bluuebunny/crossref_metadata_2025_split huggingface dataset:
The potential role for Interferon regulatory factor 6 in keratinocyte adhesion and/or migration
Authors: Olson Leah C , Schutte Brian C , Murray Jeffrey C , Dunnwald Martine 
Published: June 2021

Mutations in Interferon Regulatory Factor (IRF6) cause two orofacial clefting syndromes, Van der Woude (VWS) and Popliteal Pterygium (PPS). In addition to an oral defect, recent data show that VWS patients are more likely to have wound complications after corrective cleft surgery than patients with isolated cleft. Thus we hypothesize that Irf6 is critical to wound closure. To test this, we used keratinocytes from Irf6 null mice. We created in vitro scratches and observed a significant delay in scratch closure. To determine the cause of delay, we examined the cellular structure of Irf6−/− keratinocytes by staining for filamentous actin and found that these cells have more stress fibers. In addition, we examined cellular adhesion by staining Irf6−/− keratinocytes for E‐cadherin and vinculin. We found that these cells form junctions, but cell‐cell contacts appear in an abnormal pattern, and there appear to be more focal adhesions. Finally, we analyzed proliferation of Irf6−/− keratinocytes and found no deficiency. Together, our data suggests Irf6−/− keratinocytes exhibit characteristics of less motile cells. Thus, we speculate that the defect in Irf6−/− keratinocyte scratch closure is due to aberrant migration and/or adhesion, and that cellular proliferation is not inhibitory to closure. Broadly, these data suggest that Irf6 is necessary for events critical to proper cutaneous wound healing.Grant Funding Source: NIH‐NIAMS AR 055313
Maternal Vitamin D Status and Child Tuberculosis, Anemia, and Morbidity in Tanzania
Authors: Finkelstein Julia L , Mehta Saurabh , Manji Karim P. , Duggan Christopher P. , Mugusi Ferdinand M. , Spiegelman Donna , Msamanga Gernard I. , Fawzi Wafaie W. 
Published: June 2021

ObjectiveTo examine the association of maternal vitamin D status with child health outcomes.MethodsVitamin D status [serum 25(OH)D] was determined in 884 HIV‐infected pregnant Tanzanian women at enrolment in a trial of vitamins. Children born to these women were followed‐up until 24 months of age and morbidity was recorded. Tuberculosis (TB) was diagnosed via chart review. Proportional hazards models were used to assess the associations of outcomes with maternal vitamin D status.ResultsChildren born to women with low vitamin D (<32ng/mL) levels were 1.58 times more likely to develop pulmonary TB (95%CI: 1.01–2.47), after adjusting for age, HIV stage, CD4 count, and regimen. Low vitamin D status was associated with 1.21 times greater risk of cough (95%CI: 1.05–1.40). No significant associations were noted for diarrhea or other respiratory symptoms. For anemia outcomes, no association was observed with low vitamin D status. However, in quintile analyses, children born to women in the second quintile had lower risk of severe anemia (Hb<8.5g/dL) (RR: 0.64; 95%CI: 0.45–0.92) and severe hypochromic microcytosis (RR: 0.34; 95%CI: 0.16–0.71), compared to the lowest quintile.ConclusionVitamin D status may affect risk of child outcomes, such as tuberculosis and anemia. Further research is warranted to examine the role of optimizing vitamin D status in reducing morbidities in children born to HIV‐infected women.Grant Funding Source: NICHD R01 32257
Specific starch digestion of maize alpha‐limit dextrins by recombinant mucosal glucosidase enzymes
Authors: Lin Amy Hui‐Mei , Nichols Buford L , Quezada‐Calvillo Roberto , Rose David R , Sim Lyann , Hamaker Bruce R 
Published: June 2021

Starch digestion requires two luminal enzymes, salivary and pancreatic alpha‐amylase (AMY), and four small intestinal mucosal enzyme activities from the N‐ and C‐terminals of maltase‐glucoamylase (MGAM) and sucrose‐isomaltase (SI) complexes. AMY is not a requirement for starch digestion to glucose but dramatically amplifies α‐glucogenesis by producing α‐limit dextrins (LDx) as favored substrates for the mucosal enzymes.Aimto study substrate specificity of α‐glucogenesis by four recombinant MGAM and SI subunits.MethodsRecombinant α‐glucosidase domains were produced in drosophila (N‐) or baculovirus (C‐) cell lines and purified by histags. LDx was produced with AMY from normal maize and three mutant cultivars with different amylopectin structures: normal, waxy, ae waxy and du waxy. Glucose released in vitro was measured by GOPOD.ResultsN‐MGAM subunit releases glucose from shortest linear oligomers while the other three subunits digest longer chains or branched molecules.ConclusionsThis is the first time that the individual α‐glucogenic activities of all four mucosal enzyme subunits have been tested. The individual α‐glucosidase subunits had different activities for each cultivar LDx substrate and selective affinities for LDx from different cultivars. Combinations of α‐glucosidase subunits thus provide a broader spectrum of α‐glucogenic activities from structurally varied starch LDx.
An investigation into the profile and dynamics of neutrophil transendothelial cell migration (TEM) using high resolution in vivo real‐time confocal imaging
Authors: Woodfin Abigail , Voisin Mathieu‐Benoit , Colom Bartomeu , Nourshargh Sussan 
Published: June 2021

