bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tryptophan‐75 is a Potential Gating Residue of Cytochrome P450 2D6
Authors: McCarty Kevin D. , Furge Kyle A. , Furge Laura 
Published: April 2020

Cytochrome P450s (CYPs) are responsible for the metabolism of an estimated 70 – 80% of pharmaceutical drugs in humans. Structurally, they have deeply buried active sites. Evidence suggests that access to the active site, and the resulting metabolism of substrates, is influenced by amino acid side chains ‐ a phenomenon referred to as tunnel‐gating. Our investigation hypothesized that tunnel‐gating in CYP2D6 exists and is mediated by a tryptophan‐75 (Trp‐75) residue. Analysis in silico and in vitro was conducted to understand gating in CYP2D6 and the role of plasticity in enzyme mechanisms. Molecular Dynamics simulations measured energy landscapes of Trp‐75 and simulated substrate passage within channel 2b. It was determined that Trp‐75 was able to obstruct both entry and exit of substrates from the enzyme within channel 2b, and that the residue alternates between three stable states. A Trp‐75 to alanine (*W75A) mutant of CYP2D6 was prepared along with a reference *1 enzyme and both were tested for their Michaelis‐Menten kinetics and ligand binding properties in vitro with the substrates bufuralol, dextromethorphan, and debrisoquine. Trp‐75 was not found to impact ligand binding by the CYP2D6 enzymes. The residue was observed to significantly impact Km and vmax, but no predictable kinetic trends were found. Furthermore, Trp‐75 was observed to impact enzymatic efficiency, where the absence of the residue doubled overall efficiency. Thus, we propose that the range of conformations available to Trp‐75 function in mediating channel‐gating that is substrate specific and that can serve to trap substrates into channel 2b for shuttling into the active site.Support or Funding InformationNIH 2R15GM086767‐03 and the Sherman Fairchild Foundation
Unbiased Identification of Key Residues in Splicing Protein Dib1
Authors: Turner Caitlyn E. , Maeder Corina 
Published: April 2020

Pre‐messenger RNA (pre‐mRNA) is transcribed from a DNA segment, and then post‐transcriptionally modified before being translated into protein. To prepare the pre‐mRNA for proper protein production, the spliceosome facilitates the removal of non‐coding introns from the pre‐mRNA and the ligation of exons together, forming the mature messenger RNA (mRNA) in a process known as pre‐messenger RNA splicing. The spliceosome is a large ribonucleoprotein complex composed of 5‐snRNA and over 90 proteins that work in concert to facilitate pre‐mRNA splicing and ensure its accuracy. Due to its complexity and evolutionary importance to survival, mutations of the spliceosome have been linked to diseases. For instance, Burn‐McKeown syndrome is a craniofacial disorder caused by abnormalities in the gene for the U5‐15k protein, a small protein centered within human spliceosomes and an ortholog of Dib1 in yeast.Dib1 is a 17‐kDa thioredoxin‐like protein that is associated with the U5 small nuclear ribonucleoprotein (snRNP) in the U5/U4.U6 complex. Located in the catalytic groove of the pre‐B complex of the spliceosome, Dib1 must vacate the complex so the spliceosome can complete its assembly. However, this “gate‐keeping” mechanism controlling this departure is still unknown. This project intends to discover residues that are crucial to the function of Dib1 by an unbiased method. To identify key residues in Dib1, a randomly mutagenized dib1 plasmid library was constructed. A temperature sensitive (ts) yeast screen was then performed with this library in order to identify Dib1 residues important for cell growth. From this screen, we have identified a new dib1 mutant and will discuss the significance of its location in Dib1 and its relevance to splicing.Support or Funding InformationTrinity University, ASBMB Distinguished Undergraduate Scholarship, NIH‐NIGMS R15GM120720, and The Welch Foundation
Antimicrobial Activity of Extracts from the Leaves of Marrubium vulgare
Authors: Cravens Ashley , Krzysiak Amanda 
Published: April 2020

