bluuebunny/crossref_metadata_2025_split huggingface dataset:
Histamine Mediates the Response to Light in the Sensory Motor Integration of Gill Lateral Cell Cilia in the Bivalve Mollusc, Crassostrea virginica
Authors: Semple Danellie , Marrett Kimone , Carroll Margaret A , Catapane Edward J 
Published: November 2023

Gill lateral cells of Crassostrea virginica are innervated by serotonin and dopamine nerves from their ganglia. Most bivalves have lateral cell cilia that respond to serotonin and dopamine, with serotonin being the neurotransmitter that increases beating rates and dopamine being the neurotransmitter that decreases beating rates. The motor aspects of gill lateral cell innervation have been well studied, but not the sensory side. There is limited information about sensory inputs. Our previous work found that C. virginica can sense and adjust gill lateral cell cilia beating to the presence of food, crab extract and light, as well as a variety of chemical cues including histamine when applied to the mantle rim. Shining light on the mantle rim or applying histamine decreased gill lateral cell cilia beating rates, but did not alter cilia beating rates when applied directly to gill. In many invertebrates histamine is a neurotransmitter involved in photoreception. We hypothesize that in C. virginica, histamine is the neurotransmitter of mantle rim photoreceptors. To test this we used whole animal preparations in which the innervation of the gill from the cerebral and visceral ganglia is intact and tested the actions of a histamine H2 antagonist, famotidine, on the response to light. Cilia beating was measured by stroboscopic microscopy. An eleven lumen, ¼ inch diameter spotlight source was used to stimulate the mantle rim in the vicinity of the siphons. Stimulating the mantle rim with light decreased cilia beating in the gill. Adding the H2 antagonist famotidine caused a statistically significant, dose‐dependent blockage of this action over the range of 10−9 – 10−3. The study further demonstrates the sensory‐motor integration of beating of lateral cilia involving the sensory mantle rim and visceral/cerebral ganglia, supporting the hypothesis that histamine is the sensory neurotransmitter in mantle photoreceptor cells.Support or Funding InformationThis work was supported by grants 2R25GM06003 of the Bridge Program of NIGMS and 0516041071 of NYSDOE.
Regulation of AMPK and Hepatic Metabolism by Folic acid Supplementation in Non‐alcoholic Fatty Liver Disease
Authors: Sid Victoria , Wu Nan , Sarna Lindsei , Shang Yvette , House James , Siow Yaw L. , Karmin O 
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a worldwide epidemic associated with obesity and type 2 diabetes. Chronic high‐fat diet consumption promotes NAFLD which is characterized by hepatic lipid accumulation, hyperglycemia and hyperlipidemia. Emerging evidence suggests that metabolic aberration during NAFLD may be related to dysregulation of AMP‐activated protein kinase (AMPK). AMPK is an endogenous energy sensor involved in regulating lipid and glucose metabolism, which has been proposed as a potential therapeutic target in NAFLD and its associated metabolic disorders. Folic acid is an essential B vitamin that has been implicated in NAFLD through improvement of liver function. The aim of our study was to investigate the mechanisms by which folic acid supplementation regulated hepatic AMPK and its impact on cholesterol and glucose metabolism. Male C57BL/6J mice were fed a control diet (10% kcals fat), a high‐fat diet (60% kcals fat) or a high‐fat diet supplemented with folic acid for 5 weeks. Mice fed a high‐fat diet exhibited fatty liver, hyperglycemia, elevated activity of hepatic HMG‐CoA reductase and lower AMPK activity. Folic acid supplementation restored AMPK activity, attenuated hepatic lipid accumulation and reduced blood glucose levels. Activation of AMPK by folic acid was mediated, in part, through activation of its upstream kinase LKB1. Folic acid‐induced AMPK activation could attenuate HMG‐CoA reductase activity via AMPK‐dependent phosphorylation of the reductase and hence reduce hepatic cholesterol biosynthesis. Our results suggest that folic acid supplementation can improve cholesterol and glucose metabolism through restoration of AMPK activation in the liver, which may have a therapeutic implication in mitigating hepatic lipid accumulation and hyperglycemia in NAFLD.Support or Funding InformationThis study was supported, in part, by Natural Sciences & Engineering Research Council of Canada and St. Boniface Hospital Research Centre.
Cyclin D1 regulates adipose triglyceride lipase (ATGL) to influence hepatic lipid droplet metabolism and cell proliferation
Authors: Ploeger Jonathan , Kamarajugadda Sushama , Mashek Douglas , Albrecht Jeffrey H 
Published: November 2023

