bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Lorcaserin on Opioid‐Induced Observable Behavior in Rhesus Monkeys
Authors: Borgatti Daniel , Bergman Jack , Kohut Stephen
Published: April 2020

Serotonergic ligands that target the 5HT2‐family of receptors, such as the 5HT2C receptor agonist lorcaserin, have been shown to decrease several of the abuse‐related effects of opioids, including self‐administration and relapse‐related behavior. This suggests that lorcaserin may be an effective medication for the management of opioid use disorder. However, the impact of 5HT2C agonists on behaviors relevant to opioid withdrawal have not been reported. The objective of the present study was to determine whether lorcaserin alters behavioral signs indicative of opioid withdrawal in morphine‐dependent nonhuman primates. Adult rhesus monkeys (N=4; 3 males, 1 female) with histories of drug self‐administration received twice daily (0930 and 1700‐hr) morphine injections (intramuscularly) for a total daily dosage of 9.0 mg/kg/day over a 6–8‐month period. Behavioral observations were conducted by a blind observer over a 6.5 hr period on test days conducted twice per week. Spontaneous withdrawal was elicited by replacing the 0930‐hr morphine injection with saline while precipitated withdrawal was elicited by administering naltrexone (0.0032 and 0.01 mg/kg/inj) 3‐hr after morphine injections; test drugs were administered 15 min after saline or naltrexone. Results show that saline produced a time‐dependent increase in observable behavior throughout the observation period while morphine (1.0 and 4.5 mg/kg) dose‐dependently attenuated observable signs compared to saline, with the highest dose nearly eliminating withdrawal signs. Lorcaserin (0.3 and 1.0 mg/kg/inj) produced a modest decrease in observable signs (~30%) at the lowest dose but was ineffective at the higher dose. Naltrexone produced approximately 30% more observable signs compared to saline, particularly at the first timepoint (30 min), but this effect gradually dissipated over 3hr. Lorcaserin (1.0 mg/kg) attenuated the initial increase but was otherwise similar to naltrexone alone. Overall, these data suggest that lorcaserin does not appreciably alter opioid withdrawal signs.

Support or Funding Information

Supported by NIH grant R01DA047130
Genetics in Action: Mutation of Chromoproteins in First‐Semester Introductory Biology
Authors: Walsh Susan Jennifer
Published: April 2020

In contrast to fluorescent proteins, chromoproteins provide a quick, reliable way to assess phenotype in visible light. In this course‐based undergraduate research experience (CURE), students utilized two different strategies to mutate the chromoproteins aeBlue and tsPurple: either intentionally or randomly. After a journal club highlighting diversity in chromoproteins, the conservation of their chromophores, and the targeted mutation of amilCP, students designed their own primers for site‐directed mutagenesis of either the aeBlue chromophore itself or amino acids near the chromophore. As a complementary technique, students used isothermal, error‐prone PCR with manganese chloride to induce mutations in the tsPurple plasmid. Potentially mutated DNAs were transformed into bacteria, and candidates for mutation were selected based on a loss of color relative to bacteria expressing the parent vector. Students analyzed mutants by DNA sequencing and protein expression (via SDS‐PAGE) to verify how changes to protein sequence contributed to protein function. Based on the phenotypes from the random mutagenesis, students also identified novel mutations in the chromoprotein that prevented color development. Understanding of the project was assessed through laboratory notebooks, formal laboratory papers, and presentations. Overall, this project provided an engaging, straightforward, and affordable way to visualize the relationship between protein structure and function while teaching first‐year college students basic molecular biology techniques.

Support or Funding Information

Soka University of America
Evidence that Sympathetic Activity is Increased Selectively to the Heart in a Mouse Model of Hypertrophic Cardiomyopathy
Authors: Larson Robert A. , Lu Yongjun , Chapleau Mark W.
Published: April 2020

Hypertrophic cardiomyopathy (HCM), the most common genetic heart disease, is characterized by cardiac hypertrophy, fibrosis, and increased risk of arrhythmias and sudden death, even in the absence of heart failure. We have studied young (<20 wks) transgenic mice with cardiac‐specific expression of mutated alpha‐tropomyosin (Glu180Gly), an established model of HCM, to investigate autonomic mechanisms. In early experiments, abnormal heart rate and blood pressure responses to β‐adrenergic receptor blockade and ganglionic blockade indicated high cardiac accompanied by low vasomotor sympathetic tone in HCM vs. wild‐type (WT) mice. The aim of this study was to further characterize regional sympathetic activity in HCM vs. WT littermates by measuring expression/activity of tyrosine hydroxylase (TH, rate‐limiting enzyme for norepinephrine (NE) synthesis) and the norepinephrine transporter (NET) in sympathetic ganglia (stellate, superior cervical, celiac/mesenteric), and expression of β1 receptors in left ventricle (LV). Results (below) are expressed relative to WT (n=4 male mice each group). TH protein expression (Western blot) (standardized to neuronal content with PGP9.5) was significantly higher in stellate (cardiac) ganglia in HCM mice (HCM 162±12 vs. WT 100±7; p<0.05), but not significantly different in superior cervical (HCM 82±4 vs. WT 100±14; p>0.05) or celiac‐mesenteric (HCM 95±7 vs. WT 100±4; p>0.05) ganglia. TH/PGP9.5 protein expression was also higher in the LV of HCM (135±15) vs. WT (100±1) mice (p<0.05). LV β1 receptor protein expression was significantly lower in HCM (60±1) vs. WT (100±4) mice (p<0.05), consistent with increased local NE levels. NET protein (immunofluorescence) was lower in stellate ganglia from HCM mice vs. WT (n=3 mice per group, 3 sections/mouse, p<0.05). NET activity was assessed by measuring the rate of uptake of a fluorescent NET substrate (Molecular Devices) into individual sympathetic neurons isolated from stellate and superior cervical ganglia. NET activity measured in stellate neurons was significantly lower in HCM (4.5±0.3 a.u.*min−1, n=115 neurons, from 4 mice) vs. WT (5.3±0.4 a.u.*min−1, n=98 neurons, from 4 mice). NET activity in superior cervical ganglia did not differ between genotypes. In conclusion, increased TH expression (enhanced ‐NE synthesis) and decreased NET expression/activity (reduced NE reuptake) in stellate ganglia are consistent with increased cardiac sympathetic activity in HCM, whereas the lack of differences between HCM and WT in other sympathetic ganglia are consistent with normal sympathetic activity to non‐cardiac target organs in HCM.