Natural products have served as powerful therapeutics against pathogenic bacteria since the golden age of antibiotics of the mid‐20th century. In the United States, antibiotics are a key component of modern medicine and are one of the top written prescriptions every year. However, overuse and misuse of antibiotics has led to an increasing frequency of antibiotic‐resistant infections. These infections are difficult and expensive to treat, often resulting in increased length of illness, hospital stays, and mortality rates. This clearly demonstrates that new antibiotics are critical for modern medicine. Murrubium vulgare (White Horehound) is a shrub found in the Appalachia region that has been used in herbal medicine to treat many pulmonary aliments as an expectorant, tonic, and emmenagogue. Previous studies of this plant and its commercial products have indicated that it may contain antioxidant and antibacterial properties. The aim of this study is to determine if whole leaf extracts of M. vulgare contain antimicrobial properties. Five different plants were sampled for this project. The leaves were dried and then underwent extract in 95% ethanol in a 1:10 (m/v) ratio. The solvent was removed using a rotavapor at 60°C and hot plate evaporation. The crude product was left in a desiccator until constant weight was achieved. The crude samples were analyzed to characterize the different classes of natural products present. The crude extracts were dissolved in 5% DMSO at different ratios. The disk diffusion method was applied to determine the antimicrobial activity.Support or Funding InformationBellarmine University, Student Government Association.
A Cellular Based Assay to Monitor the Cleavage of the Extracellular Matrix by Matrix Metalloproteinases as a Tool for Drug Discovery Against Metastasis
Authors: Doyle Ryan , Canter Ben , Nader Joy , Wolkowicz Roland , Tandon Ruby 
Published: April 2020

One of the most important genes up‐regulated in cancer is known as Matrix Metalloproteinase (MMPs). MMP’s are enzymes critical for remodeling the protein matrix, called extracellular matrix (ECM), that surrounds and supports cells. This protein can be secreted or membrane‐bound as it remodels the extracellular environments in healthy individuals. However, in cancerous cells, its overexpression leads to the metastasis of cancer cells.To investigate novel ways to block metastasis, our lab engineered a new cellular Two‐Tag Assay as a tool for drug discovery, enabling the search for inhibitors. The assay, based on a system we developed for HIV and Dengue Virus, was adapted to MMP‐14. It relies on a scaffold composed of two antibody epitopes, HA and FLAG, flanking a protein substrate. Because the FLAG epitope is encoded upstream of the substrate and HA, it is cleaved while HA remains attached to the cell membrane. We then use flow cytometry to monitor the presence of HA and FLAG. Based on the presence of one or two tags, we can determine the robustness of cleavage. Previously, we inserted viral proteases between the tags and showed autocatalytic cleavage or lack of while the protein travels through the classical secretory pathway. The MMP‐14 assay has a consensus substrate of MMP‐14 instead of a viral protease, as well as a cell line obtained through retroviral technology. In addition, we expressed MMP‐14 enzyme in cells expressing substrate, as well as naïve cells. Comparing substrate‐expressing cells with substrate and enzyme expressing cells, we showed that MMP‐14 cleaves the substrate. We performed mixing experiments with substrate‐expressing and enzyme‐expressing cells to specifically corroborate cleavage at the cell surface. Results were corroborated with confocal microscopy and western blotting. Decrease in FLAG expression in the presence of MMP‐14 proved the assay’s ability to discover new inhibitors of MMP‐14.Support or Funding InformationThis research is supported by fellow researchers in the Wolkowicz Lab and the National Cancer Institute of the National Institutes of Health under award numbers: U54CA132384 & U54CA132379
Immunohistochemical analysis of macrophage polarization in aged kidneys and spleen
Authors: Moyer Eric , Bloomer Steven 
Published: April 2020

Aging is accompanied by a pro‐inflammatory state that adversely affects physiological function in many organs. Macrophages, innate immune cells of the myeloid lineage, exist on a phenotypic spectrum ranging from pro‐inflammatory (M1) to anti‐inflammatory (M2), and play a major role in tissue homeostasis throughout life. Previously, we have shown that aging increases the number of macrophages of both phenotypes (M1 and M2) in the liver; however, in other organs, there are conflicting findings as to the effects of aging on macrophage number and polarization. Therefore, the purpose of this study was to determine the effects of aging on macrophage abundance and phenotype in the kidneys and spleen. We hypothesized that aging would increase the number of M1 macrophages, and decrease the number