bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein Binding to Artificial Lipid Droplets Demonstrates the Importance of Phospholipid Composition
Authors: Paal Matthew , Gillingham Jacob , Furcich Justin , Brown Margaret , Listenberger Laura 
Published: April 2020

All cells and tissues store excess lipid in intracellular structures known as lipid droplets. Each lipid droplet consists of a neutral lipid core (primarily made of triacylglyceride and cholesteryl ester) surrounded by a phospholipid monolayer. Several proteins interact specifically with the surface of the lipid droplet and control their growth and degradation. Here, we share the development of an in vitro binding assay, that utilizes synthetic lipid droplets and partially purified lipid droplet proteins, to study protein binding to lipid droplets. We separate bound from unbound proteins with sucrose gradient ultracentrifugation and detect lipid droplet proteins following SDS‐PAGE and western blotting. We have used this assay to show that changing the phospholipid composition of the lipid droplet surface alters interactions with perilipin 2, a ubiquitously expressed lipid droplet protein that is predicted to interact with lipid droplets through an amphipathic alpha helix. Specifically, perilipin 2 association with synthetic lipid droplets was increased when the amount of phosphatidylethanolamine relative to phosphatidylcholine was increased. We hypothesize that the smaller headgroups of phosphatidylethanolamine allows additional interaction with the underlying neutral lipid. These results may be relevant to the formation of lipid droplets in alcoholic fatty liver disease, where lipid droplets form with altered levels of phosphatidylethanolamine and phosphatidylcholine.Support or Funding InformationFunding for this research was provided by an ASBMB Undergraduate Research Award and the Lowell and Doris Kispert Endowment at St. Olaf College.
Metformin Treatment Attenuates Non‐Small Cell Lung Carcinoma Cell Viability In Vitro.
Authors: Stott Nicole L. , Beach Cassandra , Marino Joseph S. 
Published: April 2020

Lung cancer, the second most common cancer, is one of the largest causes of cancer‐related mortalities worldwide. Non‐Small Cell Lung Carcinoma (NSCLC) comprises 80–85% of all new lung cancer diagnoses, suggesting a great need to continue finding measures to combat this mounting disease. Metformin induces an anti‐proliferative effect in many other cancers, including breast, prostate and obesity‐activated thyroid cancer. The purpose of this study was to determine how Metformin treatment influences NSCLC cell viability in vitro. NSCLC cells were grown for 24 hours and serum‐starved overnight with serum‐free media to induce quiescence. Cells were treated with equal volumes of fresh growth media followed by Metformin treatment with a range of concentrations from 5mm to 15mM. NSCLC cell viability was assessed every 24 hours for 3 days via an MTT assay using 3‐(4,5‐Dimethyltiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide. Proliferating NSCLC treated with Metformin showed significantly reduced cell viability (p=0.0109), after 48 hours of treatment, irrespective of Metformin concentration. The results of this study indicate that Metformin is effective at decreasing NSCLC viability. Experiments are ongoing to investigate alterations in cell cycle regulators and metabolic signaling pathways.Support or Funding InformationTargeted Research Internal Seed Program
Peripheral Inflammation Drives TLR2 and IL‐1R1 Mediated Neurodegeneration in Parkinson’s Disease
Authors: Bearoff Frank , Johnson Marguerite , Dhavale Dhruva , Nonnemacher Michael , Kotzbauer Paul , Kortagere Sandhya 
Published: April 2020