Obesity is well‐documented to promote the development of nonalcoholic fatty liver disease including its more advanced stages such as non‐alcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma. Cyclin D1 (D1) is a cell cycle protein that regulates G1/S transition, the rate‐limiting step in cell division. D1 is highly expressed in liver cancer and its overexpression (OE) leads to lipid droplet (LD) accumulation in hepatocytes. Liver‐specific knockdown of adipose triglyceride lipase (ATGL), the rate‐limiting enzyme in triglyceride (TG) catabolism, promotes LD accumulation. In order to characterize a potential link between D1 and ATGL we employed studies in primary hepatocytes and mouse immortalized AML12 cells. As expected, in the absence of mitotic stimuli, D1 OE was sufficient to drive DNA synthesis in primary hepatocytes. However, these effects are abrogated with ATGL. Knockdown of D1 in the presence of mitogens inhibited DNA synthesis and increased ATGL mRNA expression, but knockdown or chemical inhibition of ATGL recovered DNA synthesis. Moreover, cell cycle analysis using flow cytometry confirmed that D1 knockdown increased accumulation of cells in the G0/G1 phase and reduced cells in the S and G2/M phase; these results were reversed with ATGL knockdown or chemical inhibition. Taken together, these data illustrates for the first time that cyclin D1 regulates ATGL to alter hepatic LD metabolism and proliferative capacity in primary hepatocytes and AML12 cells.Support or Funding InformationThis project was supported by NIDDK (DK054921) to JHA and (DK085008) to DGM, NCI T32CA132670 to JMP and the Minnesota Obesity Center (DK050406).
Tracking Diacylglycerol Pools in Budding Yeast
Authors: Ganesan Suriakarthiga , Ponce Maria Laura Sosa , Zaremberg Vanina 
Published: November 2023

Many diacylglycerol (DAG) sensing probes have been developed to monitor DAG signalling in mammalian cells. Several C1 domains have been fused to fluorescent proteins that can sense DAG in cellular membranes. The C1 domain of PKCδ is very well characterized and known to bind DAG with high affinity, and has been successfully used as a probe fused to GFP in order to monitor DAG in live cells. We have cloned the C1 domain of PKCδ (C1δ) into a centromeric yeast expression vector under a constitutive promoter for in vivo visualization of DAG pools. Localization of C1δ‐GFP was first monitored in different wild type strains grown under standard conditions. The main localization of this DAG‐binding reporter in actively growing cells was the vacuolar membrane, further confirmed by co‐localization with FM4‐64. This localization is consistent with studies done using similar probes to show that DAG localizes to the membrane of isolated vacuoles from yeast cells. Importantly, no changes in vacuolar morphology of wild type cells due to expression of C1δ‐GFP were detected. In addition to vacuolar membrane localization, the C1δ probe also associated with the plasma membrane of daughter cells and at the bud neck. In contrast to the phosphatidic acid (PA)‐binding reporter GFP‐Spo2051–91, C1δ‐GFP did not show nuclear localization as determined by co‐localization analysis using the nucleolar marker Sik1‐RFP. We then tested if the localization would change in conditions where lipid homeostasis and levels of DAG are known to be altered. For this, we used cells lacking the four enzymes responsible for the final step in the synthesis of triglycerides in yeast, namely DAG acyltransferase Dga1, the transacylase Lro1, and the steryl acyltransferases Are1 and Are2 (4Δ quadruple mutant), which accumulate DAG and have defects in both nuclear and peripheral ER membrane organization. Localization of the C1δ‐GFP probe drastically changed when expressed in 4Δ quadruple mutant cells, clearly revealing its association with the expanded nuclear and cortical ER in addition to the vacuolar membrane. Additionally, the polarized distribution of C1δ‐GFP to the plasma membrane of buds and daughter cells significantly decreased in the quadruple mutant. After validating the use of this probe in yeast, we investigated the localization of DAG pools in response to lysophosphatidylcholine (LysoPC) analogues, as we have determined they induce a 5 times increase in DAG levels. Interestingly, incubation with LysoPC analogues induced a drastic change in the localization of DAG while no differences were observed regarding the distribution of PA. These results further support the use of C1δ‐GFP as a valuable tool to track cytosolic accessible pools of DAG in yeast.Support or Funding InformationThis work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to VZ
Cysteinyl Leukotriene 2 Receptor Enhances Angiogenesis, Vascular Permeability and Tumor Metastasis
Authors: Duah Ernest , Kondeti Vinay , Al‐Azzam Nosayba , Adapala Ravi , Patil Prachi , Thodeti Charles , Paruchuri Sailaja 
Published: November 2023

