bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Information

Hampden‐Sydney College Honors Council
Development of Novel Small Molecule Antibiotics against T‐box Riboswitches in Gram‐positive Bacteria
Authors: Väre Ville Yrjö Petteri , Todd Gabrielle C. , Frohlich Kyla M. , Bell Janeen T. , Weintraub Spencer F. , Agris Paul F.
Published: November 2023

T‐box riboswitches are RNA regulatory elements located at the 5′‐untranslated region (5′‐UTR) found in numerous gram‐positive bacteria that control several genes involved in tRNA aminoacylation, as well as amino acid metabolism and transport. T‐boxes function by recognizing the charge status of their cognate tRNA ligand: uncharged tRNA stabilizes an antiterminator complex when bound, allowing transcription of the downstream operon, whereas charged tRNA is unable to fully bind the T‐box, allowing a terminator loop to persist and halt transcription. Given that T‐boxes are found exclusively in gram‐positive bacteria and share highly conserved regions necessary for tRNA binding, this RNA regulatory element represents a prime target for antibacterial drug development. Using docking studies to known atomic‐resolution T‐box structures, 43 putative antibacterial compounds representing distinct chemical families were selected for further characterization. Antibacterial microdilution studies resulted in a moderately active hit compound (PKZ18) that has been selected for antibacterial drug development. Characterization studies demonstrate that PKZ18 is refractory to resistance, displays low cytotoxicity, and possesses activity against a wide range of gram‐positive bacteria, including clinical isolates of Staphylococcus aureus, Streptococcus pyogenes, and Clostridium difficile. Mechanism of action studies confirm binding of PKZ18 to both glycine and tyrosine T‐boxes. Structure‐activity relationship (SAR) studies have revealed essential moieties for both selectivity as well as activity. Hit‐to‐lead optimization of PKZ18 by medicinal chemistry and further SAR studies is on‐going.
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 Information

Supported 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 Information

This 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 Information

Support 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 Information

NIGMS/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 Information

Program 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 Information

Marshall 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 Information

Supported 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

Objectives

To 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.

Methods

Study 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.

Results

We 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.

Conclusions

Changes 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 Information

L.K. Whittier and the Helen Posthuma Foundations
In good taste: An investigation of lactate metabolons in the mouse tongue
Authors: Ochrietor Judith D , Zahir Haaris , Castillo Luz , Seddeik Rita , Gelsleichter Jim D
Published: November 2023

Recent studies of sensory receptor tissues in the mouse suggest the importance of lactate metabolons within those tissues for proper function. A lactate metabolon consists of a monocarboxylate transporter (MCT) and a member of the immunoglobulin superfamily, such as Basigin gene products. In the neural retina, a lactate metabolon consisting of Basigin gene products and MCT‐1 and MCT‐4, has been deemed necessary for photoreceptor maturation and function. Basigin‐null mice do not express these metabolons and are blind from the time of eye opening. Basigin and MCT‐1 are also expressed on the myelin of olfactory nerve fibers, presumably to form a lactate metabolon required to support saltatory conduction. Basigin‐null mice have olfactory deficits, in that they do not respond to the smell of acetic acid as normal mice do. The absence of the metabolon likely accounts for the slower response rates observed in the null animals. The purpose of the present study was to investigate the presence of a lactate metabolon consisting of Basigin gene products and MCTs in the mouse tongue. Tongues were obtained from mice using an accepted protocol for use in immunohistochemical and quantitative RT‐PCR analyses. It was determined that Basigin is expressed on the mouse tongue epithelium, but not in the underlying muscle. No significant expression of MCT‐1 or MCT‐4 was detected in the tissue; however, MCT‐2 expression was observed within the taste buds. The data suggest that a lactate metabolon like those associated with olfactory nerve fibers and in the neural retina is not present in the mouse tongue.