Current evidence suggests a causal role of inflammation in the pathogenesis of Parkinson’s Disease (PD). PD is a progressive neurodegenerative disorder that is associated with loss of dopaminergic neurons and the aberrant accumulation of alpha‐synuclein fibrils in Lewy bodies present in surviving dopaminergic neurons in the substantia nigra. Importantly, alpha‐synuclein fibrils can interact with toll‐like receptor 2 (TLR2) on both neurons and microglia to induce a neuroinflammatory response. Previous work has investigated the effects of pre‐existing inflammation on neurodegeneration. However, the effect of pre‐existing peripheral inflammation on alpha‐synuclein fibril induced neuroinflammation in PD has not been well understood. We hypothesize that peripheral inflammation promotes crosstalk between TLR2 and IL‐1R1 receptors when activated by alpha‐synuclein fibrils. Our preliminary results in SH‐SY5Y cells demonstrate that activation of either TLR2 or IL‐1R1 promotes synergistic interaction between the receptors leading to an increase in p‐ERK1/2 levels in a MYD88 dependent manner. In the in vivo model, prior exposure to a low dose of lipopolysaccharide (LPS) systemically increased the expression of proinflammatory cytokines such as IL‐1b and TNFα, and nuclear factor of kappa light polypeptide gene enhancer in B‐cells, alpha (IkBa) compared to animals that received saline. Treatment with alpha‐synuclein fibrils post LPS treatment significantly enhanced IL‐1b, TNFα, p‐ERK1/2 expression suggesting a synergistic effect from TLR2 and IL‐1R1 activation by alpha‐synuclein and IL‐1b respectively. In addition, these animals showed a significant increase in GM‐CSF expression compared to LPS only or saline treated animals suggesting alpha‐synuclein fibril treatment lead to an increase in infiltration of T‐cells, neutrophils and macrophages that may be causal for aberrant neuroinflammatory response in PDSupport or Funding InformationDrexel Coulter Foundation
Insight into Parkinson’s Disease from Yeasts: Combined Impact of Covalent Modifications and Familial Mutations on α‐Synuclein
Authors: Borland Carris Temera , Ganev Yoan , Thomas Rosemary , Roman Alexandra , Grauer Thea , Mwale Chisomo , Jones Paul , Balaram Ariane , Tcaturian Estella , Debburman Shubhik 
Published: April 2020

Parkinson’s Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are composed of misfolded α‐synuclein protein. α‐Synuclein is highly post‐translationally modified in healthy and diseased states. While phosphorylation and nitration of α‐synuclein are well‐studied as contributors to PD pathology, less is known about SUMOylation, acetylation, and glycation. Also, the combined effects of these modifications remain largely unclear, on both wildtype α‐synuclein (linked with sporadic PD) and the six mutant forms (A30P, E46K, H50Q, G51D, A53T, A53E) linked with early‐onset familial PD. We first evaluated the effects of blocking SUMOylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more toxic and aggregated, losing its membrane localization. Second, we found that SUMOylation and phosphorylation counteract each other in toxicity and localization. Third, we expanded our investigation to two newly reported modifications – acetylation and glycation. We found that acetylation is protective and glycation is harmful. When we combined acetylation or glycation manipulations with SUMOylation and phosphorylation alterations on the α‐synuclein level, we found that the effects of these modifications are not additive – the impacts of acetylation and glycation depends on phosphorylation status. Furthermore, we found that decreased nitration protects against toxicity and reduces α‐syn aggregation in the mutants modifying SUMOylation and phosphorylation. Finally, we investigated the effects of the familial mutants in tandem with altered acetylation, glycation, or nitration. We report two familial mutant‐specific effects: H50Q is surprisingly sensitive and non‐toxic in a hyper‐acetylation environment, while A53E is highly toxic with hypo‐glycation. We also found that too much nitration exacerbates toxicity of the familial mutants relative to low nitration. Specifically, mutant A53T is highly sensitive to excess nitration while H50Q is non‐toxic in such environment. These studies show the relevance of covalent modifications in sporadic and familial PD, and their in‐tandem effects that underlie toxicity mechanisms.Support or Funding InformationYG= supported by Parkinson's foundation‐American Parkinson's disease association, A.B,C.M, A.R and P.J: Research grants from Neurosci
Disease‐Causing Mutations Shift the pK of the pH‐Sensitive H+‐Conductor Slc4a11
Authors: Quade Bianca Natalia , Marshall Aniko , Parker Mark Daniel 
Published: April 2020