Support or Funding Information

NIH F32HL140880
Experimental Investigation of Profibrotic Cell Signaling in Pulmonary Arterial Adventitial Fibroblasts
Authors: Wang Ariel , Valdez-Jasso Daniela
Published: April 2020

Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during pulmonary arterial hypertension (PAH). PAAFs can be activated into myofibroblasts that produce more extracellular matrix (ECM), which contributes significantly to pulmonary vascular remodeling and stiffening in disease progression. There are currently no therapies that reverse the matrix remodeling and vascular stiffening that occurs during PAH. In order to better identify treatments to target adventitial fibrosis, we sought to investigate the interplay between major pathways that have been reported to regulate myofibroblast transformation and ECM expression in PAAFs.

Compared with other fibroblast types, there is a scarcity of experimental data on mechano‐signaling in PAAFs, therefore more in vitro experiments are needed to determine which pathways are crucial to creating a systems biology model that describes mechanosignaling in PAAFs.

We have developed techniques to decellularize and reseed pulmonary arteries from Sprague‐Dawley rats to investigate the effects that purely the ECM have on the phenotypic transition of PAAFs. We surmise that pulmonary artery ECM alone from rats with PAH will have a significant impact on the transition of PAAFs into myofibroblasts and will investigate this difference using quantitative RT‐PCR. We will measure collagen I, collagen III expression, and alpha smooth muscle actin to measure the level of myofibroblast transition. In addition, levels of matrix proteins such as elastin and fibronectin will be measured via histology to characterize the level of fibrosis.

Combining these results with a profibrotic signaling network model, we can use these methods to identify potential combinatorial therapeutics to prevent or inhibit wall remodeling to treat PAH.

Support or Funding Information

This work was partially funded by the American Heart Association Scientist Development Grant 16SDG29670010, and the National Institutes of Health NHLBI 1 T32 HL105373 and NHLBI 1 R01 HL137100.
Defining the Regulatory Roles of Glucan Phosphatases in Transitory Starch Degradation
Authors: Mak Claudia , Raththagala Madushi
Published: April 2020

Plants generate starch during the day and metabolize it at night to store and use for cellular energy. The glucan phosphatase Starch EXcess4 (SEX4) plays a crucial role in transitory starch degradation via its involvement in the reversible starch phosphorylation mechanism. Previous structural studies have shown that SEX4 preferentially binds to and dephosphorylates starch at the C6 position of glucosyl residues, but there remains little data on how SEX4 navigates and interacts with the complex starch granule. Further, enzyme kinetics and regulatory mechanisms of SEX4 are currently not well understood. We employed both generic and substrate specific phosphatase assays to determine kinetics and substrate binding mechanisms of SEX4. Upon use of a generic phosphatase substrate, p‐nitro phenyl phosphate (p‐NPP), SEX4 followed Michaelis‐Menten kinetics. A sigmoidal curve observed for the physiological substrate amylopectin suggests a non‐ Michaelis‐Menten behavior for SEX4 and provides experimental evidence for a possible allosteric regulatory mechanism of SEX4. The enhanced dephosphorylation rates obtained for generic phosphatase assays with SEX4 preincubated with different domains of starch structure strongly suggest a conformational change within the active site of SEX4 upon glucan binding. Collectively, our finding suggests a novel posttranslational regulatory mechanism of SEX4 that is necessary for the regulation of transitory starch degradation at night.
Oroxylum indicum extract prevents chemotherapy‐induced cognitive impairment in mice
Authors: Pondugula Satyanarayana R. , Majrashi Mohammed , Almaghrabi Mohammed , Abbott Kodye L. , Govindarajulu Manoj , Ramesh Sindhu , Gill Kristina , Fahoury Eddie , Narayanan Natasha , Desai Darshini , Nadar Rishi , McElroy Edwin , Moore Timothy , Nagabhushanam Kalyanam , Majeed Muhammed , Dhanasekaran Muralikrishnan
Published: April 2020

