bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationOmni International, Inc935 Cobb Place BlvdKennesaw Ga 30144
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 InformationBurroughs Welcome Fund: Post Doctoral Enrichment Program awardNIH/NHLB T32 Cardiovascular Physiology and Pharmacology Training Grant ‐NIGMSR01GM116597 (Trejo)
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 InformationNational 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 InformationThis 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.
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 InformationThis 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 InformationThis 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 InformationThis 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 InformationNational Institutes of Health (HL091071, AG052722) and Veterans Administration (BX001963).
A Semester Long Teaching Practicum For Graduate Students In Biomedical Sciences
Authors: Towse Theodore Francis , Thomas Derek 
Published: April 2020

IntroductionTeaching assistants are often given little or no guidance before receiving their first teaching assignment. As a result they feel underprepared and overwhelmed by the experience, which undoubtedly reduces their effectiveness in the classroom. Therefore we developed a one‐semester teaching practicum to better prepare new graduate students for the rigors of college teaching.PurposeTo provide students with a mentored experience in college teaching while simultaneously exploring topics in the pedagogy of college teaching.MethodsWe developed a 15‐week course for first year graduate students in the Department of Biomedical Sciences at Grand Valley State University. Student teaching assistants were assigned to shadow a faculty‐mentor teaching an introductory laboratory course in Anatomy and Physiology, or Microbiology. Students performed basic teaching duties including grading and assisting students during the lab. In addition they completed a curriculum, which included weekly readings and writing assignments on topics in college teaching pedagogy. Selected readings, links to external content, and writing reflections were managed through a course website on Blackboard.Learning ObjectivesAfter completing the course the student should be able to: critically read and apply current concepts in the pedagogy of college teaching, facilitate student‐centered learning activities, make effective use of visual aids in classroom instruction, plan, execute, and evaluate classroom instruction.Evaluation and AssessmentWeekly assignments included readings on topics in college teaching pedagogy ranging from – setting the right tone in the classroom on day one, managing the classroom, to developing a teaching philosophy. Each assignment required a brief writing reflection and a discussion with the faculty‐teaching mentor. As part of the course students completed two teaching demonstrations. The first teaching demonstration was video taped and later reviewed and evaluated by the student and faculty mentor. Both teaching demonstrations were graded and the grading rubric was provided to the student prior to the demonstration. The course culminated with the student drafting a statement of teaching philosophy.SummaryWe developed a 15‐week teaching practicum designed to provide students with a mentored experience in college teaching. The course was initially piloted with two graduate students in the Department of Biomedical Sciences at Grand Valley State University but could easily be scaled up to a larger cohort of students and different academic disciplines.
The Role of Innate Immunity in Dystrophic Diaphragm Pathology
Authors: Petrof Basil 
Published: April 2020

The pathophysiology of Duchenne muscular dystrophy (DMD) is complex and incompletely understood. However it is clear that the primary defect, lack of dystrophin, is necessary but not sufficient on its own to fully account for the onset of muscle fiber destruction, leading to eventual replacement by fibrotic tissue. Dystrophin restoration strategies are currently under clinical development, but the only treatment clearly shown to slow disease progression thus far in human DMD patients is the use of anti‐inflammatory corticosteroids. This presentation will focus on the role of the innate immune system in promoting the dysregulated inflammatory and aberrant muscle repair responses found in DMD. Using the mdx mouse diaphragm as a preclinical model due to its resemblance to the human DMD phenotype, the importance of monocyte‐derived macrophages in driving early disease progression will be outlined. This is exemplified by the therapeutic benefits (improved force generation and reduced fibrosis) noted when monocyte/macrophage recruitment to the dystrophic diaphragm is prevented through genetic or pharmacologic inhibition of the chemokine receptor CCR2. In addition, preventing activation of Toll‐like receptors (TLRs) is similarly beneficial and suggests significant involvement of damage‐associated molecular patterns (DAMPs) in DMD pathogenesis. Data will also be presented indicating that epigenetic alterations occur at the level of macrophage precursors in the bone marrow, which are likely stimulated by TLR4 recognition of DAMPs, and that subsequently play a role in dictating monocyte/macrophage phenotypic characteristics following their entry into the diseased muscles. Although the ideal treatment for DMD would be restoration of dystrophin to all muscles of the body including the diaphragm, significant hurdles remain for this ultimate therapy. In the meanwhile, there are serious drawbacks to the current practice of treating DMD children with corticosteroids, which have major side effects in DMD such as obesity, diabetes and osteoporosis. Future development of a more targeted therapeutic approach based on innate immune system modulation could represent an important advance in the field.Support or Funding InformationCanadian Institutes of Health Research