SLC4A11 is a H+‐conducting membrane protein that is sensitive to changes in both intracellular and extracellular pH. It is expressed in corneal endothelial cells (CECs) where it functions alongside other acid/base transporters such as MCT1 and NBCe1. These proteins function to maintain the osmotic balance and clarity of the cornea. Mutations in SLC4A11 cause congenital hereditary endothelial dystrophy (CHED) and Fuchs’ endothelial corneal dystrophy (FECD). Signs of these corneal dystrophies include loss of normal CEC morphology and corneal edema. Previously—using the Xenopus oocyte expression system, two‐electrode voltage clamp circuitry (to monitor conductance), and H+‐selective microelectrodes (to monitor intracellular pH)—we determined that the intracellular pK of wild‐type (WT) mouse Slc4a11 activity is 7.15 ± 0.04 (n=13, SD, pHe=8.5). In this study we use the same experimental setup to explore whether disease‐causing mutations can affect the intracellular‐pH dependence (pK) of Slc4a11.We chose FECD mutation E399K and CHED mutations T401K and R804H for comparison to WT due to the loss or gain of a titratable residue. We find that Slc4a11‐‘E399K’ exhibits a pK value of 7.02 ± 0.03 (n = 6, SD) and that Slc4a11‐‘T401K’ exhibits a pK value of 7.09 ± 0.03 (n = 6, SD); both significantly acid‐shifted compared to WT. On the other hand, ‘R804H’‐Slc4a11 exhibits a significantly alkali‐shifted pK value of 7.29 ± 0.04 (n = 6, SD) [Statistics are the result of two‐tailed unpaired t‐tests with Bonferroni correction].Thus we describe three disease‐causing mutations that cause a shift in the intracellular pK value of Slc4a11. If the pH‐dependence of the H+ conductance mediated by Slc4a11 is critical to the physiological role of Slc4a11, these shifts in pK could contribute towards the etiology of corneal dystrophy.Support or Funding InformationThis work was supported by NIH R01‐EY028580 to MDP.
Anticancer Properties of Secondary Metabolites Isolated from Lichen Parmelia vagans
Authors: Bondarenko Vladimir , Raymond Ky 
Published: April 2020

This study aims to analyze the antiproliferative properties of the secondary metabolites extracted from lichen Parmelia vagans collected from the semi‐deserts of Russia. Identification of novel natural compounds that can selectively slow down or stop proliferation of cancer cells can enhance development of anticancer therapeutics.Various lichen extracts were prepared using either water or organic solvents. We assessed the antiproliferative activity of the extracts against several human cancer cell lines; human malignant melanoma (A375), human mammary gland adenocarcinoma (MCF7). Human dermal fibroblasts (HDFa) were used as control. The same number of cells of different cell lines were plated into a 96‐well plate and allowed to adhere overnight in CO2 incubator. Different dilutions of the lichen extracts were added to the cell cultures next day, and the plate and incubated at 37°C for 6–72 hours. By the end of the incubation time a picture of each well was taken to assess cell confluency and morphology. To asses the effect of the lichen extracts on cell growth and proliferation we used a Resazurin assay. After Resazurin assay, the cells in each well were treated with trypsin, suspended in growth medium and counted using Bio‐Rad cell counter to evaluate a percent of live cells.Our experimental data from cell viability assay and live cells count indicate that ethyl acetate extracts of P.vagans significantly suppress (30% – 60%) the growth of human malignant melanoma but have a limited effect on human mammary gland adenocarcinoma. We also found that cancer cells subjected to lichen extract change their morphology, and these changes resemble an activation of apoptosis.Our findings indicate that Parmelia vagans extracts contain secondary metabolites that slow cancer cells growth and proliferation. Furthermore, the extracts affect cancer various cells differently, suggesting that the active compound(s) utilizes a distinct mechanism of action on different types of cancer. Future experiments will focus on the isolation and characterization of the compounds from P.vagans extracts that are responsible for the inhibition of cancer cell growth. Another important direction of future research is to identify the cellular target(s) of the antiproliferative compound(s) and its molecular mechanism of action. In summary, the lichen’s secondary metabolites may hold vast medicinal potential and could be a viable source of a novel of anticancer drugs.Support or Funding InformationThe funding for this project was provided by Touro University Nevada research grant.
Impact of heat therapy on exercise performance, collateral artery growth and skeletal muscle capillarization in a peripheral artery disease model
Authors: Kim Kyoungrae , Ro Bohyun , Damen Frederic , Gramling Daniel P. , Lehr Trevor D. , Song Qifan , Goergen Craig J. , Roseguini Bruno T. 
Published: April 2020