Support or Funding Information

University of North Florida Office of Undergraduate Studies and the Office of Academic Affairs
Exploration of Plasmodium Kinases
Authors: Eubanks Amber L. , Derbyshire Emily R.
Published: November 2023

Malaria is a neglected tropical disease that affects millions of people worldwide. The World Health Organization reported almost 600,000 deaths in 2013 alone, with most of these among children and pregnant women. Malaria is caused by protozoan parasites from the genus Plasmodium and is transmitted to human hosts through Anopheles mosquitoes. Eradication of this devastating disease is complicated by the complex life cycle of the parasite and increasing drug resistance to first‐line treatments. In order to combat Plasmodium infections and drug‐resistance, a deeper understanding of the parasite biology is required such that better therapeutic targets can be elucidated. Protein kinases are becoming increasingly popular as druggable targets in many diseases due to their essential function in eukaryotic organisms. Unfortunately relatively little is known about the malaria kinome. More than 3% of the Plasmodium parasite proteome is made up of kinases and offers a wide array of targets. The goal of our work is to biochemically characterize malaria protein kinases that are involved in key biosynthesis and signaling pathways. These efforts will allow further biological understanding of parasite biology while also providing potential therapeutic targets. Screening compounds against these kinases will also aid in the discovery of small molecule probes and antimalarial agents.

Support or Funding Information

NIH

Duke University Chemistry Department
Expression profile of Monocarboxylate Transporters and Basigin gene products in the mouse cornea
Authors: Maniccia Randall G. , Fong Joseph D , Ochrietor Judith D
Published: November 2023

Monocarboxylate transporters (MCTs) transport lactate, pyruvate, and ketone bodies. Expression of MCTs has been linked to the expression of Basigin gene products, to form a lactate metabolon, that has been determined to be essential for proper photoreceptor function in the neural retina, and hence vision. Since the cornea is the outer, protective portion of the eye, this research group was curious if a similar lactate metabolon is present in that tissue. The group is also curious about the role of MCTs in the wound healing process. Therefore, the purpose of the present study was to determine whether Basigin gene products and MCTs found within the neural retina are also expressed in the corneal epithelium of mice. Corneas were isolated from mice using an accepted protocol and pooled so that detergent‐solubilized proteins and RNA could be isolated from epithelial cells. ELISA analyses and quantitative reverse transcriptase polymerase chain reaction (q‐RT‐PCR), were performed to test for the presence of Basigin gene products, MCT1, MCT2, and MCT4. It was determined via ELISA that Basigin gene products, MCT2, and MCT4 were found within the corneal epithelium; however, MCT1 was not detected. Analyses of RNA from corneal epithelial cells indicate that the ubiquitously expressed short form Basigin is present in the tissue, but the photoreceptor‐specific long form Basigin‐2 was not. In addition, no MCT1 transcript expression was observed in the cornea sample. These data suggest that a lactate metabolon may be present in mouse cornea. In addition, the data are consistent with data obtained by other laboratories using human and rabbit corneas, with the exception of MCT1, which was found in those other species. Future studies will determine the role played by the lactate metabolon during wound healing of the mouse cornea.

Support or Funding Information

University of North Florida Office of Undergraduate Studies and the Office of Academic Affairs
High Fructose Corn Syrup‐55 Alters Hepatic Metabolism to Increase Risk of Non‐Alcoholic Fatty Liver Disease in Female Sprague‐Dawley Rats
Authors: Lateef Sundus , Mock Kaitlin , Benedito Vagner , Tou Janet
Published: November 2023

