bluuebunny/crossref_metadata_2025_split huggingface dataset:
Time Course Metabolic Profiling of Senescing Flight Muscles in The Hawk Moth, Manduca sexta
Authors: Wone Beate , Kinchen Jason M. , Kaup Elana R. , Wone Bernard W. M. 
Published: November 2023

Biological aging profoundly impairs skeletal muscle function, performance, and metabolism. Because aging muscle in vertebrates has parallels with aging muscle in insects, we conducted a high‐resolution time course metabolic profiling of middle‐age to advanced age dorsolateral flight muscle in the hawk moth, Manduca sexta, to characterize the metabolic changes associated with aging muscles. These muscle samples were analyzed using non‐biased, global metabolomics profiling technology based on UHPLC/MS/MS2 and GC/MS platforms. A total of 535 metabolites were characterized across diel time (daytime/nighttime) and from middle‐age to advanced age in senescing muscle. We identified a number of differences, including changes in biochemicals related to energetics, extracellular matrix turnover, and polyamine metabolism. Metabolite over representation analyses indicated a greater than 2‐fold enrichment in biotin, fatty acid, purine, polyamine, branched chain amino acid, TCA cycle, glycolipid, and monoacylglycerol synthesis or function. Increased abundances of glycolytic metabolites suggest shifts toward activated glycolysis with age, whereas decreased abundances in acylcarnitines potentially reflect declining beta‐oxidative use. We have also seen a shift towards decreased polyamine metabolism with age, which might have downstream effects such as an age‐related decline in lipid metabolism because polyamines appear to regulate lipid metabolism through autophagy. Significant metabolite changes were detected between the metabolic profiles of males and females, revealing both similarities and differences across diel time and ages. Notably, 43 metabolites showed diel oscillations in their abundance levels in females and this pattern attenuated in advanced age. Males showed much dampened diel oscillations regardless of age, but overall abundances of these metabolites decreased with age for both sexes. Interestingly, biotin levels were elevated in senescing muscle of males. Collectively, our results obtained uncovered a cascade of biochemical regulation events coordinating senescence in muscle.Support or Funding InformationThis material is based upon work supported by the University of South Dakota start up funds.
Inhibition of Glycogen Metabolism as a Potential Strategy for Anticancer Therapy
Authors: Dukhande Vikas V , Barot Shrikant , Husein Suemaya , Palaguachi Christian 
Published: November 2023

Cancer cells reprogram their energy metabolism to suit their growth and proliferation needs. Cancer cells upregulate glycolysis even in the presence of oxygen, a phenomenon termed Warburg effect. Glycolysis thus emerged as a promising target to kill cancer cells. Glycogen, a storage cache of glucose in the cell, could fuel the metabolic reprogramming in cancer cells. However, little is known about glycogen metabolism and its contributions to carcinogenesis, cancer growth, and metastasis. We aim to inhibit glycogen metabolism to arrest the growth of cancer cells and want to study molecular mechanisms of glycogen inhibition‐induced cancer cell death. We studied cell viability, apoptosis markers, autophagy markers, and cellular metabolism after glycogen metabolic inhibition using pharmacological agents in hepatoma and glioblastoma cells. Our results indicate that inhibition of glycogen metabolism induces cancer cell death and enhances the effects of anticancer drugs. Our ongoing studies will investigate the roles of apoptotic and autophagic processes in cancer cell death by glycogen metabolic inhibition. Our study is highly relevant for the understanding of the metabolic reprogramming in cancer cells and can elucidate potential targets for anticancer drugs.Support or Funding InformationSupported by startup funds from the Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University to Vikas Dukhande.
Targeting Mitochondrial Molecular Chaperones as a Potential Therapeutic Target in Leukemia Kelly G. Bryant and Dario C. Altieri Prostate Cancer Discovery and Development Program and Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia PA 19104
Authors: Bryant Kelly G. , Altieri Dario C. 
Published: November 2023