of M2 macrophages in each organ. To investigate this, we utilized immunohistochemistry to detect the M1 marker, inducible nitric oxide synthase (iNOS), and the M2 marker, cluster of differentiation 206 (CD206), in kidney and spleen sections from young (6 months) and old (24 months) male Fischer 344 rats. The number of cells were counted in 6–7 visual fields, the area of the field was determined, and the results are expressed as the number of cells per square millimeter. In the kidney, we detected diffuse iNOS staining in tubular cells, however there was no visible staining in cells with morphologic characteristics of macrophages in either age group. Cells positive for CD206 were detected most frequently in the medulla, with no significant differences in number between young (3.3 cells/mm2) and old (4.1 cells/mm2) animals. In the spleen, macrophages resided in the marginal zone, and in the red pulp. We observed a trend (p=0.07) for fewer splenic iNOS+ macrophages in young animals, compared to the old (0.015 cells/mm2 vs. 0.072 cells/mm2). However, there was no difference in splenic CD206+ macrophages between young and old groups (59 vs 57 cells/mm2). Unlike the liver, we have observed similar numbers of M2 macrophages between young and old animals in the kidneys and spleen, which reflects the heterogeneous nature of aging. Our results have also identified a unique M2 macrophage population in the kidney, which resides in the renal medulla.Support or Funding InformationFaculty Development Grant, Penn State Abington. Erickson Discovery Grant, Penn State University.
Transforming Growth Factor‐α Acts via Epidermal Growth Factor Receptor to Increase p44/42 Mitogen‐Activated Protein Kinase Signaling and Expression of Excitatory Mediators in the Hypothalamic Paraventricular Nucleus in Rats
Authors: Yu Yang , Wei Shun-Guang , Weiss Robert M. , Felder Robert B. 
Published: April 2020

Myocardial infarction (MI)‐induced heart failure (HF) in rats leads to activation of brain p44/42 mitogen‐activated protein kinase (MAPK) signaling, which may increase proinflammatory cytokine expression and upregulate renin‐angiotensin system (RAS) activity in the brain, contributing to sympathetic excitation. We recently reported (Hypertension, 2019; 74: AP2010) that activation of the epidermal growth factor receptor (EGFR) in the hypothalamic paraventricular nucleus (PVN), a critical cardiovascular and autonomic center, increases p44/42 MAPK signaling in rats with HF. Both transforming growth factor (TGF)‐α and TGF‐β1 can activate EGFR and p44/42 MAPK signaling in peripheral tissues. Both are expressed in the PVN. In the present study, we first examined whether HF alters TGF‐α and TGF‐β1 expression in the PVN. Male rats underwent coronary artery ligation (CL) to induce HF or sham surgery, and brains were collected 4 weeks after CL. Compared with sham rats, HF rats had significantly (*P<0.05) increased TGF‐α mRNA expression (1.93±0.83* vs 1.03±0.29, fold change) in the PVN, but TGF‐β1 mRNA expression in the PVN was comparable between HF and sham rats (1.42±0.63 vs 1.01±0.20). We next examined whether TGF‐α increases p44/42 MAPK activity in the PVN via EGFR. Male rats received bilateral PVN microinjections of a lentiviral vector encoding a small interfering RNA (siRNA) for EGFR or a scrambled siRNA. One week later, these rats received an intracerebroventricular (ICV) injection of TGF‐α (10 μl, 0.1 μg/μl over 5 min) and the brains were collected 4 hours after the TGF‐α injection. Normal rats without any treatments served as control. Compared with control rats, scrambled siRNA+TGF‐α rats had significantly increases in protein levels of phosphorylated p44/42 MAPK (0.70±0.15* vs 0.23±0.11, corrected by total p44/42 MAPK) and in mRNA expression of the proinflammatory cytokines tumor necrosis factor‐α (5.45±2.10* vs 1.24±0.77, fold change) and interleukin‐1β (2.58±0.33* vs 1.00±0.09, fold change), with a tendency toward an increase in mRNA expression of the RAS component angiotensin II type 1a receptor (1.40±0.29 vs 1.02±0.24, P = 0.08, fold change) in the PVN. All of these findings were ameliorated in the EGFR siRNA+TGF‐α rats (by 21–48%), in which EGFR mRNA expression was reduced by 57%. These results indicate that TGF‐α, an endogenous ligand for EGFR, increases in the PVN in HF and may increase p44/42 MAPK signaling, thereby contributing to the augmented inflammation and RAS activity that drives sympathetic excitation in HF.Support or Funding InformationSupported by NIH grant R01HL136149 and the Department of Veterans Affairs.