Studies have reported that high fructose intake promotes de novo lipogenesis (DNL). In turn, DNL stimulation can promote non‐alcoholic fatty liver disease (NAFLD), a metabolic condition that can progress to cirrhosis and liver failure. A frequent alternative to sucrose in calorically sweetened beverages, high fructose corn syrup‐55 (HFCS‐55) contains a higher percentage of free fructose (55%) compared to sucrose (50%, all of which is combined with glucose as a disaccharide). The objective of this study was to determine the effect of HFCS‐55 sweetened solution intake on hepatic lipid metabolism and risk of developing NAFLD in a rodent model. Female Sprague‐Dawley rats (age 28 days) were randomly assigned (n=7/group) to one of four treatment groups consisting of distilled deionized water (ddH2O) or ddH2O sweetened with sucrose, fructose or HFCS‐55 for 8 weeks. Caloric sweeteners were administered at a concentration of 13% w/v, the level typically found in sugar‐sweetened beverages. Gene expression of key transcription factors and enzymes involved in DNL including sterol regulatory element‐binding protein 1c (SREBP‐1c), fatty acid synthase (FAS), and stearoyl‐CoA desaturase 1 (SCD1) as well as peroxisome proliferator‐activated receptor alpha (PPARα), a transcription factor activating lipolysis, were measured using real‐time quantitative polymerase chain reactions (RT‐qPCR). Liver fatty acid composition was determined using gas chromatography and serum clinical markers were measured by enzymatic assays. Expression of SREBP‐1c (P=0.02) and FAS (P=0.001), molecular markers of DNL, were up‐regulated in all caloric sugar treatment groups compared to the ddH2O group. Transcript abundance of SCD1, which is involved in synthesizing palmitoleate (C16:1n‐7) and oleate (C18:1n‐9) from palmitate (C16:0) and stearate (C18:0), was also induced in all caloric sugar treatment groups compared to the ddH2O group (P=0.001). Hepatic palmitoleate content was higher in the sucrose and HFCS‐55 groups compared to the ddH2O group (P<0.001), and hepatic oleate content was highest in the HFCS‐55 group (P<0.001). PPARα expression was lower in the sucrose and HFCS‐55 groups (P=0.002). Altogether, we show evidence that HFCS‐55 intake had a greater effect on fatty acid metabolism than sucrose by promoting DNL, which was not compensated for by increased lipolysis. Rats drinking HFCS‐55 solution had highest hepatic triglyceride content (P=0.03) among all treatment groups and higher low density lipoprotein (P=0.02) than rats drinking ddH2O, indicating NAFLD. Thus, HFCS‐55 intake in healthy growing rats resulted in higher risk of NAFLD indicated by hepatic lipid accumulation and dyslipidemia.

Support or Funding Information

This research was sponsored in part by the West Virginia Research Challenge Fund through a grant from the Davis College of Agriculture, West Virginia University.
Parental diet modulates gene expression in Drosphila melanogaster
Authors: Tresser Jason
Published: November 2023

Caloric restriction is known to promote longevity in various animal species presumably through modulations in various metabolic pathways. In fruit files, low yeast content in food (low protein diet) has been shown to significantly extend lifespan. Microarray studies in Drosophila indicate that in addition to changes in the expression of metabolic genes, epigenetic factors also play a role in longevity. This suggests the possibility that offspring from diet restricted flies may inherit a propensity for long life through genetic imprinting. In our study, we are using quantitative PCR to measure gene expression levels in the offspring of flies subjected to diet restricted. In particular, the NAD+ ‐dependent deacetylase Sir2 is known to be upregulated in diet restricted flies. We are measuring Sir2 levels in offspring at embryonic, larval and adult stages.
Contribution of Poly‐unsaturated Fatty Acids on Cerebral Neurobiology: An Integrated Omics Approach with Epigenomic Focus
Authors: Chakraborty Nabarun , Muhie Seid , Gautam Aarti , Hammamieh Rasha , Jett Marti
Published: November 2023

Our previous transcriptomic study [1] suggested potential positive significance of the effects of fish oil on the cerebralneurogenesis and neuroprotection, and contrasting therapeutic implications for the neurodegenerative disorders. Stimulating the calcium‐induced growth cascade and downstream PI3K‐AKT‐PKC network, fish oil augmented neuritogenesis, reinforced synapse and potentially promoted long‐term potentiation. In conjunction, this diet reduced the amyloidal burden, attenuated oxidativestress, and assisted in somatostatin activation. Therefore, we anticipated the potential therapeutic roles of fish oil in attenuating Alzheimer's disease, Parkinson's disease, and affective disorder. Motivated from this past study, we investigated the shift of the DNA methylation and microRNA profiles caused by these same dietary supplements and potential consequences on cerebralfunctionalities.

N‐3 PUFA‐enriched diet (ERD, n3:n6= 7:1) and balanced diet (BLD, n3:n6= 1:1) were customized to contain caloric content equivalent to the standard lab diet (STD, n3:n6= 1:6). The diets were administered to three groups of C57BL/6J male mice from their weaning age until late adolescence. At the study endpoint, the transcriptomic and epigenomic profiles of the hemi brains were probed.

Our primary focus was on hypermethylated probes and microRNAs which were causally related to transcriptional silencing. We found 18 overexpressed microRNAs in the ERD‐fed mice that inhibited the transcriptional expressions of 580 genes.. Likewise, there were 40 overexpressed microRNAs targeting 800 suppressed gene in BLD‐fed mice; a majority of these microRNAs are functionally associated with the neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.

