bluuebunny/crossref_metadata_2025 huggingface dataset:
A Time Course Analysis of the Effects of Arachidin 1 and Arachidin 3 on a Rotavirus‐Infected Human Intestinal Cell Line
Authors: Witcher Caleb Michael , Lockwood Hannah Nicole , Napier‐Jameson Rebekah , Mattila Macie N , Strange Essence B , Taylor Josephine , Clack Beatrice , Medina‐Bolivar Fabricio , Ball Judith M
Published: November 2023

Rotavirus (RV) causes severe gastroenteritis in infants and children worldwide. To prevent RV induced diarrhea, research efforts are focusing on new vaccines and novel therapeutic approaches. Previously, our laboratory has demonstrated that arachidin 1 (A1) and arachidin 3 (A3) have significant effects on the amount of infectious RV particles released from a human intestinal cell line, HT29.f8, at 18 hours post infection (hpi). Morphometric analyses have suggested changes in programed cell death pathways. The objective of this study was to observe the effects of A1 and A3 on RV infected HT29.f8 cells at 12, 14, 16, 18 hpi. A time course morphometric analysis was performed to visualize the effects of the arachidins on cellular ultrastructure and RV particles using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Additionally, changes in ratios of nucleus to cytoplasm during the time course were determined with/without RV and arachidin treatments. RV particles (with/without envelope) in infected cells (with and without the arachidins) were measured and statistically compared to determine if the observed virus particles represented different populations. To quantify the infectious virus particles released from the RV infected cells, plaque forming assays were performed. Additionally, tunable resistive pulse sensing technology (TRPS) using the qNano system from Izon was used to quantify total infectious virus particles and determine the size distribution of the released RV particles from HT29.f8 cells with/without the arachidins during the time points of collection. qRT‐PCR was performed to further identify changes in the expression of key apoptotic and autophagic genes, and western blots assays were used to confirm changes in the expressed proteins that are essential for the two cell death pathways. The data suggests that the arachidins have a significant role in influencing HT29.f8 cells to enter apoptotic or autophagic cell death pathways, and inhibit the maturation and production of progeny RV populations. The plaque assays confirmed a change in the amount of infectious virus particles produced with the addition of thee arachidins. This implies that A1 and A3 arrest RV particles at the enveloped stage of development. Thus, these small molecules have the potential to be developed as anti‐RV therapeutic agents.

Support or Funding Information

This work was supported by: 1) The Office of Research and Sponsored Programs at Stephen F. Austin State University (RPS # 107552‐26112‐150); 2) The NSF‐EPSCoR (grant# EPS‐0701890; Center for Plant Powered Production‐P3).
Peptide B7–33, a Single‐Chain Derivative of the Relaxin Hormone Protects Cytotrophoblasts from Marinobufagenin‐Induced Preeclampsia Phenotype
Authors: Pantho Ahmed Faiaz , Afroze Syeda Humayra , Dean DeShaundra M , Hossain Mohammed Akhter , Bathgate Ross , Xia Sijie , Zawieja David C , Kuehl Thomas J , Uddin Mohammad Nasir
Published: November 2023

Background

Preeclampsia (PreE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. Recently, a digitalis‐like factor, marinobufagenin (MBG) has been implicated as a causative factor in preE. We demonstrated that MBG inhibits the proliferation, migration, and invasion of cytotrophoblast (CTB) cells. Relaxin is a peptide hormone that allows vasodilation and plays an important role in the process of parturition. The literature suggests potential therapeutic role of H2 relaxin in PreE, however, there is a controversy on hypotensive action of the peptide. Due to the complex insulin‐like structure of relaxin (A‐ and B‐ chains, 53 amino acids, 3 disulfide bonds), a novel H2 relaxin B‐chain‐only peptide variant B7–33 (27 amino acids without any disulfide bonds) has recently been developed. This single‐chain peptide displayed equivalent efficacy to the natural H2 relaxin in several functional assays both in vitro and in vivo. Importantly, B7–33 was shown to have H2 relaxin‐like RXFP1 specific effects, particularly in endogenously expressing RXFP1 cells, thus we hypothesized that B7–33 could be an alternative and cost‐effective treatment option for PreE compared with H2 relaxin.

Methods

Human CTBs were treated with DMSO (vehicle) or 0.1, 1, 10 or 100 nM of MBG for 48 h and were co‐treated with B7–33 (25 nM) with MBG exposure, while some cells were treated with 5, 10, 25 and 50 nM B7–33 alone. Levels of vascular endothelial growth factor (VEGF), placental growth factor (PlGF), soluble fms‐like tyrosine kinase‐1 (sFlt‐1), and soluble endoglin (sEng) were measured in culture media using ELISA kits. Cell lysates were utilized to evaluate the expression of mTOR, pAKT and MMP‐9 by western blotting. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.

Results

Secretion of sFlt‐1 and sEng were increased while VEGF and PIGF were decreased in CTBs treated with ≥1.0 nM MBG (*p < 0.05 for each). B7–33 co‐treatment significantly rescued CTBs from MBG‐induced anti‐angiogenic profile (p < 0.05 for each). There is no effect of B7–33 on sFLT‐1, sEng and PlGF; however, it increases the expression of VEGF, while CTBs were treated only with B7–33. B7–33 also causes increased expression of mTOR, pAKT and MMP‐9 in CTBs.