Chemotherapy‐induced cognitive impairment (CICI) is an emerging concern among cancer patients undergoing chemotherapy treatment. Several mechanisms including neuronal oxidative stress and mitochondrial dysfunction have been proposed as the underlying mechanisms of CICI. While a few synthetic and natural compounds with different mechanisms of action have been proposed for targeting the CICI, their beneficial effects are outweighed by severe adverse effects including reduced efficacy of chemotherapy drugs. Therefore, search for novel and safer neuroprotective avenues to target CICI is needed. We sought to determine whether Oroxylum indicum extract (OIE, Sabroxy®) prevents CICI in a rodent model by suppressing chemotherapy induced neuronal oxidative stress and mitochondrial dysfunction. Male C57BL/6J mice were injected intraperitoneally with saline or doxorubicin (2mg/kg) and cyclophosphamide (50mg/kg) once a week for 4 weeks. While control mice received powdered rodent food alone, OIE treatment group received the powdered rodent food mixed with OIE (250 or 500mg/kg) daily for 4 weeks. After the treatment period, the mice were assessed for cognitive function using novel object recognition, Y‐maze, and Morris water maze analyses. Biochemical assays were performed using brain tissues to determine the underlying mechanisms of CICI. While the chemotherapy treatment impaired the performance of the mice in the novel object recognition, Y‐maze and Morris water maze tests, OIE significantly prevented the chemotherapy impaired performance in these tests. Additionally, OIE treatment prevented the chemotherapy‐induced changes in oxidative stress and mitochondrial dysfunction in the cortex and rest of the brain. Mechanistically, OIE enhanced mitochondrial function and counteracted chemotherapy‐increased reactive oxygen species formation and lipid peroxidation. Furthermore, OIE treatment increased the monoamine oxidase activity in the cortex and rest of the brain. Our results support the hypothesis that neuronal mitochondrial dysfunction induced by doxorubicin and cyclophosphamide treatment is an underlying cause of CICI, and that OIE may be offered as an adjuvant therapeutic strategy to treat CICI.

Support or Funding Information

OIE (Sabroxy®) and funding was provided by Sabinsa Corporation.
Evaluation of Novel Epibatidine Analogs in Rats Discriminating Nicotine
Authors: Wilkerson Jenny , Felix Jasmine , Restrepo Luis , Reeves Morgan , McMahon Lance
Published: April 2020

Tobacco use is a persistent public health issue. One of every three cancer deaths is linked to tobacco use, including nearly 90% of all deaths related to lung cancer. Nicotine is the primary psychoactive component in tobacco, and nicotine has been identified as the primary compound responsible for maintaining tobacco dependence in humans. Drug discrimination is a pharmacologically selective bioassay, utilized in the past for elucidating the receptor pharmacology of nicotine. If a test compound shares discriminative stimulus effects with nicotine, then it might serve as an effective substitution pharmacotherapy. Epibatidine reliably substitutes for nicotine in the drug discrimination assay, but has a side‐effect profile that limits its therapeutic potential. Thus, considerable efforts are underway to produce derivatives of epibatidine. Here we tested three epibatidine derivatives, 2′‐fluoro‐3′‐(4‐nitrophenyl)deschloroepibatidine (RTI‐7527‐102; i.e., RTI‐102), 2′‐fluorodeschloroepibatidine (RTI‐7527‐36; i.e., RTI‐36), and 3′‐(3″‐dimethylaminophenyl)‐epibatidine (RTI‐7527‐76; i.e., RTI‐76). A total of 4 male and 4 female Sprague‐Dawley rats were trained on a fixed‐ratio 10 schedule to discriminate nicotine (0.32 mg/kg base) from vehicle. All compounds dose‐dependently substituted for nicotine, without significant decreases in response rates. The rank‐order potency in the discrimination assay was RTI‐36 > nicotine > RTI‐102 > RTI‐76. There is abundant evidence that the α4β2* subtype is of particular importance to the abuse potential of nicotine. Thus, we examined the relative contribution of the β2 subunit with the antagonist by dihydro‐β‐erythroidine (DHβE). DHβE antagonized the discriminative stimulus effects of nicotine. However, relative to antagonism of nicotine, DHβE produced less antagonism of RTI‐102, RTI‐36 and RTI‐76. Thus, it is likely that RTI‐102, RTI‐36 and RTI‐76 possess differing activity at nicotinic receptor subunits.

Support or Funding Information

This work was supported by the National Institutes of Health National Institute on Drug Abuse [DA25267] and [DA48353].
Construction of the mycobacterium phage Giles genome through transformation‐associated recombination (TAR) cloning in Saccharomyces cerevisiae
Authors: Pozsgai Riana , Laverty Carly , Roecklein-Canfield Jennifer
Published: April 2020