In addition, there were ~4,000 probes hypermethylated in either ERD‐ or BLD‐fed mice. Of particular interest, ERD‐fed mice exhibited ~300 genes concurrently imprinting hypermethylated CpG islands and silenced transcripts. Similarly, 131 genes displayed hypermethylated CpG islands and transcriptional suppression in BLD‐fed mice. All these hypermethylated CpG islands were mapped within 50kb from their respective transcriptional startsites, indicating their rather archetypal effects on transcriptional silencing. Functional annotation of these genes indicated a pattern of neurological implications distinguishing the positive effects related to ERD and BLD. ERD primarily influenced the developmental phase of neuronal and cerebral activation, while the behavioral manifestations were potentially stimulated by BLD. A validation study is underway.

Disclaimer: Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.
The role of 12/15‐Lipoxygenase in a Mouse Model of Alcoholic Liver Disease
Authors: Kirpich Irina , Miller Matthew , Ramsden Christopher , Feldstein Ariel , McClain Craig
Published: November 2023

Introduction/Aim

12/15‐Lipoxygenase (12/15‐LOX) catalyzes the oxidation of free and esterified polyunsaturated fatty acids, predominantly, linoleic and arachidonic, generating the whole spectrum of bioactive lipid mediators. This enzyme is involved in the regulation of various homeostatic processes as well as in the pathogenesis of multiple diseases. Recently published studies from our group and others have shown that experimental and clinical alcoholic liver disease was associated with elevated oxidized linoleic acid metabolites (OXLAMs), specifically 9‐ and 13‐hydroxy‐octadecadienoic acids (9‐ and 13‐HODEs). The aim of the present study was to examine the role of 12/15‐LOX in an animal model of ALD.

Materials and Methods

Eight‐week‐old male 12/15‐LOX knockout mice (12/15‐LOX−/−), and their genetically unaltered wild type (WT) counterparts were fed Lieber–DeCarli control (isocaloric maltose‐dextrin) or ethanol (5% w/v) liquid diets ad libitum for 10 days plus a single binge ethanol administration (5 g/kg) by gavage, whereas mice in control groups were gavaged with isocaloric maltose dextrin. Liver histopathological changes were estimated by hematoxylin and eosin staining. Liver triglyceride accumulation was assessed by biochemical assay and Oil Red O staining. Liver injury was evaluated by plasma ALT and AST activity. Liver inflammation was determined by hepatic proinflammatory gene expression. Chloroacetate esterase staining was used to evaluate necroinflammatory changes. TUNEL staining was used to determine hepatic apoptotic cell death. Plasma OXLAMs were analyzed on LC‐MS and GCxGC/TOF‐MS platforms.

Results

Chronic‐binge alcohol administration resulted in a marked increase in plasma OXLAM levels, specifically 9‐HODE and 13‐HODE, in parallel with up‐regulation of hepatic 12/15‐LOX in WT animals. These effects were associated with hepatic steatosis, inflammation and injury. 12/15‐LOX genetic depletion did not reduced hepatic steatosis caused by EtOH, but did ameliorate hepatic injury as assessed by ALT (231.2+32.8 U/L in WT vs 148.4+48.6 U/L in 12/15‐LOX−/−) and AST (279.1+36.7 U/L in WT vs 177.1+28.3 U/L in 12/15‐LO−/−) levels. 12/15‐LOX deficiency did not affect hepatic apoptosis; while neutrophil infiltration was significantly reduced in 12/15‐LOX deficient mice compared to WT animals. 12/15‐LOX deficiency prevented EtOH‐induced pro‐inflammatory cytokine and chemokine expression, including Tnf‐α and Mcp1 mRNA levels.