Excessive or insufficient angiogenesis can lead to number of diseases including atherosclerosis, diabetic retinopathy, age‐related macular degeneration and cancer. Although long‐standing inflammation secondary to chronic infection or irritation mediated by immune cells has been implicated in cancer progression, the contribution of endothelial cells (EC) in the generation of inflammatory mediators and their effects on angiogenesis is relatively unknown. One of the pro‐inflammatory mediators produced by EC are cysteinyl leukotrienes (Cys‐LTs: LTC4, LTD4 and LTE4), and they mediate their effects through two main receptors, CysLT1R and CysLT2R. Cys‐LTs have been shown to be involved in several human cancers including breast cancer and melanoma. Tumor microenvironment comprises numerous signaling molecules and pathways that influence angiogenic response leading to increased and aberrant vascularization. Further, angiogenesis is required for the supply of nutrients and oxygen for tumor growth and also serves as a route of tumor cells metastasis. Therefore, we investigated the role of CysLTR in angiogenesis, tumor progression and metastasis, using mice lacking CysLT1R (CysLT1RKO) and CysLT2R (CysLT2RKO).We observed enhanced CysLT2R expression, and not CysLT1R in wild type (WT) tumors and tumor growth as well as angiogenesis were significantly decreased in CysLT2RKO mice compared to WT and CysLT1RKO mice in Lewis lung carcinoma (LLC) tumor model. Further, though few in number, tumor vessels in CysLT2RKO mice showed intact pericyte coverage and reduced permeability with simultaneous reduction in tumor cell metastasis to the lung. Furthermore, we found that activation of CysLT2R, but not CysLT1R increased EC contraction leading to junctional destabilization and permeability in vitro. Importantly, our results show that CysLT2R activates Rho kinase and inhibition of Rho kinase significantly attenuated CysLT2R‐induced EC contraction and permeability. Further, our preliminary results revealed that pericytes express CysLT2R and display calcium influx in response to LTD4. Taken together, our results suggest that CysLT2R promotes leaky blood vessel formation, enhancing tumor growth and metastasis to the lung. Blocking CysLT2R could offer novel CysLT2R‐targeted, therapeutic candidates for the treatment of cancer and other angiogenic disorders.Support or Funding InformationJames Foght Professorship Support, AHA GIA 15GRNT 25670004
Is the site of influenza virus assembly and budding enriched with cholesterol and sphingolipids?
Authors: Kraft Mary L , Yeager Ashley N. , Weber Peter K. , Zimmerberg Joshua 
Published: November 2023