The rise of antibiotic resistance in pathogenic bacteria has lead to a renewed interest in bacteriophages. These naturally occurring antimicrobial agents can potentially be exploited as novel therapeutic agents. Existing limitations such as bacterial resistance and narrow host range; however, are problematic. The advent of new techniques in genome science and synthetic biology have provided mechanisms to build unique phage genomes that confer new functions that may overcome these difficulties. Building upon methods developed in the Build‐a‐Genome course at Johns Hopkins University and the Synthetic Yeast Project (
www.syntheticyeast.org), we present the complete synthetic genome of the mycobacterium phage Giles. We adapted the novel assembly line approach introduced by Oldfield et al in 2017. We set out to construct the complete genome from 12 overlapping DNA fragments of the phage using the yeast based transformation‐associated recombination (TAR) system. Individual mini‐chunks of DNA were incorporated via polymerase chain reaction (PCR) with a TAR cloning vector using specifically designed primers. The DNA fragments were then assembled into the full length phage genome inside yeast. Genetic modifications were attempted by inserting novel DNA sequences into one or more fragments. The final objective of this project is to transform the synthetic phage into bacterial cells and measure its infectivity. The results of this project are expected to show successfully engineered and infectious clone of phage Giles. The significance of which will be to demonstrate the optimization of novel methods to construct a synthetic phage genome.
Comparing the DNA‐Binding Specificity of Glucocorticoid and Androgen Receptors
Authors: Henze Tyler , Boulanger Kyle , Pufall Miles A. , Hall Bonnie L.
Published: April 2020

Prostate cancer is the second most common cancer among men, with an estimated 175,000 diagnoses expected in 2019. Androgen hormones fuel prostate tumor growth through the actions of the androgen receptor (AR), a member of the nuclear hormone family of transcription factors. Therapies for prostate cancer include surgery and inhibition of AR with antagonists. Patients, however, often relapse with castration‐resistant prostate cancer (CRPC) in which the cancer grows in the absence of AR ligand action. In some cases, the need for androgens is subverted by overexpression of the AR‐related glucocorticoid receptor (GR). GR effectively replaces AR by regulating genes that drive prostate growth and survival. We have previously used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method along with the SelexGLM computational algorithm to demonstrate that GR and AR have similar, but distinct DNA sequence specificities. Upon overexpression, GR is able to bind genomic loci once preferentially bound by AR and regulate associated genes. To better understand differences in AR and GR DNA recognition, we are now measuring DNA sequence preferences of both AR and GR using the No Read Left Behind (NRLB) method. NRLB yields high resolution models of specificity after just one round of selection, allowing rapid screening of both wildtype and hybrid AR and GR DNA binding domains. We have over‐expressed and purified both the AR and GR DNA binding domains, and are designing hybrid mutants. Exploring the similarities and differences between AR, GR and the hybrid molecules should help us understand how GR might be playing a role in CRPC.

Support or Funding Information

• Grant: NSF:CAREER (MCB‐1552862) to M.A. Pufall
Identifying Post‐translational Regulatory Strategies of Glucan Phosphatases in Transitory Starch Metabolism
Authors: Raththagala Madushi
Published: April 2020

Plant glucan phosphatases remove phosphates from the outer most layers of the starch granule, which is already partially solubilized and phosphorylated by glucan dikinases. Thus, the activity of glucan phosphatase is at an interface between semi‐crystalline starch structure and soluble chains of amylopectin. Recent structural studies of glucan phosphatases bound to a soluble glucans have provided important insights into how glucan phosphatase family of enzymes engage with the linear portion of the amylopectin chains of the complex starch structure. However, our understanding of how these enzymes interact with the insoluble or partially soluble semi‐crystalline starch granule is not clear. Using enzymatic assays coupled with Differential Scanning Fluorometry (DSF) and X‐ray crystallographic studies, we have identified the physical mechanisms glucan phosphatases utilize to bind, activate and regulate their enzymatic activity at the starch granule. In this work, we also investigate how glucan phosphatases respond to the metabolic and energy changes during day and night. The findings explore novel posttranslational regulatory mechanisms of glucan phosphatase family of enzymes in transitory starch metabolism.
Essential Anatomy for General Internal Medicine in the Undergraduate Medical Curriculum
Authors: Latacha Kimberly , Keim Sarah , Harmon Derek , Marzban Hassan , Lopez Lisa , Topping Daniel , Royer Danielle , Carpenetti Tiffany , Downs Mary Beth , Davies David , Harrell Kelley , Brooks William , Cotter Meghan , McNary Thomas , Mork Amy , Wineski Lawrence , Lewis Steven , Giannaris Eustathia Lela , Farias Anna , Martindale Jim , Hankin Mark
Published: April 2020

Introduction

To prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.

Aim

To address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in Internal Medicine clerkships and electives to identify the anatomy they consider essential.

Methods

An IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the percentage of Internal Medicine clinical educators (clerkship/elective directors and attending physicians) that considered each anatomical region important to their specialty. Second, the study examined the rank assigned to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).

Results

At the time of abstract submission, data had been collected from 31 General Internal Medicine clinical educators at 16 medical schools and 43 Hospitalist Internal Medicine clinical educators at 16 medical schools. The percentage of clinical educators that considered each anatomical region important to their specialty were (highest‐to‐lowest): General Internal Medicine – Back (97%), Upper Limb (83%), Lower Limb (79%), Thorax (79%), Abdomen (79%), Pelvis & Perineum (76%), Head & Neck (79%); Hospitalist Internal Medicine – Back (87%), Abdomen (80%), Thorax (82%), Head & Neck (75%), Lower Limb (75%), Pelvis & Perineum (75%), and Upper Limb (61%). Further data analysis has identified the highest ranked anatomical topics in each body region for the General Internal Medicine and Hospitalist Internal Medicine clerkships/electives.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by General and Hospitalist Internal Medicine clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
Homogenization Does Not Alter nor Remove Post‐Translationally Modified Phosphorylations
Authors: White Taylor
Published: April 2020