Alzheimer’s Disease‐Related Pathology and Impaired Cognitive Function Associate with Reduced O‐GlcNAc Transferase in Aged Metabolic Syndrome KKAy+/− Mice
Authors: Jinka Sanjay , Lallo Jason , Gupta Shreya , Mathias Amy , Khanal Saugat , Al-Rhayyel Yasmine , Herman Danielle , Fleming Sheila , Raman Priya 
Published: April 2020

Metabolic syndrome (MetS) refers to a cluster of anomalies including type 2 diabetes, obesity, insulin resistance and dyslipidemia. Patients with MetS are 1.5 times more likely to develop late‐onset Alzheimer’s Disease (AD), with impaired glucose metabolism, defective insulin signaling, increased oxidative stress and inflammation being shared between the comorbid diseases. Notably, risk of AD is profoundly enhanced in the aging MetS population. Recent studies in AD patients and AD mouse models suggest a putative link between hyperphosphorylated tau neurofibrillary tangles, a commonly accepted AD pathology, and cerebral glucose hypometabolism. Impaired glucose metabolism is characterized by reduced glucose flux through the hexosamine metabolic pathway triggering attenuated signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), a major regulator of intracellular protein O‐GlcNAcylation. However, the role of OGT in the etiology of MetS‐induced AD remains incompletely understood. The goal of the present study was to examine the link between cognitive function, AD pathology and OGT signaling in a mouse model of MetS (KKAy+/−) that develops increased body weight, hyperglycemia, elevated total cholesterol and total triglyceride levels. Briefly, male and female obese agouti KKAy+/−, lean non‐agouti KKAy−/− and normal C57BL/6 control mice weaned at 4 wks of age on regular chow diet were subjected to periodic body weight and random blood glucose monitoring followed by a battery of behavioral tests at 12+ months of age. Plasma and brain (frontal cortex and hippocampus) tissues were then harvested from each genotype for biochemical and molecular studies. In an object recognition test of attention and memory, obese KKAy+/− mice showed a more severe impairment in discrimination between a novel and familiar object compared to lean KKAy−/− and normal C57BL/6 mice. Additionally, in a test of spontaneous activity obese KKAy+/− mice made significantly fewer rears vs lean KKAy−/− mice and wild‐type C57BL/6 controls; these results suggest diminished cognitive function in the obese KKAy+/− mice. Furthermore, immunoblotting of brain tissue lysates derived from the agouti KKAy+/− mice revealed increased ptau expression coupled with reduced pERK (signaling mediator of neuronal function) and pGSK (inactive form of tau kinase GSK3β) expression compared to non‐agouti KKAy−/−. Importantly, augmented tau phosphorylation was concomitant to attenuated OGT expression in brain lysates of obese KKAy+/− mice. Together, these data demonstrate a direct association between cognitive dysfunction, AD‐related pathology and reduced OGT expression in aged MetS KKAy+/− mice. Overall, our study implicates a role of OGT in MetS‐induced cognitive decline and AD pathogenesis.Support or Funding InformationNEOMED Start‐Up Funds
Targeting primary cilia with polymeric nanoparticles
Authors: Pala Rajasekharreddy , Nauli Surya M. 