Molecular chaperones of the Heat Shock Protein‐90 (HSP90) family play an important role in proper protein folding, protein complex formation, as well as minimizing aggregate formation. The discovery of HSP90 and its ortholog, tumor necrosis factor receptor‐associated protein‐1 (TRAP‐1) abundantly localized to the mitochondria of tumor, but not the majority of normal cells has broadened the field to include a role in cancer progression, and importantly, a therapeutic candidate in many solid tumors. We sought to determine whether targeting the mitochondrial HSP90s with small molecule inhibitors would be effective in leukemia. Fractionation studies of several different types of leukemia cell lines show both TRAP‐1 and HSP90 proteins in the mitochondria, thus providing a target for the mitochondrial targeted HSP90 inhibitor Gamitrinib (GA‐mitochondrial matrix inhibitors). Accordingly, a variety of different leukemia cell lines were treated with Gamitrinib and for each line tested, we found growth inhibition in a dose and cell‐dependent manner. Treatment also induced cell death, PARP cleavage, and caspase activation at lower doses than is required for solid tumors. Treatment with low doses of Gamitrinib did not affect protein levels of the mitochondrial electron transport chains I through V. Additionally there were not significant differences in the levels of lactate produced by these cells, and only a slight decrease in glucose consumption. Metabolic studies also show that treatment leads to a decrease in ATP levels in the leukemia cell lines, as well as a decrease in the oxygen consumption rates of these cells. Utilizing several primary Acute Myeloid Leukemia Patient derived samples, Gamitrinib treatment resulted in a dose‐dependent decrease in viability and increase in apoptosis as determined by FACS analysis for Annexin V/Propidium Iodide, as well as protein markers of cell death. Current ongoing experiments are focused on determining the efficacy of this inhibitor in vivo by utilizing whole body luciferase imaging in MV‐411 GFP/Luciferase xenografts. Taken together, Gamitrinib results in robust cell death in all of the leukemia cells tested, and these studies provide ample evidence that Gamitrinib could be a strong clinical candidate as an alternative HSP90 inhibitor for the treatment of different types of leukemia.Support or Funding InformationWistar Institute T32 Training Grant
Human Hepatocellular Carcinoma Cells Adapt to ASCT2 and LAT1 Amino Acid Transporter Silencing by shRNA and CRISPR‐Cas9
Authors: Bothwell Paige , Bode Barrie 
Published: November 2023

Amino acid transporters ASCT2 and LAT1 are coordinately enhanced in human cancers where, among other roles, they are thought to drive mammalian target‐of‐rapamycin (mTOR) growth signaling. To assess the value of ASCT2 and LAT1 as therapeutic targets in hepatocellular carcinoma (HCC), nine unique shRNA sequences in lentiviral vectors were used to produce stable transporter suppression in mesenchymal and epithelial HCC cell lines (SK Hep and Hep 3B, respectively). In addition, direct editing of the ASCT2 and LAT1 genes was performed using six CRISPR‐Cas9 vectors in SK Hep and HUH7 HCC cell lines. Glutamine and leucine transport rates were reduced proportionally to diminished transporter protein expression in each cell line, and a greater degree of knockout was achieved by the CRISPR‐Cas9 gene editing system versus the stable shRNA expression system (75–85% vs. 20–80% respectively). Contrary to expectations, diminished glutamine and leucine influx failed to affect cellular proliferation. Analysis of mTOR complex 1 (mTORC1) signaling output assessed by 4E‐BP1 showed no compromise to basal or amino acid‐stimulated mTORC1 signaling. Quantitative RT‐PCR indicated no significant upregulation of other transporters or glutamine synthetase (GLUL) to supplement intracellular availability of glutamine. Prolonged uptake of radiolabeled glutamine and leucine revealed that diminished transport rates did not affect the total intracellular accumulation of either glutamine or leucine. Further in vivo analysis will be essential to understanding upregulation of ASCT2 and LAT1 across cancers, and these findings suggest that, in an in vitro context, HCC cell lines are able to adapt to decreased rates of amino acid transport.Support or Funding InformationNIH Grant# R15CA108519 (BPB)
Role of iNKT cells in obese adipose tissue
Authors: Park Jeu , Huh Jin Young , Ji Yul , Kim Jae Bum 
Published: November 2023