Post‐translational modification (PTM) describes the covalent and generally enzymatic modification of proteins during or after translation. Proteins undergo PTMs to form the mature protein product and are important components in cell signaling. For example, protein phosphorylation is a reversible post‐translational modification in which a phosphate group is covalently added to an amino acid residue by a protein kinase. Phosphorylation alters the structural conformation of a protein, which activates, deactivates, or modifies its function and is a target for many studies. Biological tissues are routinely homogenized in order to extract proteins, despite the concerns that the forces produced through homogenization may alter or remove PTMs. Here, using murine heart and kidney samples, we show that tissue homogenization of tissue does not alter nor remove post‐translationally modified phosphates. Furthermore, we show how detergents and sonication alter post‐translationally modified phosphate when compared to homogenization.

Support or Funding Information

Omni International, Inc

935 Cobb Place Blvd

Kennesaw Ga 30144
“Genetic Predisposition to Type II Diabetes in the Hispanic Population in Relation to SNP rs7901346 on the TCF7L2 Gene”
Authors: Dominguez Jhonathan , Curiel Alejandro , Silverio Rachel , Ramirez Alexa , Calleja-Macias Itzel Evelyn
Published: April 2020

The number of people living with Diabetes today is estimated to be around 100 million within the United States. From those 100 million, the ethnic category that is the third highest in Type II Diabetes is the Hispanic Population that is at 12.1% in comparison to American Indians/Alaska Natives who are 15.1%. This disease has been found to have a genetical predisposition and has become a leading cause of death in the United States in the Hispanic population. Since there is a diverse genetic variation between different ethnicities, scientists study genes through SNPs which are single nucleotide polymorphisms, in order to locate a specific gene that is associated with a disease. Recent studies with SNP 7901346 have shown that the gene TCF7L2 has been associated with Type II Diabetes. These risk alleles increase TCF7L2 gene expression in the pancreatic B‐cell which causes a reduction in insulin which therefore leaves the patient vulnerable to obtain diabetes. Hence, the goal of this research project is to find a prevalence of SNP 7901346 in a Hispanic population of the south of California. DNA extraction from buccal samples were done followed by PCR and confirmed by gel electrophoresis. SSP1 Restriction enzyme was used to identify both variants. The data shows the C allele was found dominant in both groups: patients and controls. The allele T was found it in one diabetic patient. It is too early to make a conclusion for the association of this SNP and its relation to Diabetes type 2 in the Hispanic population. Further research must be done increasing the sample to improve our results.
Role of Protease‐activated receptors in Activated protein C‐mediated anti‐inflammatory responses in endothelial cells
Authors: Birch Cierra A. , Molinar-Inglis Olivia , Lin Ying , Trejo JoAnn
Published: April 2020

Endothelial dysfunction is a hallmark of inflammation is and associated with the pathogenesis of vascular diseases induced by infections, oxidative stress or mechanical stress. This results in endothelial cell activation, disruption of endothelial barrier function, sensitivity to apoptosis and organ failure. While there are very limited options for treating diseases associated with endothelial dysfunction (e.g. sepsis, cardiovascular diseases), Activated protein C (APC) has shown pharmacological benefits in various preclinical settings. APC mediates multiple cytoprotective effects including 1) anti‐inflammatory responses, 2) stabilization of the endothelial barrier, and 3) anti‐apoptotic responses. Protease‐activated receptor‐1 (PAR1), a G protein‐coupled receptor (GPCR), is the central mediator of APC cellular signaling. However, there is limited understanding of the signaling pathways by which APC/PAR1 and APC/PAR1/PAR3 promote endothelial anti‐inflammatory responses. The long‐term goal of these studies is to understand the pathways by which the endothelium can resist inflammation and disruption to facilitate the advancement of new targets for therapeutic development. The subsequent studies aim to provide key mechanistic insight into how PAR1 and PAR3 drive endothelial anti‐inflammatory responses. Endothelial cytoprotection is driven by APC activation of PAR1 through cleavage at an N‐terminal arginine (R)‐46 site, that is distinct from the canonical thrombin cleavage site at R41. This results in the generation of a tethered ligand that promotes PAR1 biased signaling. We hypothesize that different co‐receptors including PAR3 and S1PR1 distinctly regulate specific APC/PAR1 anti‐inflammatory responses by influencing distinct beta‐arrestin signaling pathways. Here, we will examine the requirement of PAR1, co‐receptors PAR3 and S1PR1, and beta‐arrestin adaptor proteins to modulate APC‐mediated inhibition of TNF‐alpha pro‐inflammatory signaling in endothelial cells. Other studies aimed at elucidating additional mechanisms of APC‐mediated anti‐inflammatory responses, including post‐transcriptional control of pro‐inflammatory genes.