Conclusions

B7–33 mitigates the MBG‐induced dysfunction of CTBs by attenuating anti‐angiogenic phenotype similar to that seen in PreE. This study supports the importance of continuing research of B7–33 in preE prevention.
Inhibition and Dispersion of Biofilms: Targeting Bacterial Response Regulators to Resensitize Multidrug Resistant Bacteria to Antibiotics
Authors: Milton Morgan E , Draughn G Logan , Feldmann Erik A , Thompson Richele J , Jung David , Kang Brian , Theisen Kelly E , Zeng Daina , Lucas Julie L , Melander Christian C , Cavanagh John
Published: November 2023

Biofilms provide cells with protection against numerous physical and chemical assaults. Bacteria utilize biofilms to avoid antibiotics, leaving the bacteria resistant to antibiotic treatments. The rise of antibiotic resistance and lack of new viable treatments threaten to usher in a post‐antibiotic era if new approaches towards drug development are not made. Many of the currently available antibiotics target a narrow spectrum of metabolic processes required for cellular growth. These traditional approaches no longer offer long‐term protection due to the acquisition rate of resistance; new mechanisms must be pursued. One such mechanism is through the two‐component systems (TCSs) used in bacterial communication. TCSs allow the cell to detect and respond to changes in its environment and are used to trigger quorum sensing and virulence factors. Drugs that target the TCS signaling process could inhibit pathogenic behavior, making them a potent new approach that has not yet been exploited. The TCS response regulator BfmR from Acinetobacter baumannii is the master controller of biofilm formation, making BfmR an attractive drug target. BfmR is targeted by 2‐aminoimidazoles (2AIs), compounds that inhibit and disperse biofilms in Gram‐negative and Gram‐positive bacteria. This work focuses on the interactions between 2AI based compounds and the response regulators BfmR and PmrA/QseB, from Fancisella tularensis, which also regulates biofilm development. Understanding the impact of 2AIs on response regulators and their mechanism of interaction will lead to the development of more potent inhibitors.
Kinetic evaluation of the reaction of glutamate racemase with computationally derived inhibitors provides validation of a successful pharmacophore model for a new line of antibiotic therapy
Authors: Young Michael , Wells Emily , Dinsmore Olivia , Marchiano Joe , Mahfouz Tarek , Stockert Amy
Published: November 2023

As increasing antibiotic resistance diminishes the effectiveness of once‐valuable drug therapies; research efforts must seek novel targets for new antibiotics compounds. Glutamate racemase, the enzyme responsible for the cofactor‐independent racemization of glutamic acid in bacteria, remains an untargeted step in the peptidoglycan synthesis pathway. Glutamate racemase has been shown to be essential for the viability of several pathogens including Helicobacter pylori, Escherichia coli, and Streptococcus pneumoniae, making it an ideal target for developing a new class of antibiotics. Crystal structure data for S. pneumoniae glutamate racemase is not available, thus comparative modeling was employed to develop a three‐dimensional structure for this enzyme using Prime® software. Several potential glutamate racemase inhibitors were docked into the model's active site to investigate binding modes and develop derivations to enhance for strong binding to the enzyme active site. Synthesis of the MurI gene, the gene encoding glutamate racemase, and placement into the pBluescript plasmid has allowed successful expression of functional glutamate racemase. However cellular components from the lysis of E.coli cells transformed with an inducible plasmid containing MurI have made reliable enzyme assays difficult. Addition of a His tag linker to the MurI containing plasmid allowed for purification prior to kinetic assays, thereby eliminating potential cellular contaminants that interfere with our coupled enzyme assay. Our results validated the development of a structure‐based pharmacophore model for glutamate racemase inhibitors. Previous kinetic assays have demonstrated successful inhibition with our potential drugs, but reliable determination of Ki was not possible due to interference with our coupled assay. Use of the purified enzyme for kinetic assays has removed this interference and provide more reliable and accurate Ki determination.

Support or Funding Information

Department of Pharmaceutical and Biomedical Sciences, Ohio Northern University
It takes two to tango: The interaction of male and female genital traits mediate sperm and ejaculate transfer during mating
Authors: Friesen Christopher R.
Published: November 2023

Genitalia are the most differentiated morphological traits in sexually reproducing organisms. Natural selection, sexual selection and sexually antagonistic coevolution contribute to genital diversification, but the relative importance of each of these evolutionary processes is unknown in most taxa, especially vertebrates. In addition, female genital morphology and behavior has been neglected in most taxa but are clearly crucial to our knowledge of genital coevolution. A critical first step toward understanding how these processes shape the evolution of specific genital traits is to determine how female and male genitalia interact during copulation. Female‐male interactions during copulation may affect sperm and ejaculate transfer in ways that influence male fitness via cryptic female choice (CFC: females biasing paternity toward or away from particular males when they mate with more than one male in a reproductive cycle). Here I present data from experimental manipulation of genitalia aimed to understand female x male interactions during copulation and how these interactions affect the fitness of both sexes. We use garter snakes as a model for genital evolution because males display great variation in form and accessory features (e.g., spines). I will also present some comparative work on garter snake genitalia, copulatory behavior, seminal fluids and future directions for that research.

