a single homolog exists, but the human genome contains four such homologs (DPY‐19L1, DPY‐19L2, DPY‐19L3, DPY‐19L4) that are increasingly more divergent. We are particularly interested in DPY‐19L1 and DPY‐19L4, which have not been isolated biochemically for activity studies. The purpose of this research is to determine the enzymatic mechanisms of these two homologs with a particular interest in expression levels among a mammalian tissue panel for DPY‐19L4. Our first objective is to clone and express hsDPY‐19L1 and –19L4. To do so for DPY‐19L1, a HEK‐293 cell line was transiently transfected with purified DPY‐19L1 plasmid using calcium phosphate nanoparticles. Once the protein had been expressed, it was purified by nickel‐affinity chromatography. For DPY‐19L4, a gene insert of DPY‐19L4 was amplified, DPN digested, and mixed with DPN digested, linearized pcDNA3.1 plasmid as described by Benoit et al. (Benoit et al., 2006, Protein Expr. Purif.). In Fusion enzymes mediated the mixture of the insert and plasmid and the cells were cultured on agar plates to produce a plasmid colony that can be used to tag spun down RNA samples from a eukaryotic cell panel that contain DPY‐19L4. Thus far, the amplification of the DPY‐19L4 insert and linearization of the pcDNA3.1 plasmid have been successful. Our second objective is to evaluate C‐mannosyltransferase expression in vivo. We performed real time polymerase chain reaction (RT‐PCR) for the mammalian DPY‐19 homologues in a panel of cell lines and tissues and probed the expression of these four transferases in lipopolysaccharide‐stimulated macrophages.Support or Funding InformationMarshall University College of Science
Regular Supplementation of Dietary Compounds Modulates Longevity and Aging in Drosophila melanogaster
Authors: Holohan Liam , Rogers Ryan Patricia 
Published: November 2023

Each time a cell divides telomeres shorten until a critical number of cell divisions is reached. This shortening of telomeres is associated with cells entering a post‐mitotic state of replicative senesce, which has been related to both cancer and aging. Previously, it has been shown that metals such as zinc and magnesium can modify cell replicative longevity as a result of improved metabolic homeostasis. Actively dividing cells can be characterized by the expression of specific genes associated with growth and proliferation; therefore, cells in a state of replicative senescence can be identified by variation in such gene expression patterns. Mitotic stem cell populations maintain several adult Drosophila tissues; therefore, these organisms are used to investigate the relationship between gene expression, telomere maintenance, and aging at the cellular and organismal levels.Adult yellow‐white Drosophila (n>100) were exposed to varied levels of ZnSO4 to determine optimal effects of supplementation. Survival curves were produced over the course of 52.5 hours and it was concluded that 0.125mM ZnSO4 had an effect on lifespan without lethality. Similar trials suggest similar outcome with respect to a dose‐dependent effect on longevity following Mg SO4 supplementation. Flies were exposed to food (Carolina Biologic Supply 4–24, plain) supplemented with 0.125mM ZnSO4 daily for two weeks. RNA was isolated from whole flies and RT‐qPCR was used to measure changes in telomerase fusion protein (tefu) mRNA, which is a homolog to the mammalian enzyme. Female flies showed increased tefu mRNA levels following prolonged ZnSO4; whereas male flies were found to have less than their respective controls.The results from these experiments may reveal therapeutic applications to enhance our understanding of the relationship between cell division and organismal longevity. Furthermore, these data can be applied to cancer presentation and progression.Support or Funding InformationSupported by the funds from the Wentworth Institute of Technology Department of Sciences
Detection of glycerophospholipids and sphingolipids in urine and their correlation with cerebrospinal fluid beta‐amyloid 42
Authors: Fonteh Alfred N. , Cipolla Matthew , Chiang Jiarong , Castor Katherine , Harrington Michael 
Published: November 2023

ObjectivesTo test the hypothesis that, a defective blood‐brain barrier in Alzheimer's disease (AD) results in increased brain lipid release, and that urine lipid levels correlate with cerebrospinal fluid (CSF) Aβ42.MethodsStudy participants (70–100 years) were enrolled into an IRB‐approved aging study and CSF and urine were obtained after an overnight fast. After a neuropsychological assessment, the cognitively normal (CN) group was subdivided based on CSF Aβ42 and tau levels; CN study participants with normal Aβ42/tau (CN‐nAT, n=36), and CN with pathological Aβ42/tau (CN‐pAT). Urine lipids were extracted and liquid chromatography tandem mass spectrometry was used to quantify glycerophospholipids and sphingolipids.ResultsWe detected glycerophospholipids [phosphatidylcholine (PC), lysophosphatidylcholine (LPC), platelet activating factor (PAF)], sphingolipids [sphingomyelin (SM), ceramide (CM), dihydroceramide (dhCM)] in urine. When normalized to total lipid content of CSF or to creatinine, PC, LPC, and PAF levels were higher in AD than in CN. PC, but not levels of the other lipids, significantly correlated with Aβ42 levels in CSF.ConclusionsChanges in glycerophospholipids and their correlation with CSF Aβ42 suggest that urine lipids are potential biomarkers of AD pathology that may be used for non‐invasive screening for dementia in the general population.Support or Funding InformationL.K. Whittier and the Helen Posthuma Foundations