IntroductionRepeated exposure to heat therapy (HT) has been shown to improve exercise tolerance in patients with symptomatic peripheral artery disease (PAD), but the mechanisms underlying this effect remain unknown. We investigated the effects of repeated exposure to HT on endurance running capacity, collateral growth, and skeletal muscle capillarization in a preclinical model of PAD.MethodsMale C57Bl/6 mice (32‐week‐old) were fed a high‐fat diet for 10 weeks. At 42 weeks of age, the animals underwent bilateral ligation of the femoral artery to induce hindlimb ischemia. After two weeks of recovery, the animals were randomly allocated to receive HT (n=12) or a control intervention (n=13). Heat therapy was applied by placing the animal in a flat bottom restrainer and immersing the lower half of the body in a glass container filled with water at 39°C for 30 min daily over 3 consecutive weeks. Animals assigned to the control group were also restrained, but were placed in an empty container. At baseline and one day after the last treatment session, the animals completed a treadmill exercise test (10 m/min for 5 min at 10% grade followed by an increase in speed by 1 m/min every minute until exhaustion) for the assessment of endurance exercise performance. One day after the final treadmill test, the animals were anesthetized, the infrarenal aorta was cannulated with polyethylene‐50 tubing, and a vascular casting compound (Microfil) was injected into the cannula after perfusion‐fixation. Microcomputed tomography (micro‐CT) was used for the measurement of the average diameter of the anterior and posterior gracilis arteries. Skeletal muscle capillarization was assessed in the soleus and extensor digitorum longus (EDL) muscles in a subset of animals (Control: n=8 vs. HT: n=7).ResultsThe change from baseline to 3 weeks in maximum running speed (Control: 1.0±1.1 m/min vs. HT: 3.5±0.7 m/min, p=0.072), running distance (Control: 25.7±23.5 m vs. HT: 82.2±15.8 m, p=0.065) and total running time (Control: 62.7±63.5 s vs. HT: 217.3±39.0 s, p=0.056) tended to be greater in the animals exposed to HT as compared to the control group. However, there were no differences between groups in the average diameter of the upper and lower gracilis arteries (Control: 0.14±0.00 mm vs. HT: 0.13±0.00 mm, p=0.119) or the number of capillaries around a fiber in both the EDL (Control: 4.61±0.24 vs. HT: 4.64±0.16, p=0.923) and the soleus (Control: 5.17±0.33 vs. HT: 5.28±0.39, p=0.834) muscles.ConclusionThese findings indicate that the improvement in exercise tolerance following repeated exposure to HT does not appear to stem from collateral artery enlargment or enhanced muscle capillarization in a mouse model of PAD.Support or Funding InformationFunding: Ralph W. and Grace M. Showalter Research Trust Award
Isolation and Single Cell Chemical Biological Analysis of Natural Product Producing Cave Microorganisms
Authors: Algoo Jonathan David , Penton Kathryn , Bachmann Brian 
Published: April 2020

Microbial secondary metabolites (natural products) constitute a large fraction of pharmaceuticals, with applications as antibiotics and anti‐cancer therapeutics. However, the discovery of new natural product drug leads is increasingly difficult as isolation studies using traditional methods, and from traditional sources, have resulted in progressively fewer novel compounds discovered. Historically, searching for new sources of biodiversity from unique ecosystems has resulted in increased discovery rates. The cave environment, a previously uninvestigated ecosystem, is a promising terrain to discover bacteria with novel natural products because of its unique conditions, which include low light, relatively constant temperature year‐round, abundant oxygen content, and low nutrient content (oligotrophy). We hypothesize that the oligotrophic environment present in caves forces microorganisms to compete or cooperate with each other, which require the development of natural products as communication, offensive, or defensive chemicals. Non‐Streptomyces actinomycetes are a group of bacteria found in caves that represent good candidates for the isolation of new natural products. We acquire bacterial samples by swabbing rock formations and collecting sediment inside a cave. These samples are diluted and incubated on a number of different agar plating conditions. Individual colonies are then isolated and identified by their 16S rRNA sequences. Isolated actinomycetes are stimulated using a battery of chemical and biological conditions known to elicit secondary metabolism (coculture strains, heavy metals, and antibiotics), and then culture extracts are analyzed for possible cellular activity using multiplexed biological assays against mammalian cells. Specifically, extracts are tested using single cell analysis via a flow cytometry platform termed Multiplexed Activity Metabolomics (MAM), which identifies compounds for isolation by analyzing extracts and metabolomic arrays against multiple cell status markers.Support or Funding InformationNational Cancer Institutes R01 CA226833
Renal Olfr1393 may contribute to glucose metabolism
Authors: Hotait Zahraa S. , Shepard Blythe D. 
Published: April 2020