Conclusion

12/15 LOX plays an important role in the pathogenesis of experimental ALD, in part, via increased production of OXLAMs. 12/15‐LOX and OXLAMs may represent novel therapeutic targets in ALD management

Support or Funding Information

Study supported by the NIH and Veterans Administration.
Development of a Python‐Based Algorithm for Comparative Analysis of Multiparticipant Next Generation Sequencing Data
Authors: Martz Flora G. , Forst Thomas M. , Ryan Sean M. , Murphy Patrick J. M.
Published: November 2023

The goal of this study was to develop a computational approach using the Python programming language to identify the extent to which genetic variation, and single nucleotide polymorphisms (SNPs) in particular, identified by whole exome sequencing correlate with previously observed phenotypic variations. Using previously obtained whole exome data from otherwise healthy volunteers possessing extreme glucocorticoid‐response phenotypes, an open source algorithm was developed. Python was selected as the preferred programming language for this purpose because of its cross‐platform operability, open source code, and precedent for managing omics‐level biological data. The program was designed to parse multiple participants’ whole exome sequencing files in sequence and allow for the selection of user‐defined filtering parameters to generate comparative analyses of polymorphisms unique to, or shared among, participants of known phenotype. In addition, a proofreading mechanism and basic user interface were incorporated into the code to enhance the scope and capability of the program. Further analytical parameters include data selection based mutation type, significance of mutation, zygosity, sequence coverage, confidence interval, gene/transcript information, and coding significance. Creation of this program allowed for enhanced analytical evaluation of systems‐wide observations regarding phenotypic variance.
Development of a Python‐Based Algorithm for the Quantification and Analysis of Multivariate Next Generation Sequencing Data Following Genome‐Wide Mutagenesis and Cell Survival Screening
Authors: Forst Thomas M. , Murphy Patrick J. M.
Published: November 2023

Hsp90 is a highly conserved eukaryotic chaperone protein responsible for mediating a myriad of intracellular signaling pathways, including those associated with the glucocorticoid receptor (GR) and the intracellular immunoinflammatory response. Both hsp90 inhibitors such as geldanamycin and glucocorticoids such as prednisone have been shown to possess potent anticancer activity, which can be overcome at the cellular level by hereto unknown genetic modifications. The purpose of this study was to develop a computational approach for interpreting and analyzing next generation sequencing data following genome‐wide mutagenesis and anticancer cell survival screening assays. In order to assess genome mutation sites, an open‐source computer program was developed. The program allows for the reading of raw sequencing and annotated data files and permits the user to select filtering parameters to quantify the incidence and prevalence of identified mutations and mutation insertion sites. Further analysis parameters include annotation and extrapolation of the surrounding gene landscape and anticipated mutation influence. Additional parameters, including filtration based on mutation type and significance, sequencing confidence and reads, mutation location and neighboring gene profiles are also included, thus allowing for prediction of potential causal candidate mutations and genetic perturbations affecting cell viability subsequent to cell chaperone inhibition and glucocorticoid‐mediated transactivation.
Effects of Synthetic Ligands on Heterodimer Pairs Regarding PPARα, RXRα and LXRα
Authors: Delman Emily Erin , Rider Stanley Dean , Hostetler Heather Ann
Published: November 2023

In this study, we hypothesize that ligand binding determines the binding partners and subcellular localization of nuclear receptors involved in atherosclerosis and diabetes. Specifically, we are interested in the effects of synthetic ligands on the peroxisome proliferator‐activated receptor alpha (PPARα), the liver X receptor alpha (LXRα) and the retinoid X receptor alpha (RXRα) and corresponding effects on heterodimer formation. Binding assays using a photon counting spectrofluorometer indicate promiscuity of protein‐ligand interactions where ligand binding is not solely exclusive. Additionally, Forster Resonance Energy Transfer (FRET) assays indicate that synthetic ligands affect the protein binding capability of the novel heterodimer PPARα:LXRα. Changes in secondary structure are anticipated, which would argue certain synthetic compounds can induce conformational changes that alter heterodimer formation. Bimolecular Fluorescence Complementation (BiFC) allows us to view protein‐protein interactions in vivo using fluorescence microscopy. We show that synthetic ligands influence subcellular localization of these nuclear receptors in stably transfected living cells. Due to the complex regulation of these nuclear receptors, it becomes necessary to study synthetic ligands with each receptor, individually and in heterodimeric form. Our findings will contribute to further the understanding of the clinical implications of synthetic ligands on disease states such as atherosclerosis and diabetes.