Support or Funding Information

Burroughs Welcome Fund: Post Doctoral Enrichment Program award

NIH/NHLB T32 Cardiovascular Physiology and Pharmacology Training Grant ‐NIGMS

R01GM116597 (Trejo)
“Dementia Biomarker Discovery Through Differential Immune Response Analysis”
Authors: Gaxiola Christian , Reyes Jorge , Glabe Charles
Published: April 2020

Alzheimer’s disease (AD) is the most common cause of dementia and it is estimated that 13.5 million people will be living with AD by 2050. AD begins years to decades before any symptoms begin to first appear in most patients which calls for the need of diagnostic tools that allow early detection. The goal of this research was to test and compare the immunological responses between individuals with dementia and healthy individuals by means of antibody immunoprecipitation and computational analyses. Phage display was performed to isolate the DNA of phages that bind to the antibodies in human serum from both dementia and healthy individuals. The isolated phage DNA was sequenced by NGS. Bioinformatics was performed to find a differential immunological response between dementia patients and healthy individuals to possibly discover a biomarker for early detection of AD. Early detection of AD can allow for better treatment opportunities before progression of cognitive degeneration.
SPARC’s Open Online Simulation Platform for Computational Modeling of the ANS’s Physiological Role and its Modulation by Electroceutical Devices: o2S2PARC
Authors: Neufeld Esra , Chavannes Nicolas , Zhuang Katie Zhi , Cassara Antonino , Lloyd Bryn , Crespo-Valero Pedro , Guidon Manuel , Maiz Odei , Anderegg Sylvain , Pascual Ignacio , Kainz Wolfgang , Kuster Niels
Published: April 2020

The NIH SPARC initiative aims to “transform the understanding of nerve‐organ interactions” and “advance the neuromodulation field towards precise disease treatment.” To this end, a freely‐accessible online platform (o2S2PARC) has been designed to complement and leverage in vitro and in vivo physiological experiments with computational modeling. Computational models validated on existing data, can be used to investigate manifold subjects including organ electrophysiology, neural dynamics and electroceutical device interactions.

At the core of o2S2PARC are detailed human and animal models, which include functionalized and dynamic peripheral nerves. These models serve as integration centers for simulations and measurement data and as contexts for physical modeling. The o2S2PARC platform also allows users to create, host, connect, share and execute simulations without requiring any software other than a web browser. A full‐stack system production version of the platform has been implemented and deployed in the cloud. It includes a scalable network of computational services deployed inside Dockers, essential solvers (e.g., for coupled electromagnetic‐neuronal dynamics modeling) and a flexible, user‐friendly graphical user interface. In addition, users can select from feature‐rich post processing services including 3D visualisations, plotters, and the possibility to include the user’s own data analytics tools.

Selected models from SPARC teams (cardiac physiology & regulation, compound action potential prediction, enteric nervous system) can now be freely accessed and executed through the SPARC data portal website. In addition, solvers implemented on the platform have already successfully applied to study a range of therapeutic applications, such as vagus nerve stimulation. In short, o2S2PARC has established a freely accessible, extendible, and intuitive online simulation platform for neuromodulation that facilitates the integration of organ models and simulation of nerve‐organ interactions within the complex environment of the body. The platform is constantly progressing and amassing collaborators, becoming an important contribution for translational research.

Support or Funding Information

National Institutes of Health SPARC award OT3OD025348
The Enzymatic Function of KVβ2 Contributes to Resistance Artery Vasodilation
Authors: Raph Sean M. , Bhatnagar Aruni , Nystoriak Matthew A.
Published: April 2020

Acute increases in tissue metabolic activity require concurrent enhancement of blood flow to meet the added demand for oxygen and energetic substrates. The cytosolic levels of nicotinamide adenine dinucleotides reflect cellular metabolic status and changes in intracellular NAD(P)(H) are associated with altered blood flow, although the contributing mechanisms are unknown. Smooth muscle voltage‐gated potassium channels (KV) are key regulators of vascular tone and consist of Kvα pore‐forming subunits in association with intracellular Kvβ proteins, which belong to the aldo‐keto reductase (AKR) superfamily. It is currently unclear whether the AKR enzymatic properties of KVβ proteins contribute to the regulation of resistance arterial tone. Here, we tested the hypothesis that resistance artery vasodilation to elevated smooth muscle [NADH]i:[NAD+]i requires the AKR function of Kvβ2. Using ex vivo pressure myography, we found that fourth order mesenteric arteries preconstricted with the thromboxane analogue U46619 (100 nM) exhibit robust vasodilation in response to application of lactate (e.g., 40±7% at 20 mM), used to acutely elevate [NADH]i:[NAD+]i. Vasodilation to lactate was not affected by endothelial denudation, but was abolished by either preconstriction with elevated external K+ (60 mM) or application of the KV1‐selective inhibitor psora‐4 (500 nM), suggesting a role for KV1‐mediated smooth muscle hyperpolarization. To determine whether KVβ proteins contribute to lactate‐induced vasodilation, we compared the dilatory responses in mesenteric arteries from either Kvβ1‐null (Kvβ1.1−/−) or Kvβ2‐null (KVβ2−/−) mice with those from strain‐matched wild type (wt) mice. Whereas lactate‐induced dilations were not significantly impacted by loss of KVβ1.1, these effects were abolished by ablation of KVβ2 (Kvβ2−/−: 3±6%, wt: 40±7%; p=0.0025). Conversely, no differences in dilation to the classical vasdilatory agent adenosine (100 uM) were observed between the groups. We next tested whether vasodilation to increases in [NADH]i:[NAD+]i require the catalytic function of Kvβ2 by using arteries from genetically modified mice in which the active site tyrosine of KVβ2 is mutated to phenylalanine (KVβ2Y90F), which abolishes catalytic function. Remarkably, similar to our observations in arteries from KVβ2−/− mice, we observed that lactate‐induced dilations were eliminated in arteries from KVβ2Y90F mice (KVβ2Y90F:1±2%, wt: 40±7%; p=0.0006). Taken together, our data suggest that the AKR catalytic function plays a functional role in resistance artery vasodilation upon changes in intermediary metabolism and altered smooth muscle [NADH]i:[NAD+]i.