Support or Funding Information

National Science Foundation Grants [IOS IOS‐0920344, IOS‐0620125, IOS‐1011727]
Resveratrol Shortens the Chronological Life Span of Saccharomyces cerevisiae under Dietary Glucose Restriction
Authors: Madrigal‐Perez Luis Alberto , Olivares‐Marin Ivanna Karina , Canizal‐Garcia Melina , Gonzalez‐Hernandez Juan Carlos , Nava Gerardo , Ramos‐Gomez Minerva
Published: November 2023

The resveratrol (3,4′,5‐trihydroxystilbene, RSV) supplementation in mammalian systems has been related with a decrease of symptoms of diseases related with aging and the increase of longevity. However, notwithstanding the growing number of studies performed with RSV in biomedical area, the molecular mechanism by which RSV acts still remains unknown. Lately, it has been proposed that inhibition of the oxidative phosphorylation activity is the molecular mechanism of RSV action. This mechanism suggests that RSV might induce mitochondrial dysfunction resulting in oxidative damage to cells with a concomitant decrease of cell viability and cellular life span. Additionally, RSV might also have a negative impact on the ATP production via mitochondrial respiration; consequently under energy restriction the RSV effect in cellular viability could be more severe. To prove this hypothesis we measured oxygen consumption, mitochondrial membrane potential and hydrogen peroxide (H2O2) release to determine the extent of mitochondrial dysfunction, as well as the chronological life span (CLS) of Saccharomyces cerevisiae, which has been accepted as an important model of oxidative damage and aging. All the experiments were performed in a dietary glucose restriction (0.5% glucose) and with five levels of RSV (0, 10, 30, 50 and 100 μM). As expected, we found that RSV supplementation inhibits the basal respiration in all the concentrations assayed. However, we did not found any change in the mitochondrial membrane potential, except in the high‐level of RSV (100 μM) that showed an increase in this parameter. In the H2O2 release experiment, we found an inverted U‐shape dose‐response behavior, with the highest value in the 10 μM RSV and the lowest in 100 μM RSV. Altogether this data indicate that 100 μM RSV might produce a mitochondrial dysfunction, since inhibits the oxygen consumption and increases the mitochondrial membrane potential. Finally, as anticipated, we found that supplementation of S. cerevisiae cultures with 100 μM RSV decreased CLS. Collectively, this data supports the hypothesis that RSV supplementation decreases CLS in a dietary glucose restriction condition as a result of mitochondrial dysfunction.

Support or Funding Information

Luis Alberto Madrigal‐Perez (PRODEP scholarship ITESCH‐002).
Teniposide reduces vascular calcification by inhibiting BMP2 expression
Authors: Liu Lipei , Zhang Xiaomeng , Chen Yuanli , Duan Yajun , Han Jihong
Published: November 2023

Vascular calcification, characterized by ectopic calcium deposition, is clinically associated with the development of chronic kidney disease, atherosclerosis and diabetes mellitus. Bone morphogenetic proteins 2 (BMP2) regulates orthotopic bone formation physiologically. However, high expression of BMP2 in atherosclerotic lesions suggests that BMP2 may play an important role in vascular calcification under pathological conditions. Teniposide, a well‐defined DNA topoisomerase II (Topo II) inhibitor, is widely prescribed to patients with various types of cancers. In addition, we previously reported that teniposide is able to activate macrophage ABCA1 expression and free cholesterol efflux, and hepatic CETP expression indicating that Top II inhibitors may demonstrate multiple anti‐atherogenic properties. Herein, we investigated if teniposide can influence calcification in aortic root, and BMP2 is involved in this process. In vitro, we employed the β‐glycerophosphoric acid method to induce calcification in human aortic smooth muscle cells (HASMCs). We determined that treatment of HASMCs with teniposide attenuated the phosphate‐induced calcium deposition. Mechanistically, we found that teniposide suppressed HASMC calcification by decreasing the BMP2 and Smad1/5/8 signaling cascade in a p53‐dependent manner. To determine the effect of teniposide on vascular calcification, apoE deficient (apoE−/−) mice were randomly divided into 3 groups and received the following treatment: Group 1 (control), mice were fed high‐fat diet (HFD) for 4 months; Group 2 (prevention group), mice were fed HFD containing teniposide [0.5 mg/kg bodyweight/day, (mpk)]; and Group 3 (therapy group) mice were pre‐fed with HFD alone for 3 months followed by one month of HFD containing teniposide (0.5 mpk). At the end of experiment, we determined the development of lesions and calcification. We observed that teniposide reduced atherosclerotic lesions and vascular calcification in lesion areas, in both prevention and therapy groups. The immunofluorescent staining demonstrates that BMP2 expression in the calcified vascular areas was also inhibited by teniposide. In contrast, the results of microCT analysis on the distal femur show no effect of teniposide on bone structure indicating the tissue‐type specific function of teniposide. Taken together, our study demonstrated that teniposide treatment ameliorates vascular calcification both in vitro and in vivo which was completed by regulating p53‐BMP2 pathway. Our study also demonstrates another biological function of TopII inhibitors.

Support or Funding Information

The National Science Foundation of China Grants 81473204 to J Han and 81573427 to Y Duan
Hepatic Metabolic Reprogramming by Caloric Restriction in Rhesus Monkeys
Authors: Rhoads Timothy W. , Burhans Maggie S. , McIlwain Sean J. , Hutchins Paul D. , Chen Vincent B. , Eghbalnia Hamid R. , Ong Irene M. , Markley John L. , Coon Joshua J. , Anderson Rozalyn M.
Published: November 2023