A high resolution real‐time 3D imaging technique was established for analyzing the characteristics and dynamics of neutrophil transmigration in vivo. Our model involves rapid confocal microscopy imaging of the cremaster muscle of lys‐EGFP mice (which exhibit EGFP+‐neutrophils) in conjunction with fluorescently labeled endothelial cell (EC) junctions using an anti‐PECAM‐1 mAb. With this method we have examined the profile and dynamics of neutrophil TEM as elicited by several stimuli (eg IL‐1β, FMLP and ischemia/reperfusion injury). In all reactions the majority of the TEM events occurred via the paracellular route where the formation of EC junctional pores at bi‐ and tri‐cellular junctions could be clearly imaged and analyzed in terms of their frequency (no significant difference noted between bi‐ and tri‐cellular junctions) and duration (6 ± 0.5 minutes in IL‐1β‐stimulated tissues). Clear evidence for the formation of transcellular pores was also obtained (9.3 ± 2.1 % for all reactions). Collectively the high spatial and temporal resolution of our approach has led to novel qualitative and quantitative analysis of neutrophil TEM in vivo.Funded by the Wellcome Trust (Ref: 081172/Z/06/Z to SN)
CORM‐3‐derived CO attenuates oxidative stress in vascular endothelial cells in SOD‐activity independent manner
Authors: Mizuguchi Shinjiro , Stephen Jancy , Katada Kazuhiro , Capretta Alfredo , Potter Richard F , Fraser Douglas F , Cepinskas Gediminas 
Published: June 2021

Our previous studies indicate that CO derived from CO‐releasing molecule, CORM‐3, while suppressing PMN transendothelial migration at the same time amplifies oxidative stress in PMN, whereas oxidative stress in vascular endothelial cells (HUVEC) is effectively suppressed by CORM‐3. However, the mechanisms of such opposing effects of CO with respect to ROS production are unknown.In this study we assessed the effects of CORM‐3‐derived CO on modulation of the antioxidant enzyme, superoxide dismutase (SOD), activity in an in vitro model of endotoxemia. To this end, HUVEC were stimulated with LPS (1μg/ml) for 1–4h in the presence or absence of CORM‐3 (100μM) and assessed for production of ROS, activation of NFκB and expression of adhesion molecule ICAM‐1. In parallel, SOD activity in cell lysates (cell‐associated SOD) and supernatants (soluble SOD) were also assessed.The obtained results indicate that LPS‐induced increase in ROS production, activation of NFκB and ICAM‐1 expression were effectively reduced by CORM‐3. Interestingly, CORM‐3‐dependent suppression of ROS production was not associated with increased SOD activity. On the contrary, CORM‐derived CO attenuated SOD activity in both, cell lysates and supernatants, and in an assay employing purified SOD1 enzyme, indicating that CORM‐3‐derived CO negatively regulates SOD activity in cultured vascular endothelial cells (HSFO‐NA6171; IRF‐025‐09).
Biological Insights From Quantitative Analysis of Tyrosine Kinase Signaling Networks
Authors: White Forest M 
Published: June 2021

To effectively monitor protein phosphorylation events governing signaling cascades, we have developed a mass spectrometry‐based methodology enabling the simultaneous quantification of tyrosine phosphorylation of specific residues on dozens of key proteins at multiple time points under a variety of perturbations. We have recently applied this technique to identify key signaling nodes regulating EGFR, Insulin Receptor, and T Cell Receptor signaling network response to stimulation.We have also performed an in‐depth characterization of the network‐wide effects of single‐point mutations on the oncogenic, constitutively active, EGFRvIII receptor tyrosine kinase. This study quantifies the adaptive capacity of cellular signaling networks and the pleiotropic effects of knocking out individual phosphorylation sites with the network. Computational analysis of the phosphorylation data relative to phenotypic data highlights the role of selected protein phosphorylation sites in regulating biological outcome. We also demonstrate the utility of a data‐driven computational model that is capable of fully describing glioblastoma cell growth based on just 10 phosphoproteins. Overall, we have now demonstrated that the combination of mass spectrometry‐based analysis of protein phosphorylation with phenotypic measurements and computational modeling yields novel insights into the regulation of cellular signaling on a network scale.
Dynamics of PKA Signaling
Authors: Taylor Susan S. 
Published: June 2021