Support or Funding Information

This work was supported by the University of Louisville, Department of Pharmacology and Toxicology, Integrated Programs in Biomedical Sciences (IPIBS) and the Diabetes and Obesity Center, HL142710, GM103492.
Effect Of Peppermint Oil On The Gastric Restriction Model With Phytobezoar In Wistar Rats
Authors: Carioca Levi Costa , Campos Leticia Cavalcante , Rocha Paulo Victor Pinheiro , de Menezes Victor Matheus Lean , de Aguiar Lemmuel Fagnus Linhares , Pires Barbara Barbosa , Barbosa Maria Lucianny Lima , da Siva Francisco Rafael Oliveira , Cerqueira Gilberto Santos
Published: April 2020

Obesity is a public health problem and many patients resort to bariatric surgeries as a form of treatment. However, surgical weight loss mechanisms, histopathological changes and long‐term consequences of bariatric surgery remain unclear. Among the main histopathological changes in the intestine we have increased expression of neutral mucins, acid mucins, caliciform cells and collagen in animals submitted to the gastric restriction model. Currently there is no therapeutic tool to reduce histopathological and oxidative changes promoted by gastric restriction surgeries. Based on these premises, the aim of this work was to investigate whether Peppermint essential oil reduces histopathological and oxidative changes in the experimental gastric restriction model. Adult male Wistar rats weighing between 250 and 400 grams were used. The animals were submitted to surgical gastrostomy and a cylindrical natural sponge (1.5 cm in diameter) was inserted into the stomach. The rats were separated into 3 groups, namely: Control (treated only with saline), Sham (surgical procedure only) and treatment (peppermint oil 100mg/Kg orally). Weight, water and food consumption were measured daily. On the last day all animals were euthanized and had the biological material collected (stomach, duodenum and jejunum) for histopathological evaluation and malondialdehyde (MDA) concentration in tissue. It was found that peppermint oil was able to reduce histopathological changes caused by the phytobenzoar in duodenum and jejunum, as well as to reduce the concentration of MDA in the jejunum (Figure 2). Regarding feed intake, the control group presented lower average consumption (98.7g/animal) when compared with Sham (134g/animal) and treatment groups (132g/animal). Regarding the average weight of the animals, it was observed that there was no statistically significant difference. It was found that peppermint essential oil presented moderate protective effect on phytobenzoar‐induced gastric restriction and reduced the loss of the architecture of intestinal viles.
CYPs and complex transformations in bacterial gibberellin phytohormone biosynthesis
Authors: Peters Reuben
Published: April 2020

Gibberellins (GAs) are structurally complex diterpenoids that serve as important hormones in plants, with crucial roles in growth and development, but also in modulating defense. Beyond their endogenous production by vascular plants, GAs also are produced by plant‐associated microbes, both fungi and bacteria. We have recently elucidated the biosynthetic pathway by which bacteria produce GA, revealing convergent evolution, yet striking similarities in the underlying (bio)chemical transformations. We are currently engaged in mechanistic studies of the cytochrome P450 monooxygenases CYP112 and CYP114, which catalyze particularly complex reactions. CYP112 carries out ring contraction, with extrusion of aldehyde, in a reaction that requires a dedicated ferredoxin, while CYP114 mediates a coupled demethylation and lactone ring forming transformation that requires multiple sequential reactions. We have carried out labeling studies to probe the catalyzed reactions, spectral binding studies to examine substrate specificity, and have solved crystal structures for both CYPs in the presence and absence of substrates/stable intermediates. Our most recent results will be discussed.

Support or Funding Information

This work was supported, in part, by grants from the NSF (CHE‐1609917) and NIH (GM131885).
Spatiotemporal Control of ERK Activity by b2‐adrenergic Signaling
Authors: Kwon Yonghoon , Clark Mary J. , Keyes Jeremiah , Walters Geneva , Sunahara Roger K. , Zhang Jin
Published: April 2020

G‐protein coupled receptors (GPCRs) play vital roles in regulating physiological functions, such as sympathetic nervous regulation, and senses of vision and olfaction. Signaling of the β2‐adrenergic receptor (β2‐AR), which is a prototypical class A GPCR, has been proposed to control activation of ERK, subsequently regulating different processes including gene expression. However, the molecular mechanism of ERK activation downstream of β2AR signaling is not clearly understood. Here we investigate the molecular mechanism of β2‐AR mediated ERK activation using improved version of FRET‐based ERK biosensors targeted to various subcellular regions which enable us to visualize spatial and temporal regulation of ERK activity in living cells. Epinephrine stimulation leads to increases of ERK activity at the endosome and in the nucleus but not at the plasma membrane. An Nb37 nanobody that stables the active Gαsconformation enhances and prolongs the endosomal and nuclear ERK activity. We propose a new model that an endosome‐localized active β2AR recruits critical signaling components to facilitate ERK activation on the endosome. These findings provide a molecular basis for location selective activation of ERK by β2‐AR signaling.