Caloric Restriction (CR) without malnutrition delays aging and the incidence of age‐related diseases. Adult‐onset CR enhances survival and reduces age‐related mortality in rhesus monkey (Macaca mulatta) compared to ad libitum fed controls, suggesting that the longevity benefits of CR can apply to primates, including humans; however, the mechanisms responsible for the longevity benefits of CR are to date unknown. We used five high‐throughput, high‐resolution techniques to generate hepatic molecular profiles for monkeys at baseline and 2 years after introduction of the CR diet. Approximately 20,000 biological molecules were detected and quantified across two time points, enabling us to determine the trajectory of response to CR. We find that CR upregulated many metabolic pathways, including fatty acid metabolism, the citric acid cycle, and branched chain amino acid degradation, and downregulated immune and inflammation pathways at the transcriptional and proteomic levels. Our data are suggestive of a novel role for the processing of mRNA in the mechanisms of CR. We report significant remodeling of the non‐histone acetylome, especially on proteins localized to the mitochondria. Hepatic lipid composition analysis showed increased levels of highly unsaturated trigylcerides and increased prevalence of odd‐chain phospholipids as a result of CR, while metabolomics analysis revealed increases in aspartate and 3‐hydroxybutyrate, the first ketone body to be synthesized under fasting conditions. These data reveal that CR activates a distinct hepatic metabolic program, suggesting that the metabolic adaptation to CR sets the stage for downstream longevity benefits.

Support or Funding Information

This work was supported by NIH grants R01AG040178, R56AG047358, and R01AG037000.
3D Vasculature Structure in Breast Cancer on a Chip Approaches through Micromilling
Authors: Wan Li , LeDuc Philip R , Neumann Carola
Published: November 2023

The 3D cell culture environment that is found including extracellular matrix (ECM) such as collagen is essential for the beautiful biophysically governed behavior and response of cells in tissues such as in breast cancer. This 3D ECM environment also has close interplay within the vasculature structures allowing for nutrient and oxygen supply. Building cancer on a chip systems with embedded vascular networks allow for more realistic 3D physiological responses and potential testbeds for drug screening. Our approach is to build a 3D vascularized breast cancer on a chip system. Conventional soft lithography methods unfortunately have been mostly limited to 2D and rectangular channel cross‐sectional morphology due constraints in fabrication technology. We introduce a novel approach to construct microfluidic channels with curved cross‐section in a collagen matrix. Mechanical micromilling was applied to fabricate channel patterns on a polymethylmethacrylate (PMMA) master mold, followed by two‐stage micromolding to create a sacrificial gelatin template. We successfully embedded these circular gelatin vascular templates within collagen to create perfusable microfluidic channels. We used thermo‐reversible polymers including gelatin to remove the sacrificial template simply by raising temperature slightly while limited effects on the collagen matrix. Breast cancer cells were then pre‐seeded into collagen matrix and we then investigated response of breast cancer cell response to chemical stimuli with respect to motility. The 3D vascular system constructed in our work provides a novel approach for further understanding breast cancer behavior.
Development and standardization of methodology for diagnosis and detection of response to chemotherapy in patients with colorectal cancer
Authors: Assis Jéssica Vieira , Coutinho Lucélia , Oliveira Matheus , Cruz Renata , Grenfell Rafaella
Published: November 2023

Colorectal cancer (CRC) is a heterogeneous disease. Subtyping in gene expression is recognized as a relevant criterion for classification of the disease, but its clinical usefulness is hampered by the lack of standard and methodology to be used between the clinical studies and analyze the responsiveness of each patient to the chemotherapeutic treatment. CXCR4 (CD184) when highly expressed generates proliferation and cell survival, angiogenesis and metastatic tumor migration. When the CD26 gene is less expressed, there is a decrease in local chemokine levels, thereby controlling the tumor in situ. Patients responsive to 5‐FU, FOLFOX and FOLFIRI present a significant reduction in CXCR4 expression and progressive increase of CD26, where the inverse ratio is seen in nonresponsive patients. Thus, in order to standardize high sensitivity methodology for diagnosis and prognosis, we developed the use of technology of detection of circulating antigens through the use of nanotechnology associated with antibodies. Thus, following in vitro methodology, anti‐CXCR4 (CD184) and anti‐CD26 pAbs were due to producing clones of IgM or IgG subtypes for each marker, since the difference between the expressions of such genes suggests an interesting imbalance, generating a possible response. After standardization of tumor induction in nude mice, HT‐29 cells (mutation at codon 273 of the p53 gene) were performed, four inoculations of these proteins were carried out and throughout the production process, immunoenzymatic assays were performed for identification and selection of clones Positive. The purified antibodies were tested to SDS‐PAGE and Western Blotting for confirmation of specificity. Thereafter, the HT‐29 cells removed from mice will be labeled to verify the expression of CXCR4 and CD26 on their surfaces from the inoculated pAb and thereafter, tissues from patients at different stages of the disease will also be labeled. We thus sought, in clinical cases, to assist the medical team in the decision of the treatment leading the patient to the surgical intervention and/or the conventional chemotherapies.

Support or Funding Information

Financial support: Fiocruz, PDTIS, FINEP (Brazil).
MED28 Mediates Glucose Metabolism in Human Colorectal Cancer Cells
Authors: Hsieh Nien‐Tsu , Huang Chun‐Yin , Weng Yu‐Ting , Lin Yi‐Chang , Lee Ming‐Fen
Published: November 2023