Support or Funding Information

This research is supported by USPHS NIH grant DK77573 and the Emerging Science Seed Grant provided by the Boonshoft School of Medicine and Wright State University.
Critical Role of Residues in Helices 9 and 10 of the Liver X Receptor Alpha (LXRα) in Mediating Heterodimer Choice between Heterodimeric Complexes
Authors: Bedi Shimpi , Hostetler Heather A. , Rider Stanley Dean
Published: November 2023

The Liver X Receptor Alpha (LXRα) is a ligand‐activated nuclear transcription factor that is activated upon binding to oxysterols and plays an important role in lipid homeostasis. Heterodimerization with a common heterodimeric partner, the retinoid X receptor (RXR), regulates the transcription of target genes. LXRα‐RXRα activation has been shown to inhibit cardiovascular disease and reduce atherosclerotic lesions in mice. Additionally, LXRα can also form heterodimers with the Peroxisome Proliferator‐Activated Receptor Alpha (PPARα), another ligand‐activated nuclear receptor involved in fatty acid (FA) metabolism. Key residues located in helices 9 and 10 of LXRα may mediate dimerization of LXRα with partner receptors. These residues show a high degree of conservation across species and among nutrient‐sensing nuclear receptors. Little is known about the effects of charge disruption at the LXRα interface on protein‐protein and protein‐ligand associations. Using overlap PCR, we generated LXRα mutants in which the interface had been modified to determine whether heterodimerization selective variants of LXRα can be generated. Fluorescence spectroscopy in cells and in vitro demonstrated that charge reversal at the LXRα dimerization interface has the ability to influence LXRα protein and ligand binding. Using the intrinsic quenching of the aromatic amino acids located in the ligand binding pocket of LXRα, we have demonstrated that LXRα R415A does not bind T‐0901317, but retains its ability to bind 25‐hydroxycholesterol and palmitic acid. Direct protein‐protein binding experiments using the fluorescently labeled proteins suggested that LXRα R415A binding to RXRα is unaffected whereas binding to PPARα is enhanced. Another mutant of LXRα leaves LXRα‐PPARα binding intact but reduces LXRα‐RXRα binding. This indicates that key residues at the interface differentially regulate protein‐protein and protein‐ligand interactions of LXRα. Key contact regions of LXRα may represent a new drug target for modulating LXRα signaling in lipid‐related disorders.

Support or Funding Information

This research was supported by USPHS NIH grant DK77573 and the Emerging Science Seed Grant provided by the Boonshoft School of Medicine and Wright State University.
Characterization of the Functional Significance of PPAR/LXR Heterodimers via a Novel Response Element
Authors: Klingler Andrea M , Rider S. Dean , Hostetler Heather
Published: November 2023

Peroxisome proliferator‐activated receptor alpha (PPARα) and liver X receptor alpha (LXRα) are nuclear receptors known to regulate opposing processes in energy homeostasis through heterodimerization with the retinoid X receptor alpha (RXRα). Dysregulation of these proteins has been linked to such metabolic disorders as atherosclerosis, hypercholesterolemia, and hypertriglyceridemia. Among the more intriguing recent discoveries in the field is that PPARα and LXRα are also able to interact directly with each other and bind DNA as a heterodimer. Although this interaction is widely thought to be inhibitory or otherwise non‐functional, we hypothesize that PPARα‐LXRα heterodimers are indeed capable of carrying out regulatory function via previously uncharacterized response elements. Gel shift analysis of several designed direct repeat sequences revealed that PPARα‐LXRα heterodimers bind preferentially to a DR4 motif, and supershift assays confirmed both PPARα and LXRα were bound to the oligonucleotide probes. Similarly, intrinsic fluorescence quenching assays determined that PPARα‐LXRα binds to these DR4 sequences with high affinity. The sequence bound with the highest affinity was found to occur naturally in the human genome, including near the promoter regions of known PPARα and/or LXRα target genes, such as apolipoprotein A‐1 (APOA1), cytosolic sulfotransferase 2A1 (SULT2A1), and the sterol regulatory element binding protein 1‐c (SREBP‐1c). Dual luciferase reporter assays showed that overexpressing PPARα and LXRα in COS‐7 cells leads to transactivation of the endogenous APOA1 and SULT2A1 genes, but not of SREBP‐1c. These findings illuminate the complex regulatory mechanisms modulated by PPARα and LXRα, and provide a potential new avenue for targeting these receptors for treatment of metabolic disorders.

Support or Funding Information

This work was supported by USPHS NIH grant DK77573 and the Emerging Science Seed Grant provided by the Boonshoft School of Medicine and Wright State University.