Immune cells actively regulate inflammatory responses and affect whole‐body energy metabolism. Various immune cells are present in adipose tissue and they affect adipose tissue homeostasis. Obesity‐induced inflammation is considered as one of the key factors to promote insulin resistance. Immune cells also contribute to control adipose tissue remodeling, adipocyte differentiation, and lipolysis. In particular, adipose iNKT cells have various roles in protecting against obesity‐induced inflammation, weight gain, and hypertrophic adipocyte. Upon high fat diet iNKT cell deficient mice such as Ja18−/− and Cd1d1−/− show increased pro‐inflammatory responses, more body weight gain with larger adipocytes. To date, most studies in adipose iNKT cells have focused on increased pro‐inflammatory responses. In this study, we have investigated the direct crosstalk between iNKT cells and adipocytes to maintain adipose tissue homeostasis.Support or Funding InformationThis work was supported by grants from the National Creative Research Initiative Program (2011‐0018312) funded by the Ministry of Education, Science and Technology. J.P. and Y.J. were supported by BK21 program.
Improvement of diabetic cardiomyopathy by Eryngium carlinae
Authors: García‐Cerrillo Diana , Noriega‐Cisneros Ruth , Salgado‐Garciglia Rafael , Manzo‐Avalos Salvador , Clemente‐Guerrero Mónica , Montoya‐Pérez Rocío , Saavedra‐Molina Alfredo 
Published: November 2023

Diabetes mellitus (DM) is a metabolic disease that has increased significantly in recent decades. DM and its complications are powerful risk factors for cardiovascular diseases. Diabetic heart damage is closely related to mitochondrial dysfunction and an increase in the generation of reactive oxygen species. The external supply of antioxidants can counteract the effect of such species on the body, and prevent the onset of many underlying diseases.Natural sources of antioxidants may be safer to use with regard to synthetic antioxidants, due to lower toxicity and side effects. Eryngium carlinae (EC) is a plant used in traditional Mexican medicine, whose hexanic extract has been shown to possess in vitro antioxidant capacity. The serum and heart mitochondria of male Wistar rats were analyzed. Previously experimental diabetes was induced with streptozotocin (STZ) and they were treated with hexanic extract of EC for seven weeks (30 mg/kg body weight). Cholesterol, triacylglycerides and glucose and lipoperoxidation levels were determined in serum. In addition, in the left ventricle heart mitochondria, oxidative and nitrosative stress levels were determined through the quantification of parameters such as thiobarbituric acid reactive substances (TBARS) and nitric oxide (NO) levels, and tissue histological analysis was performed. The results of the biochemical parameters of glucose and triacylglycerides show a significant decrease of the diabetic group treated with extract with respect to the diabetic group without treatment. Similarly, there was a significant decrease in lipoperoxidation levels in the diabetic group treated with extract compared to the untreated diabetic group.Histological observation shows signs of hypertrophy of the left ventricle of diabetic rats which appears to be diminished by treatment with EC extract. In conclusion, EC hexanic extract possesses antioxidant activity in vivo in the groups in addition to possessing the ability to lower glucose and triglyceride levels in diabetic rats, which may prevent or delay the development of diabetic cardiomyopathy.Support or Funding InformationAcknowledgments: Special thank for the grants of CIC‐UMSNH: 2.10 to RSG; 2.16 to ASM, and 2.37 to SMA.
Generating a Transgenic Mouse Line Containing the Plasma Membrane Phosphatidylinositol 4,5‐Bisphosphate Depletion System
Authors: Gulyás Gergő , Szalai Bence , Geiszt Miklós , Balla Tamás , Hunyady László , Várnai Péter 
Published: November 2023