Most normal cells mainly rely on mitochondrial oxidative phosphorylation to generate energy, whereas cancer cells preferentially rely on aerobic glycolysis or so‐called “Warburg effect” even in the presence of sufficient oxygen. Albeit inefficient energy production, cancer cells compensate for this disadvantage by accelerating the uptake of glucose and increasing the rate of glycolysis. To increase the rate of glycolysis also provides the glycolytic intermediates for the synthesis of macromolecules, DNA, protein, and lipid, to keep up with the rapid proliferation mode. Recent studies have shown that Mediator Complex is involved in human diseases, including cancer, cardiovascular disease, metabolic disorder, and neurological disease. MED28, a Mediator subunit for transcriptional activation, is also an interactor with multiple proteins including Grb2, Src kinases, actin cytoskeleton, and merlin. MED28 was found highly expressed in colorectal cancer. In addition, our laboratory has previously reported that MED28 mediates Wnt/β‐catenin signaling in human colorectal cancer cells. This study is to investigate if MED28 plays a role in cell growth in the aspect of glucose metabolism in colorectal cancer. Expression profiling upon MED28 knockdown in SW480 colorectal cancer cells showed that MED28 suppression down‐regulated genes involved in glucose metabolism including glucose transporters and glycolytic enzymes, and the effect on several proteins and enzymes have been confirmed at both transcriptional and translational levels by RT‐PCR and Western blotting, respectively. The underlying mechanism is currently under investigation. In summary, MED28 regulates cell growth, at least in part, by mediating Warburg effect in colorectal cancer.

Support or Funding Information

This work was supported by the grants NSC102‐2320‐B‐309‐001‐MY3 and MOST105‐2320‐B‐309‐001 to M‐F Lee.
An Integrated Biology Approach to Determine Metabolic Dysfunction in Retinoblastoma
Authors: Guha Nilanjan , Winer Lisa , Babu Vishnu Suresh , Deepak SA , Lateef Syed , Gundimeda Seetaraman , Padmanabhan Arunkumar , Dranka Brian , Ghosh Arkasubhra
Published: November 2023

Cancer cells, unlike in normal cells exhibit altered metabolic activity especially with respect to oxidative phosphorylation (OXPHOS) and glucose metabolism. In this study, we looked into the transcriptomics and metabolomics profile of retinoblastoma, a pediatric eye cancer from different sample types viz. tissue, aqueous humor, vitreous humor and tear. The samples were collected from enucleated eyes of 9 patients and 2 deceased controls, whose cause of death is not due to any eye related disease. There are 5 males and 4 female patients and comprises of both unilateral and bilateral cases. The results were also corroborated with cell line models of the cancer. Combined pathway analysis of transcriptomics data from patient tissue samples and retinoblastoma cell line WERI revealed a significant overlap in the differentially regulated genes belonging to both glycolysis and OXPHOS pathways. Similarly, combined analysis of gene expression and LC‐MS data showed many glucose metabolism genes and its corresponding metabolites to be differentially regulated in patient samples. The results also revealed decreased TCA cycle activity. To better understand the implications of these two major pathways in the progression of retinoblastoma, metabolomics study using Seahorse analyzer is in progress, which can simultaneously measure the two major energy pathways of the cell ‐ mitochondrial respiration and glycolysis ‐ in live cells, in real time. Overall the study demonstrates the utility of applying an integrative biology approach to determine the severity of metabolic dysfunction in retinoblastoma and also to identify signatures that are unique to the disease.
Thimet Oligopeptidase (EP24.15), a Neuropeptide Processing Enzyme Regulating Xenin Signaling
Authors: Glucksman Marc , Philibert Keith , Mizuno Tooru , Lew Piesan
Published: November 2023

Xenin is a 25 amino acid neuropeptide with sequence homology and actions similar to hypothalamic and ileal neurotensin (a satiety factor) as well as binding to the neurotensin receptor. Xenin is produced by a subgroup of chromogranin A+ cells in the duodenum and this neurohormone is involved in glucose homeostasis and increases the response to glucose‐dependent insulinotropic peptide (GIP). Upon its release after ingestion Xenin inhibits secretion of pancreatic exocrine peptides in the gastrointestinal tract. The mode of regulating the peptide hormone Xenin remains elusive. The metalloendopeptidase EC 3.4.24.15 (thimet oligopeptidase, EP24.15) has been demonstrated to play a key role in the cleavage and subsequent regulation of several neuropeptides that also exist in the gut such as neurotensin and somatostatin. A systems biology approach to regulating signaling beginning from high throughput structural analyses, in silico molecular modeling and potential substrate screening implicated Xenin as a substrate of EP24.15 and thus, this enzyme as a possible regulator of Xenin signaling.

To substantiate a potential regulatory mechanism for the functioning of Xenin, we first identified potential active site binding and cleavage sites, measured enzymatic parameters, performed structural studies, and determined if EP24.15 and Xenin are co‐expressed in gut regions relevant to glucose homeostasis. After proprietary in silico modeling paradigms identifying a potential favorable interaction, EP24.15 and Xenin were co‐incubated and subjected to matrix‐assisted laser desorption ionization time‐of‐flight (MADLI‐TOF) mass spectrometry to confirm that EP24.15 can cleave Xenin in vitro. Enzyme kinetics were performed with standards via high performance liquid chromatography yielding results consistent with known substrates. Notably, Xenin is cleaved in a homologous manner as neurotensin. Double‐label immunohistochemistry demonstrated Xenin and EP24.15 immunoreactivity within the mouse small intestine and specifically co‐expression within the intestinal mucosa and submucosa. Furthermore, there is co‐expression of EP24.15 and Xenin in cells of both human and mouse stomach in the gastric mucosa. Taken together this data suggests that EP24.15 may act to cleave Xenin in vivo and represents an additional facet of the control mechanism of glucose homeostasis. As a potential pharmacological therapeutic target, understanding regulation of Xenin by the neuropeptide processing enzyme, EP24.15, may provide insight into an alternative strategy for glucose regulation and diseases such as diabetes and obesity.