Support or Funding Information

This work was supported by the National Institutes of Health (R01 DK073368 to J.Z.)
Effect of Sichuan Pickle Lactobacillus on Preventing Non‐Alcoholic Fatty Liver by Regulating Intestinal Function in Mice
Authors: Tan Fang , Zhao Xin , Liao Wei , Li Qin
Published: April 2020

NAFLD is a kind of liver disease with great harm. It is caused by excessive nutrition that leads to excessive weight gain and overweight. The incidence of NAFLD in obese people is much higher than that in normal weight people. Therefore, weight loss is the first choice for most NAFLD patients. Traditional Sichuan pickle is a kind of food made from pickled vegetables with a certain concentration of salt water, which is mainly fermented by lactic acid, acetic acid and alcohol. Lactic acid bacteria play a key role in its fermentation process. In this study, the NAFLD (nonalcoholic fatty liver disease) model of C57BL/6N mice was established by high‐fat diet, and the effect and mechanism of a Lactobacillus isolated from Sichuan pickle on the prevention of NAFLD by regulating the intestinal function of mice were observed. In this study, 16S rDNA technology was used for microbial identification. After establishing the NAFLD model, the serum, liver, epididymis, small intestine and feces of mice were detected by biochemical and molecular biological methods. The identification results showed that the target strain was a Lactobacillus plantarum strain, named as LactoLactobacillus plantarum CQPC11 (LP‐CQPC11). The in vivo results showed that LP‐CQPC11 could effectively reduce the body weight, liver weight and liver index of NAFLD mice. The results of serum test showed that LP‐CQPC11 could reduce the levels of ALT, AST, AKP, TC, TG, LDL‐C and increase the levels of HDL‐C in the serum of NAFLD mice (model group); meanwhile, LP‐CQPC11 could also reduce the cytokine levels of IL‐1β, IL‐4, IL‐6, IL‐10, TNF‐α and INF‐γ in the serum. The pathological results also showed that LP‐CQPC11 could improve the pathological changes of liver, epididymis and small intestine caused by NAFLD, and protect the body tissues. Further qPCR and Western blot results showed that LP‐CQPC11 could up regulate the mRNA and protein expression of LPL, PPAR‐α, CYP7A1, CPT1 and down regulate the expression of PPAR‐γ, C/EBP‐α in the liver of NAFLD mice; in addition, LP‐CQPC11 could also up regulate the expression of occludin, ZO‐1 and down regulate the expression of CD36, TNF‐α in the small intestine of NAFLD mice. The results showed that LP‐CQPC11 could reduce the level of Firmicutes and improve the level of Bacteroides and Akkermansia in the feces of NAFLD mice. At the same time, LP‐CQPC11 could also reduce the proportion of Firmicutes/Bacteroides and regulate the intestinal microecology. It can be seen that LP‐CQPC11 can improve the intestinal environment of NAFLD mice, so as to prevent NAFLD, and the effect is positively correlated with its dose.

Support or Funding Information

This work was supported by Research Project of Chongqing University of Education (KY2015TBZC) and Science and Technology Research Project of Chongqing Education Commission (KJZD‐K201901601), China.
Caveolin‐1 frame‐shift mutation induce changes in cellular morphology and function
Authors: Hertz Juan Pablo Zuniga , Patel Hemal H.
Published: April 2020

Caveolins, Cav1–3, are oligomeric proteins critical for the formation of membrane caveolae that regulates cell physiology. A frameshift mutation (Cav1F160X) in Cav1 (Cav1M) was described in a patient with a progeria‐like phenotype, which presents nuclear morphology alterations. We hypothesize that Cav1M presents dominant negative function that perturb cellular physiology, particularly nuclear morphology, mitochondrial function, exocytic behavior a plasma membrane structure. Transmission electron microscopy (TEM) of patient‐derived dermal fibroblasts (PCav1M) showed abnormal nuclear shape, increased rough endoplasmic reticulum, abnormal mitochondrial size/shape, increased number of autophagosomes and reduce number of membrane caveolae. Patient‐derive serum shown an increase in exosomes and extracellular vesicles, compared with the unaffected family members. PCav1M fibroblasts showed 4‐fold increase in exosome secretion; PCav1M present also increased exosome secretion compared with Hutchinson‐Gilford progeria patient dermal fibroblasts. Nanotracking and TEM analysis revealed a significant reduction in nanoparticle size in PCav1M‐derived exosomes. Human dermal fibroblasts (HDF) treated to express Cav1M or exogenously exposed to exosomes from PCav1M fibroblasts showed altered nuclear morphology, exosome secretion profile, proteomics, and miRNA analysis. Cellular membrane structure analysis by means of membrane fractions and electron paramagnetic spectroscopy shows an altered localization of proteins commonly found in caveolae fractions and increased membrane fluidity. Our results indicate that mutations in the C‐terminus of Cav1 lead to significant membrane alterations regulating exosome secretion and other cellular morpho‐functional features. Our findings identify a novel role of caveolin in linking plasma and nuclear membrane dynamics that have potential implications for cellular communication, impacting physiology and pathophysiology.

Support or Funding Information

National Institutes of Health (HL091071, AG052722) and Veterans Administration (BX001963).