Phosphoinositides are key regulators in many signaling cascades and several cellular processes. These lipids are located in cellular membranes containing different forms of these lipids. The plasma membrane (PM) is enriched in two phosphorylated variants, phosphatidylinositol 4‐phosphate and phosphatidylinositol 4,5‐bisphosphate (PIP2).To discover the role of these lipids, several applications have been developed. One of them is the rapamycin inducible heterodimerization system, based on the rapid and irreversible interaction of the FRB and FKBP12 proteins. In our system FRB was targeted to the PM using the localization sequence of the Fyn tyrosine kinase, whereas the FKBP was fused to an enzymatically active cytoplasmic 5‐phosphatase. After rapamycin treatment, the enzyme recruited to the PM, where rapid depletion of PIP2 could be achieved. Our goal was to generate a genetically modified mouse line containing this lipid depletion system, which can be highly capable to investigate the physiological role of PIP2 in primary prepared cells or in intact tissues and organs.Our plasmid used for the transgenic application contained both protein coding sequences of the depletion system in the same vector. To reach the stochiometric expression of the two distinct fragments, a viral T2A sequence was inserted between the protein coding sequences. For further detection of the transgene the PM targeted protein was fluorescently tagged. The transgenic construct was driven by a CMV promoter to reach a general expression in various animal tissues. The transgenic modification was carried out by applying the Sleeping Beauty transposase in FVB/Ant mouse strain. Based on the genotyping data of the founder generation, two distinct lines with low and high copy number were selected for the further investigations. Interestingly, transgene showed detectable expressions only in pancreatic acinar cells and in keratinocytes.To investigate the functional effect of the PIP2 depletion we carried out cytoplasmic calcium ion measurements. Using freshly prepared pancreatic acinar cells of the F2 generation rapamycin treatment did not resulted a reduced cytoplasmic calcium signal upon stimulation the cells with carbachol, indicating the low expression level of the PIP2 depletion system. A mathematical model was created, which predicts of the increase of protein expression, so we decided to select the animals with the highest copy numbers in each generation and use them as parents of the further generations. The efficiency of lipid depletion is monitored by measuring the translocation of PLCdelta1‐PH‐domain based PIP2 sensors, as well as by measuring other PIP2‐dependent cellular responses.Support or Funding InformationNational Research, Development and Innovation Fund (NKFI K105006)
Glucose Transporter (HXT) Homeostasis in Saccharomyces cerevisiae
Authors: Swain Evelyn 
Published: November 2023

The proposed research explores the regulation of glucose metabolism in Saccharomyces cerevisiae. Specifically, the focus is on how the loss of endocytic function results in aberrant glucose homeostasis through the deregulation of glucose transporter localization/function. By combining methods of yeast genetics, molecular biology, and cell biology, novel features regulating transporter function and localization will be studied to uncover mechanisms for maintaining glucose homeostasis. Data show that the BAR domain protein Rvs161 is required for proper transporter localization and function.1 Recent reviews describes BAR domain containing proteins as curvature inducing/stabilizing proteins essential for affecting a wide variety of membrane remodeling processes, including endocytosis.2 Endocytosis and degradation of transporters is critically important in maintaining glucose homeostasis in yeast as well as mammals. Saccharomyces cerevisiae is a well‐established model organism for the study of endocytosis.3 Additionally, yeast share similar components of glucose signaling and glucose transporter pathways with mammalian counterparts making yeast a useful model system for studies of glucose transporter biology.4, 5 Yeast strains lacking the RVS161 gene fail to grow under conditions of glucose limitation and harbor endocytosis defects. Our data indicate that overexpression of specific glucose transporters can suppress this defect. Additionally, our data suggests that glucose transporter interactions are required for proper transporter function and that Rvs161p is required for proper transporter localization and function.Support or Funding InformationNA
Regiospecificity of Galactan Polymerization by Divergent GlfT2 Orthologs
Authors: Sadecki Patric W. , Justen Alexander M. , Ho Jordan S. , Kiessling Laura L. 
Published: November 2023