Support or Funding Information

Supported by NIH OD010662 (MJG) and CIHR MOP123208 (TMM)
Effect of Surfactin Administration on Glucose and Lipid Metabolism in Mice Fed a High Fat Diet
Authors: Santos Maria Emilia Soares Martins , Porto Marcos Augusto Nascimento , Marinho Pablo Rayner Sousa , Oliveira Michael Eder , Granjeiro Paulo Afonso
Published: November 2023

The high intake of high fat diet has been related with several changes on glucose and lipid metabolism and also on gut microbiota composition. Surfactin is an active compound produced by a wide variety of bacteria, fungi, and yeast that have important biological activities as anti‐tumour, anti‐microbial and anti‐inflammatory. Oral probiotics or their secreted factors are becoming the novel strategy for treatment of metabolism alterations. The aim of this study was to determine the influence of surfactin produced by Bacillus subtilis (ATCC 19659) on glucose and lipid metabolism in mice fed a high fat diet. Male mice (n=6/group) were fed with a control (C) or high fat (HF) diets and water ad libitum for 10 weeks. One group (HFS) was orally administrated with 5 mg/kg body weight of Surfactin once a week for 4 doses. Surfactin was isolated and purified from Bacillus subtilis culture. It was lyophilized and later dissolved in phosphate buffered saline (PBS) before the administration. Food (g) and water intake (ml) were measured daily. Body weight (g) was evaluated weekly. After 10 weeks, mice were euthanized and blood was collected. Using commercial kits the levels (mg/dl) of glucose (GLU), triglycerides (TG), total cholesterol (CHO), HDL and VLDL cholesterol were measured. Epididymal (EAT) and retroperitoneal (RAT) adipose tissue depots were collected and weighted (g). Results were expressed as mean ± SEM. Data were analyzed using T‐test. P<0.05 was considered significant for all data.

No difference was observed in body weight between the groups however HF and HFS groups eat and drink less than C. An increase of ~47% at EAT was observed in HL (2.69±0.31) vs C (1.83±0.18). There was no difference at RAT in any group. There was an increase of ~27% in HF (362.7±11) vs C (284.9±30) and a decrease of ~20% in HFS (289.3±34) vs HF (362.9±11) at GLU. An increase of ~47% at CHO in HF (155.5±10.9) vs C (105.6±3.4) and also in HDL (~59%) HF (112.4±6.5) vs C (70.4±3.9) was observed. TAG was decreased ~32% in HF (109.8±2.9) vs C (162.9±19.1) and also in HFS (~30%) (76.4±11) vs HL (109.8±2.9). Same pattern of results were observed in VLDL.

In conclusion, we observed that HF diet intake was able to disrupt glucose and lipid metabolism and surfactin seems to be efficient in the control of glycemia and tryglyceredemia. We hipothesize that these effects achieved are due surfactin action on anti‐inflammatory cytokines production and/or changes in gut microbiota composition.

Support or Funding Information

CNPq
Relation of Hypoglycemic Activity and the Antioxidant Capacity of Justicia Spicigera Leaf Extracts in Diabetic Rats
Authors: Martínez‐Mora José A. , Murillo‐Villicaña Marina , Salgado‐Garciglia Rafael , Manzo‐Avalos Salvador , Cortés‐Rojo Christian , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo
Published: November 2023

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia with disturbances in the metabolism of carbohydrates, lipids, and proteins, where there is an absolute or relative deficiency in the secretion and/or insulin action, or both, reducing the quality and life expectancy of those who suffer, as it promotes the generation of various diseases such as renal disease and neuropathies, among others; hyperglycemia being the cause of complications of diabetes by a mechanism involving oxidative stress. Since ancient times, diabetics have used medicinal plants to maintain normal blood sugar levels. Justicia spicigera (J. spicigera) is a branched and medicinal erect shrub that is used to treat diabetes. The objetive of the project is: To analyze the effect of extracts from the leaves of J. spicigera on levels of glucose, urea, creatinine, cholesterol, triglycerides and activity of mitochondrial antioxidant enzymes in diabetic rats. The plant J. spicigera was used to prepare 3 extracts with solvents of different polarity (hexane, ethyl acetate and ethanol) at 100 mg/kg, and administered for 30 days on a daily basis (1ml) orally to diabetic rats streptozotocin induced. Therefore eight groups of rats were assigned: 1) Normoglycemic control, 2) Diabetic control, 3) Normoglycemic + hexane extract, 4) Diabetic + hexane extract, 5) Normoglycemic + extract ethyl acetate, 6) Diabetic + acetate ethyl, 7) Normoglycemic + ethanol extract, 8) Diabetic + ethanol extract. Our study revealed that the administration of ethyl acetate and ethanol extracts of leaves of J. spicigera have hypoglycemic activity in diabetic rats. The intake of ethanol extract increases levels of urea in normoglycemic rats and increases triglyceride levels in diabetic rats. Meanwhile, administration of the hexane extracts, ethyl acetate and ethanolic did not modify urea levels in diabetic rats, however, they did increase creatinine levels of normoglycemic rats. Hexane and ethyl acetate extracts decrease levels of cholesterol and triglycerides in diabetic rats. It can be concluded that, because each solvent drag different types of compounds expected the different effects obtained in normoglycemic and diabetic rats.