The observation that the influenza viral envelope is enriched with cholesterol and sphingolipids contributed to the hypothesis that the influenza virus assembles and bud from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane. Protein imaging experiments have demonstrated that the clustering of influenza virus proteins in the plasma membrane is sensitive to depletion of cellular cholesterol. However, the precise distributions of cholesterol and sphingolipids in relation to the site of influenza budding remains speculative due to an inability to directly image lipids without the use of potentially perturbing labels. We have used high‐resolution secondary ion mass spectrometry (SIMS), which is performed on a Cameca NanoSIMS 50, to image the distributions of stable isotope‐labeled lipids in the plasma membranes of intact cells with ~100 nm lateral resolution. Here we report using this approach to directly test the long‐standing hypothesis that the influenza virus buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. We imaged metabolically incorporated 15N‐sphingolipids and 18O‐cholesterol and immunolabeled influenza virus envelope proteins on the surfaces of influenza‐infected MDCK cells with high‐resolution SIMS. The sites of virus assembly and budding in the plasma membrane were identified as the regions on the MDCK cell that were enriched with the immunolabeled influenza virus envelope protein, hemagglutinin. High‐resolution SIMS imaging revealed the sites of influenza virus assembly and budding were not enriched with cholesterol and sphingolipids. Although sphingolipid domains were detected in the plasma membranes of these MDCK cells, they had little co‐localization with the hemagglutinin‐rich membrane patches. These findings argue against the hypothesis that the influenza virus buds from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane.Support or Funding InformationThis research was supported by the U.S. National Science Foundation under CHE 15‐08662.
Autism‐linked Mutations in the Human Serotonin Transporter Induce Distinct Structural Changes that may Account for the Altered Function of the Transporter
Authors: Krout Danielle , Henry L. Keith 
Published: November 2023

BackgroundPhysiological studies show that 20–25% of individuals with autism spectrum disorder (ASD) exhibit elevated platelet serotonin (5‐HT) levels suggesting a link between regulation of the serotonergic system and behaviors associated with ASD. Furthermore, multiple ASD‐linked serotonin transporter (SERT) coding variants (ASVs) have been identified which augment 5‐HT transport function through enhanced catalytic activity (Gly56Ala and Lys605Asn) or elevated surface density (Ile425Leu, Phe465Leu, and Leu550Val). We propose that ASV‐induced hyperactivity is a result of altered SERT tertiary structure thereby affecting phosphorylation through the PKG and p38 MAPK signaling pathways and/or modulating plasmalemmal SERT turnover.ObjectiveIdentify SERT tertiary structure alterations mediated by ASV modifications that contribute to changes in catalytic activity or surface expression.ResultsWe utilized the substituted cysteine accessibility method to assess conformational changes by comparing the sensitivity of one native (Cys109) and two engineered cysteine residues (Cys277 and Cys404) to methanethiosulfonate reagents (MTS). Cys277 displayed decreased reactivity in the ASVs Gly56Ala, Ile425Leu, Leu550Val, and Lys605Asn indicating reduced accessibility as a result of ASV‐induced conformational changes. Evaluation of reactivity changes at Cys109 and Cys404 is currently underway. In addition, we are characterizing structural changes in the ASVs after activation of the PKG or p38 MAPK pathways to determine if the functional changes stem from a common mechanism.ConclusionThe reactivity changes we have observed at Cys277 support alteration of SERT tertiary structure by the ASVs Gly56Ala, Ile425Leu, Leu550Val, and Lys605Asn. In addition, the intracellular position of the Cys277probe in SERT and its decrease in MTS sensitivity suggests an ASV‐induced outward‐facing conformation.
Unraveling the Structural Basis of P‐glycoprotein Transport of Serotonin 5‐HT1B/1D Receptor Agonists: Eletriptan and Sumatriptan
Authors: Nguyen Diana , Wilt Laura , Roberts Audie 
Published: November 2023

P‐glycoprotein (Pgp) acts as the gatekeeper at the blood‐brain barrier by preventing the entry of toxic insults; however, drugs that are Pgp substrates cannot penetrate the blood‐brain barrier to treat central nervous system (CNS) disorders and diseases. Serotonin 5‐HT1B/1D receptor agonists are members of the triptan class of drugs that abort migraines during the onset of symptoms and are known Pgp substrates. While members of a drug class are structurally similar, eletriptan (Relpax®, ETT) and sumatriptan (Imitrex®, STT) have large differences in their Pgp efflux ratios. To understand the relationship between drug binding and efflux, ETT and STT were used to probe the structural basis of triptan transport. From ATPase activity assays, ETT stimulated Pgp ATP hydrolysis and was fit to the Michaelis‐Menten equation. In contrast, STT induced very little activation of the Pgp‐coupled hydrolysis. Protein fluorescence quenching was used to determine the binding affinities and the number of binding sites of ETT and STT. Each triptan drug was identified to have a single binding site. To test if these binding sites were overlapping, competition ATPase activity assays were performed. Conformational changes induced by each ETT and STT binding was determined by acrylamide quenching.Support or Funding InformationNIH ‐1021RR571390, AHA ‐1021RR571388, Pfizer, Inc., University of Georgia, Bioexpression and Fermentation Facility at UGA
A Ten Week, First‐semester Biochemistry Lab Course Studying Bacterial Alkaline Phosphatase
Authors: Witherow D. Scott 
Published: November 2023