Despite their ubiquity of polysaccharides, we lack detailed mechanistic understanding toward how carbohydrate polymers are assembled. Unlike the predictable linkage patterns used in other major molecules, polysaccharides can draw upon a large bevy of diverse glycosidic linkages to form complex polymers. To better understand polysaccharide assembly, our research group has turned toward using the mycobacterial galactan as a model. Composed of galactofuranose (Galf) residues, this essential polymer serves as a covalent connector between the mycolic acids and peptidoglycan of mycolated bacteria. The processive glycosyltransferase GlfT2 uses the activated form of galactofuranose, UDP‐Galf, to continuously add residues onto the galactan until reaching a mature length.Due to its virulence, one of the most extensively studied mycobacteria is Mycobacterium tuberculosis (M. tb). M. tb GlfT2 makes alternating β‐(1→5) and β‐(1→6) linkages in the galactan biosynthesis. Previous work from our research group sought to evaluate whether M. tb GlfT2 strictly adheres to this linkage pattern. When given UDP‐Galf with a floro‐ substituted 5 or 6 position, the enzyme is unable to elongate the polymer past two units.1 This suggests that the alternating β‐(1→5), β‐(1→6) linkage pattern is maintained with high fidelity. Curiously, linkage patterns within galactan isolated from other mycolated bacterium, such as Nocardia and Rhodococcus species differ from those in Mycobacterium. Data suggests that Nocardia brasiliensis (N. bras) only synthesizes β‐(1→5) linkages and Rhodococcus equi (R. equi) synthesizes β‐(1→3), β‐(1→5), and β‐(1→6) linkages.2 Since GlfT2 is responsible for much of galactan biosynthesis, we propose that GlfT2 from these organisms contain differences from M. tb GlfT2, allowing them to synthesize galactan with different glycosidic linkages.Our research group is keenly interested in gaining mechanistic insight toward what variation in the active site of GlfT2 leads to control over glycosidic linkage formation. We are specifically interested in understanding differences and similarities to the mechanism used by M. tb GlfT2. Since our research group has demonstrated two ‘DXD’ motifs are critical to GlfT2 catalysis, we seek to examine the active site of Nocardia brasiliensis and Rhodococcus equi GlfT2 by mutating these motifs. We hope that these studies will better illuminate which features of glycosyltransferases guide glycosidic linkage formation.
Three‐dimensional structure of Xyloglucan Xylosyltransferase I and proposed mechanism of catalysis
Authors: Culbertson Alan Thomas , Tietze Alesia , Smith Adrienne , Zabotina Olga 
Published: November 2023

Xyloglucan is the most abundant hemicellulose component of the primary cell wall of dicotyledonous plants. It is synthesized by multiprotein complex localized in Golgi. Three xyloglucan xylosyltansferases (XXT1, XXT2, XXT5) are essential for xylosylation of the xyloglucan backbone. Experiments on these enzymes have mainly focused on characterization of their function in vivo using reverse genetics and characterization of their products in vitro. Currently, there is no information about their atomic structure, the modes of substrate binding, catalytic mechanisms, or interacting surfaces. Here, we report a 3‐D structure of XXT1 using high‐resolution X‐ray crystallography and a homology model of XXT2. Site‐directed mutagenesis was used to investigate the catalytic mechanism and critical residues involved in substrate binding. We confirmed that the DxD motif is critical for the coordination of the manganese ion that coordinates UDP‐Xylose. Additional residues were identified as being important for UDP‐Xylose binding. We have also predicted possible points of protein‐protein interactions based on the surface characteristics of these proteins and their assessment using Isothermal Titration Calorimetry is in progress.Support or Funding InformationNSF‐MCB grant #1121163