Support or Funding Information

Acknowledgements: The authors appreciate the partial economic support by CIC‐UMSNH (2.10 to RSG; 2.16 to ASM; 2.37 to SMA).
Anti‐hyperglycemic Effect of Arginyl‐fructosyl‐galactose (AFL) in SD Rat Model Induced Lactase Expression Using Milk Administration
Authors: LEE Jungyun , Choi Hwang‐Yong , Kim Young‐Cheul , Apostolidis Emmanouil , Kim Young‐Ho , Kwon Young‐In
Published: November 2023

Inhibition of small intestinal lactase involved in the digestion and absorption of lactose can significantly decrease the postprandial increase of blood glucose level after a mixed lactose diet. In the course of screening useful these enzyme inhibitors, we purified and identified arginyl‐fructosyl‐galactose (AFL), a novel Amadori rearrangement compound from milk‐related foods using in‐vitro enzyme inhibition assay method. To evaluate the effect and mode of action of AFL on the postprandial hyperglycemia we investigated the inhibitory activities of AFL against rat intestinal lactase relevant to postprandial hyperglycemia. We also investigated the changes in blood glucose and lactase expression levels in SD rat both in lactase‐induced and non‐induced models after 4 weeks induction of lactase using milk administration. Most lactase‐induced rat showed significantly higher inhibition‐sensitivity on AFL than non‐induced rats. Furthermore lactase‐induced rat showed high lactase expression in middle part of intestine, Jejunum while non‐induced rat showed high in upper intestine, Duodenum. Results suggested that AFL inhibits small intestinal lactase effectively, as a target site of pharmacophore and can improve hyperglycemia followed by high consumption of milk‐related foods.
Development of Novel Allele Specific PCR Based Assays to Investigate the Contribution of Cortisol to Metabolic Syndrome
Authors: Godlewski Brianna R. , Salm Justin R. , Cohen Brian D
Published: November 2023

A diagnosis of Metabolic Syndrome (MSX) requires patients to present with three or more of the following symptoms: elevated fasting blood glucose levels, elevated serum triglyceride levels, low serum HDL levels, elevated blood pressure, and truncal obesity. This pathology shares many similarities with Cushing's Syndrome (CS) but a diagnosis of CS requires hypercortisolemia. This similarity has led our lab and others to hypothesize that MSX may be a Cushingoid‐like state caused by hypersensitivity of the glucocorticoid receptor (GR), a nuclear hormone receptor that is activated when cortisol binds. Using an allele specific polymerase chain reaction (PCR) protocol, previous research in our lab has found significant correlations between the BclI and N363S polymorphisms of the glucocorticoid receptor and altered patient metabolic profiles in a population of patients seeking bariatric surgery, suggesting their possible contribution to diseases such as obesity and Metabolic Syndrome. Recent work in our lab has expanded our study to include the cortisol resistance polymorphism TthIIIi (rs10052957) to test the hypothesis that there would be a decreased prevalence of this polymorphism in our subject population. The TthIIIi allele is present at a lower frequency in our research population compared to reported frequencies, consistent with our hypothesis.

In addition to investigating the glucocorticoid receptor, we have also developed an allele specific PCR assay for previously identified single nucleotide polymorphisms (rs12086634 and rs846910) in the cortisol reductase enzyme, also known as 11beta‐hydroxysteroid dehydrogenase (11β‐HSD) type 1. The 11β‐HSD1 enzyme is responsible for the conversion of inactive cortisone to cortisol, and works primarily in the adipose tissue. The two polymorphisms listed above are both associated with overactivity of the enzyme and are another possible cause for the Cushingoid‐state observed in MSX patients. In our patient population, the frequency of heterozygosity for the rs12086634 SNP was nearly 50% higher than the reported frequency in the general population, suggesting that this polymorphism may contribute to increased obesity and the MSX metabolic profile by increasing the localized cortisol in adipose tissue. Greater understanding of the interplay between these single nucleotide polymorphisms can help physicians and patients make more informed decisions about treatment options for obesity and metabolic syndrome.
FoxO1 localization changes in Cinnamonium cassia treated 3T3‐L1 pre‐adipocytes
Authors: Bova Kaitlin , Aulthouse Amy , Stockert Amy
Published: November 2023

With increased focus on diabetes treatment and prevention, characterizing the mechanisms of action of various alternative treatments becomes increasingly important. Numerous studies have shown that cinnamon from a variety of species lowers both post‐prandial and fasting blood glucose levels. Our research is focused on examining the effects of aqueous extract of Cinnamonium cassia on a variety of signaling mechanisms normally affected by insulin. One such mechanism utilized by insulin is the Akt pathway which leads ultimately to the phosphorylation of FoxO1, a transcription factor involved heavily in metabolism. Nucleation of the FoxO1 factor is dependent on its phosphorylation state. This study is aimed at determining the effects of cinnamon treatment on the FoxO1 localization. Preliminary studies indicate minimal effects on expression level of FoxO1, indicating that if involved, FoxO1 localization is likely the key to lowering blood glucose levels. We completed a multi‐time point study looking at early and later differentiation stages if the 3T3‐L1 cells both in the presence and absence of cinnamon treatment. Cells were divided into two groups, those which were chemically induced for differentiation and those which received no chemical induction. RNA samples were collected from each time point and treatment groups for synthesis of cDNA followed by quantitative PCR. Although changes in expression level were observed, large changes in protein level were not observed and it is unlikely that expression level of FoxO1 plays a role in the observed clinical outcomes. In order to explore the effects of treatment on localization of FoxO1, protein extractions were conducted isolating both the nuclear and the cytosolic proteins. These samples were analyzed by Western blot to identify what effect cinnamon treatment had on nucleation of the FoxO1. Preliminary analysis of these data suggest that cinnamon facilitates nuclear exclusion.