Published: April 2020

The field of nanomedicine is a promising future in providing huge clinical impacts on advanced disease management and personalized medicine. Nanoparticles (NPs) have been used for targeted delivery at a desired site and a sustained release of a drug, which decreases the overall toxicity by delivering a smaller drug dosage. An extensively investigated NPs is poly lactic‐co‐glycolic acid (PLGA) which is an FDA approved biodegradable polymer and they are currently used in clinical setting. Hence in our current study, we used PLGA nanoparticles labeled with dopamine receptor 5 antibody to target primary cilia. Further these NPs were loaded with fenoldopam to improve cilia structure and function. The conjugation and loading was confirmed by UV‐visible spectroscopy, transmission electron microscopy (TEM), X‐ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), zeta potential measurements and Fourier transform infrared spectroscopy (FTIR). TEM confirmed the synthesized nanoparticles were around 120 nm and spherical in shape. FTIR confirms the drug loading and antibody interactions with the PLGA. When we injected these NPs to ciliopathic hypertensive mice, significantly improved cardiac function in the ciliopathic hypertensive models, in which the hearts also exhibited arrhythmia, which was corrected with the PLGA‐NPs. Intraciliary and cytosolic Ca2+ were increased when cilia were induced with fluid flow, and this served as a cilia‐dependent mechanism of the PLGA‐NPs. Fenoldopam‐alone caused an immediate decrease in blood pressure, followed by hypertrophy with compromised functions in left ventricle pressure, stroke volume, ejection fraction. Our drug delivery profiles confirmed that the PLGA‐NPs were a potential delivery system for targeting cilia more specifically, efficiently, and effectively than fenoldopam‐alone. In conclusion, our results indicate that nanomedicine provides a possibility for targeted ciliotherapy.Support or Funding InformationThis work was supported by the National Institute of Health (HL131577), Congressionally Directed Medical Research Program (PR130153) and American Heart association (19IPLOI34730020).
Gene Expression in LEW.1WR1 Rats Indicate Insulin Resistance in Muscle and Liver Tissue
Authors: Wilkerson Vidal Quiana C. , Collins Genoah , Mercado Luis , Martin Sidney , Wright Laura Catherine , Gibson Helen , Love-Rutledge Sharifa 
Published: April 2020

FAT10 is a ubiquitin‐like protein and type 1 diabetes (T1D) susceptibility gene that may play a role in age‐related inflammation, adiposity, cancer, and kidney disease. The young T1D susceptible, LEW.1WR1 rat overexpresses FAT10 and has higher fasting concentrations of blood insulin. It is unclear how the overexpression of FAT10 directly affects insulin sensitivity. It is also unclear if the initial insulin sensitivity of this animal model plays a role in disease susceptibility. We measured the relative expression of lipid and glucose metabolism genes in skeletal muscle and liver tissue in LEW.1WR1 and LEW/ssNHsd rats to determine if 12‐week old LEW.1WR1 rats were insulin resistant. We also tested the gene expression in pancreatic islets to determine if there were changes in endocrine pancreas cell health and hormone production. The results indicate that gene expression increased in the islets for glucagon, insulin, and PLIN5 (Perilipin‐5), and cyclin D expression decreased in LEW.1WR1 rats, suggesting reduced compensation and a potentially lipotoxic environment for beta cells. The increase in glucagon and insulin also supports peripheral insulin resistance. In liver tissue, UBD (Ubiquitin‐D), a gene suspected to regulate beta‐oxidation, was increased. INSIG (Insulin‐induced gene 1) and GLUT2 (Glucose transporter 2) gene expression decreased, indicating decreased expression of insulin‐regulated gene and glucose transporters, respectively. In muscle tissues, ATGL (Adipose triglyceride lipase), GAPDH (Glyceraldehyde‐3‐Phosphate Dehydrogenase), and PNPLA2 (Patatin Like Phospholipase Domain Containing 2) gene expression decreased, indicating a reduced ability to burn fat as energy and glucose intolerance in muscle tissue. These results, in tandem with previous work, suggest that fat storage in organs may account for the difference in weight previously observed between LEW.1WR1 and LEW/ssNHsd rats, and that LEW.1WR1 rats were developing insulin resistance which further suggests these rats are a novel animal model for pre‐diabetes.Support or Funding InformationThe authors would like to thank the UAH Office of the Vice President for Research and Economic Development, The Alabama Space Grant Consortium, ALSAMP, Adriel D. Johnson Sr. Fellowship, College of Science, Department of Chemistry, Department of Biology, Honors College, and Office of the Provost for the financial support for the resources, supplies, and student salaries.