Olfactory receptor 1393 (Olfr1393), a G‐protein coupled sensory receptor most commonly associated with olfaction, has been located in the kidney, liver, and other vital organs. We have recently discovered that renal Olfr1393 has a regulatory role in glucose handling via the proximal tubules’ essential sodium glucose cotransporters 1 and 2 (SGLT1, SGLT2). These studies demonstrated that Olfr1393 knockout (KO) mice exhibited improved glucose tolerance as compared to their wild type (WT) counterparts, presenting with euglycemia and glycosuria. Furthermore, we recently determined that the KO mice also exhibit an improved diabetic phenotype when challenged with a high fat diet (type II diabetes) or streptozotocin (type I diabetes). Because of its involvement in glucose reabsorption, we postulated that Olfr1393 may also alter glucose metabolism in the kidney and liver, the two organs capable of gluconeogenesis. To begin, RNA was isolated from kidneys obtained from euglycemic and type I diabetic Olfr1393 WT and KO mice. The RNA samples were reverse transcribed into cDNA, and quantitative polymerase chain reactions (qPCR) was performed to determine the expression levels of glycolytic and gluconeogenic enzymes in each sample. The enzymes selected for this study are those involved in the rate limiting steps of these metabolic processes, and include gluconeogenic enzymes phosphoenolpyruvate carboxykinase 1 (PEPCK), glucose‐6‐phosphatase (G6Pase), fructose 1,6 bisphosphatase (FBP), and glycolytic enzymes phosphofructokinase, liver type (PFK‐L), and pyruvate kinase, liver type (PK‐L). Thus far, the qPCR data ‐ normalized to 18s rRNA for each sample ‐ shows an overall trend towards higher cycling threshold (Ct) values for the KO samples in normal chow mice, indicating lower expression of these enzymes in the murine kidney. Additionally, we find that gluconeogenesis is differentially regulated in the kidneys from diabetic WT and KO mice with an overall trend toward decreased enzyme expression in the Olfr1393 KO samples. Emphasis is currently focused on confirming these findings at the protein level. Further efforts to understand the influence of Olfr1393 on wholebody glucose metabolism will also examine hepatic glucose metabolism. Collectively, these studies suggest that Olfr1393 influences crucial glucose handling processes reaching beyond proximal tubule reabsorption and may even act as a global regulator of glucose handling.Support or Funding InformationNIDDK
Characterization of the Putative Cytosine RNA Methyltransferase TbCRMT5 from Trypanosoma brucei
Authors: Schultz William C. , Militello Kevin T. 
Published: April 2020

RNA methylation is a type of posttranscriptional modification that plays an important role in controlling gene expression. The organism Trypanosoma brucei, the protozoan parasite responsible for Human African Trypanosomiasis, does not seem to have abundant promoter regions or transcriptional regulation machinery. Thus, RNA methylation may play an especially important role in regulating gene expression in this organism. Recently, we have identified seven putative cytosine RNA methyltransferase (CRMT) genes in T. brucei. One of the putative CRMTs, termed CRMT5, is required for maximum parasite
growth. The purpose of this research is to provide evidence for genuine RNA methyltransferase activity. CRMT5 was expressed in E. coli with an N‐terminal 6x‐histidine tag and purified using a His‐affinity column. Purified CRMT5 was used in a series of methyltransferase assays using luciferase activity as a readout. CRMT5 addition resulted in luciferase activity in the presence of cytosine‐containing RNA (T. brucei total RNA and Poly‐IC RNA). There was little to no luciferase activity observed in the presence of RNA that lacks cytosine or when a mock purification from E. coli without the CRMT5 gene was used. Furthermore, a CRMT5 mutant was created changing a putative active site cysteine to alanine (C439A). We expect the mutant to show reduced luciferase activity in the methyltransferase assay. Future experiments will include performing a methyltransferase reaction with CRMT5 and subsequently isolating the RNA for bisulfite sequencing to confirm the methylation of cytosine bases, performing methyltransferase assays with different substrates to determine if there is a binding preference, and confirming our methyltransferase assay results with a radioactivity assay. Evidence for the presence of 5‐methylcytosine and RNA methyltransferases indicates the presence of a process to create an epitranscriptome in T. brucei.Support or Funding InformationNIH grant 1R15AI133428‐01 to KTM