Support or Funding Information

Ohio Northern University Academic Affairs Summer Research Support Program
Analysis of Gene Expression in a Female Rat Model of Diet‐induced Non‐alcoholic Fatty Liver Disease Using RNA‐sequencing
Authors: Bowman John James , Lee Jennifer Karin , Perets Ron A , Knabe Melina L , Guider Jamie W , Phillips Scott , Sarwadnya Uma D , Blythe Sarah N , Toporikova Natalia , Whitworth Gregg B
Published: November 2023

We have established a diet‐induced model of non‐alcoholic fatty liver disease (NAFLD) in the adult female rat, which we have characterized through RNA‐seq, histology, and physiological measurements. Furthermore, we investigated whether peripheral estrogen signaling has affected this disease. All rats were ovariectomized, with basal levels of estrogen restored in half the animals by implantation of a slow release estradiol pellet. We observed a significant effect of both diet and estrogen on animal weight gain, with animals fed a high‐fat, high‐sugar diet without supplemental estrogen demonstrating a >2‐fold increase in weight gain compared to control diet animals that received estradiol pellets. Our high‐fat, high‐sugar animals demonstrate physiological and histological markers of NAFLD, including steatosis, inflammation, and an increase in connective tissue in the liver. Additionally, we measured percent body fat, liver weight, percent liver fat, and insulin, glucose, triglycerides, and cholesterol levels. Moreover, we used RNA purified from liver tissues to quantify global changes in gene expression between our experimental groups through RNA‐seq, with the goal of identifying genes that are differentially expressed in the NAFLD induced animals. In addition, the quantification of certain promising gene targets using RT‐qPCR serves as validation for RNA‐seq. Interestingly we see the greatest effect on gene expression through the interaction between diet and estrogen. We will be discussing the in‐depth, high resolution gene analysis done on the liver tissues as it relates to the induction of this disease. A few initial pathways and processes that are upregulated in our high‐fat animals include the inflammatory cytokines and genes involved in lipid metabolism.

Support or Funding Information

Howard Hughes Medical Institute

Jeffress Trust Awards Program Interdisciplinary Research

R.E. Lee Summer Research Scholars
Micro RNAs Mediating Effects of Adipose Angiotensinogen in Adipocyte Inflammation and ER Stress
Authors: Menikdiwela Kalhara Rashmikumara , Moustaid‐Moussa Naima , Ramalingam Latha , Scoggin Shane , Kalupahana Nishan Sudheera
Published: November 2023

Obesity is a complex disease and a global epidemic associated with chronic low grade inflammation and metabolic dysregulation. The renin‐angiotensin system (RAS) plays a vital role in controlling blood pressure and fluid balance. Although RAS components are generally overexpressed in adipose tissue in obesity, mechanisms by which RAS induces adipose endoplasmic reticulum (ER) stress and inflammation, both hallmarks of obesity, are not well understood. Here, we hypothesized that overproduction of angiotensinogen (Agt, precursor protein in RAS), induces ER stress and inflammation in adipocytes, thereby, contributing to obesity‐associated metabolic alterations. To test this hypothesis, we used the aP2 promoter‐driven Agt overexpression in adipose tissue of transgenic (Agt‐Tg) mice, compared to wild type mice (Wt), with or without a RAS inhibition (captopril, ACE inhibitor). We also used 3T3‐L1 adipocytes to test direct effects of angiotensin II and RAS inhibitors in obesity‐associated inflammation and ER stress.

The serum levels of pro‐inflammatory markers such as MCP‐1 (Monocyte Chemoattractant Protein‐1) were significantly higher in Agt‐Tg compared to Wt mice. By contrast, anti‐inflammatory cytokines such as IL (interleukin)‐10 and IL‐2 were significantly lower in Agt‐Tg mice compared to Wt mice. Further, mRNA levels for ER stress markers, including BiP, ATF4 and XBP1 and cell apoptosis marker, CHOP, were significantly higher in Agt‐Tg mice compared to Wt mice. To gain further insight into mechanisms and pathways mediating Agt effects on ER stress and RAS‐associated obesity and inflammation, we conducted miRNA profiling by RNA Seq in adipose tissue from Agt‐Wt and Wt mice. We identified 3 miRNAs that were upregulated and 14 miRNAs that were downregulated in adipose tissue from Agt‐Tg vs. Wt mice. These were further validated by real time quantitative PCR, and identified miRNA 708 and 143 to be upregulated, while miRNA 30 family was down regulated in Agt‐Tg mice compared to Wt mice. These results are consistent with previously reported roles for these miRNAs in ER stress. Moreover, Agt‐Tg mice treated with Captopril had a significantly lower expression of CHOP and ATF4 mRNAs compared to untreated controls. In agreement with these findings, captopril prevented angiotensin‐II (Ang II) induced expression of ER stress genes in 3T3‐L1 adipocytes. Further studies are ongoing in cultured adipocytes to dissect molecular mechanisms linking our candidate miRNAs to RAS, ER stress and inflammation.

In conclusion, overexpression of Agt in adipose tissue increased inflammation and ER stress. These effects are possibly mediated in part by miRNAs 708, 143 and 30 and by direct Ang II actions in adipocytes. These miRNA‐regulated pathways could be used as potential therapeutic targets to reduce obesity‐linked inflammation and ER stress.