Atheroprotective Effects Induced by Glyceraldehyde‐3′‐phosphate Dehydrogenase (GAPDH) in Murine Model of Atherosclerosis
Authors: Sukhanov Sergiy , Hou Xuwei , Snarski Patricia , Yoshida Tadashi , Delafontaine Patrice 
Published: April 2020

GAPDH was shown to protect smooth muscle cells (SMC) against oxidant‐induced apoptosis via stimulation of DNA repair. GAPDH levels were reduced in atherosclerotic plaque SMC and therefore we hypothesized that SMC‐specific GAPDH overexpression will reduce atherosclerosis. GAPDH protein levels were increased >2‐fold (P<0.05) in aortas isolated from Apoe‐null mice with SM22α promoter‐driven GAPDH overexpression (SM‐GAPDH mice) compared to Apoe‐null controls. SM‐GAPDH mice have BW and BP similar to control. Aortic circumference, lumen area and media area were not changed by GAPDH overexpression. SMC GAPDH did not change pulse wave velocity (SM‐GAPDH, 1.1±0.1 m/s, control, 1.2±0.3 m/s) (ultrasound imaging) as well as endothelium‐dependent and ‐independent vascular responses to PE, Ach or SNP (organ chamber assay with thoracic aortic rings). High‐cholesterol fed SM‐GAPDH had reduced atherosclerotic burden (H&E‐stained aortic valve cross‐sections: 28±3% decrease, P<0.05), elevated plaque SMC levels (IHC for a‐SM actin, 3.1‐fold increase, P<0.01; IHC for calponin, 3.8‐fold increase, P<0.05), increased plaque collagen (Trichrome, 2.1‐fold increase, P<0.05), reduced plaque SMC apoptosis (TUNEL assay with IHC for calponin, 3.2‐fold decrease, IHC for cleaved caspase‐3, 2.6‐fold decrease both are P<0.05) without changes in macrophages (IHC with Mac3, P=NS). Aortas isolated from SM‐GAPDH have increased expression of Ape1 endonuclease (major DNA repair enzyme, 31±5% increase, P<0.05), reduced cleaved PARP (apoptotic marker, 74±12% decrease, P<0.001) and decreased oxidative DNA damage (AP sites assay, 2.5‐fold decrease, P<0.05; pSer139 histone H2AX, 33±3% decrease, P<0.05) compared to controls. Plaques in SM‐GAPDH mice had thicker SMC‐rich fibrous plaque cap (3.5‐fold increase, P<0.05) and decreased area of necrotic core (1.8‐fold reduction, P<0.05) suggesting enhanced plaque stability. These data taken together with our in vitro findings indicate that Ape1 upregulation mediates GAPDH effect on DNA and apoptosis. Our results suggest that stimulation of GAPDH/Ape1 axis is a novel potential anti‐atherosclerotic therapy.Support or Funding InformationStudy was supported by NIH/NHLBI 1R01HL142796‐01A1 (SS), AHA Grant‐in‐aid (SS) and NIH/NHLBI 7R01HL070241 (PD).