In a traditional introductory biochemistry lab, students are expected to learn a variety of standard biochemical techniques. One of the challenges in designing a lab curriculum is finding a way to engage students in a way where they not only learn the necessary skills, but see how different aspects of biochemistry work together with one another. This work describes a 10 week laboratory project studying wild‐type and mutant bacterial alkaline phosphatase, where students purifying, quantitate, and perform kinetic assays on wild‐type and selected mutants of the enzyme. Additionally, students also perform vector DNA analysis. In addition to simply learning important techniques, students acquire novel biochemical data in their kinetic analysis of mutant enzyme. The experiments are designed to build on student's work from week to week in a way that encourages students to apply quantitative analysis and reasoning skills, reinforcing traditional textbook biochemical concepts. Students are assessed through lab reports focused on journal style writing, quantitative and conceptual question sheets, and traditional exams. Students also are surveyed to obtain data relating to their perception of the efficacy of the course design and in achievement of learning outcomes.Support or Funding InformationThis work was supported by The University of Tampa Dana Foundation Grant; The University of Tampa David Delo Research Professor Grant; The University of Tampa College of Natural and Health Sciences; and The University of Tampa Department of Chemistry, Biochemistry, and Physics.
The Voelcker Biomedical Research Academy: A Longitudinal and Multi‐tiered Model for Preparing the Next Generation of Biomedical Scientists
Authors: Chapa Irene , Hensler Julie , McManus Linda , Giuffrida Andrea 
Published: November 2023

The Voelcker Biomedical Research Academy (VBRA) is a unique training program for high school students in the San Antonio area. VBRA allows talented high school freshmen (VBRA Scholars) to conduct a 3‐year long research project in biomedical sciences with faculty at the UT Health Science Center‐San Antonio. The VBRA experience incorporates personalized mentorship, guidance for preparation for college, leadership development, and awareness of career opportunities in science. VBRA applications have increased from 60 applicants in 2009 to 121 in 2015. A cohort of 20–22 selected Scholars is enrolled in the program each year. To date, 7 cohorts have been admitted to the Academy (145 students). These Scholars represent 44 different high schools, are ethnically diverse, (Hispanic, 46%; Caucasian, 32%; Asian, 13%; African American, 9%) and have demonstrated exceptional commitment to the Academy, as 97% of them have completed the program. All senior Scholars have graduated from high school scoring significantly higher on SAT exams than their high school peers. In addition, 100% of the Scholars are attending 4‐year universities and 88% of them are pursuing STEM related majors (88%). Eighty‐two VBRA Alumni serve as role models for current VBRA Scholars and interact daily with their younger counterparts. The Alumni offer presentations on the college experience (College Connection) and continue their own professional development by serving as teaching assistants and lecturers. VBRA Alumni can further grow their research experience, technical skills and scientific knowledge by becoming VBRA Summer Undergraduate Research Fellows (VBRA SURF); these Fellows work full time in a research laboratory during the summer months and participate in supplemental didactic activities. A key feature of the Academy is the contribution of ~ 40 medical students, graduate students and post‐doctoral fellows to the education and mentoring of VBRA Scholars and Alumni. These VBRA Junior Mentors are valuable members of the VBRA Admissions Committee, provide didactic activities and individualized training in specific laboratory techniques, and/or clinical perspectives relevant to translational research. In summary, the VBRA model represents a multi‐tiered approach to professional development in biomedical sciences through the integration of programs for high/professional school and college students with hands‐on research experiences, mentoring and networking opportunities. This model creates an innovative ecosystem that could be integrated or added to existing training programs to support the development of the next generation of biomedical scientists and professionals.Support or Funding InformationSupported in part by the Max and Minnie Tomerlin Voelcker Fund