The protein kinases represent one of the largest mammalian gene families and regulate in diverse and critical ways most biological processes in all eukaryotic cells. cAMP‐dependent protein kinase (PKA) has served as a prototype for understanding the structure and function of this protein kinase superfamily. The PKA catalytic subunit is assembled into an active conformation by phosphorylation of its activation loop where this single phosphate has profound effects on global structure and function. All active protein kinases share a common architecture that is built upon two hydrophobic spines comprised of residues from both the small (N‐) and large (C‐) lobes. These spines are anchored to the hydrophobic F Helix, which traverses the large helical C‐lobe. The regulatory spine is typically assembled by phosphorylation of the activation loop while the catalytic spine is completed by the adenine ring of ATP. The active kinase then opens and closes as it navigates through the catalytic cycle. The PKA C‐subunits are regulated by dimeric regulatory (R) subunits, which form an inactive tetrameric complex in the absence of cAMP. Catalytic activity is unleashed by cAMP binding with high affinity to the R‐subunits. Structures of R‐subunit monomers bound to the C‐subunit demonstrated that the R‐subunit undergoes major conformational changes as it releases cAMP and binds to the C‐subunit. The cooperative binding of cAMP then releases the C‐subunit. Specificity in PKA signaling is achieved in part by the isoform diversity of the regulatory subunits and by targeting of the holoenzymes to specific sites by A Kinase Anchoring Proteins (AKAPs). The AKAP docking site is localized to the N‐terminal dimerization/docking (D/D) domain of the R‐subunits, a four helix bundle that binds with high affinity to an amphipathic helix in the AKAP. Structures of the D/D domains from RIIα and RIα bound to the same AKAP peptide from D‐AKAP2 reveal an isoform‐specific code for binding to AKAPs. Crystallization of tetrameric holoenzymes, coupled with small angle Xray an neutron scattering, demonstrates how full length holoenzymes are assembled and anchored to AKAPs and reveals for the first time the full allosteric potential of PKA signaling.Support for this work was provided by NIH DK54441, GM19301, and GM34921.
Identification of anti‐inflammatory constituents in Hypericum perforatum and Hypericum gentianoides extracts using mouse macrophages
Authors: Huang Nan , Hauck Cathy , Rizshsky Ludmila , Murphy Patricia A. , Nikolau Basil J. , Birt Diane F. 
Published: June 2021

Ethanol extracts from eleven species and accessions of Hypericum were screened for their inhibitory properties on LPS‐induced prostaglandin E2 (PGE2) and nitric oxide (NO) in RAW264.7 mouse macrophages. All extracts inhibited PGE2 production by 19%–45% at 10 μg/ml compared to vehicle control while all but one extract also decreased NO production by 10%–35%. No cytotoxicity was noted on any extract at 30 μg/ml. Among all extracts, those from H. perforatum and H. gentianoides demonstrated stronger overall inhibitory potency. HPLC analysis of the extracts revealed that H. perforatum extract contained pseudohypericin, amentoflavone, quercetin and chlorogenic acid; previously identified as a ‘4 component system’; while H. gentianoides extract contained acylphloroglucinols including uliginosin A. H. perforatum and H. gentianoides extracts were fractionated using semi‐preparative HPLC and several fractions inhibited both LPS induced PGE2 and NO production dose‐dependently. HPLC analysis of the most active fractions indicated that ‘4 component system’ could be the major contributor to H. perforatum activity and suggested that acylphloroglucinols were potentially involved in H. gentianoides activity. This study demonstrated significant differences between two Hypericum species with regards to anti‐inflammatory compounds and potential mechanism of action, which will be confirmed by further study.Grant Funding Source: P50 AT004155‐06 NCAAM/ODS from NIH
The effect of food viscosity on markers of appetite
Authors: Hollis James , Lamsal Buddhi , Zhu Yong 
Published: June 2021

Accumulating data indicate that food form can influence appetite. In this study, a cross‐over study design was used to determine the effect of food viscosity on markers of appetite. On two separate occasions, 20 male participants reported to the laboratory first thing in the morning following an overnight fast. They were asked to consume a bowl of one of two soups: normal viscosity or high viscosity. The soups contained acetaminophen as a marker of gastric emptying rate. After consuming the meal, subjective appetite measures were recorded at regular intervals (t0+15, 30, 45, 60, 90, 120 and 180 minutes) using visual analogue scales. At each time‐point, a blood draw was also made. Following the last appetite measurement, an ad libitum lunch meal was served. There was a significant main effect of viscosity on hunger, fullness, desire to eat and prospective consumption (p<0.05). There was a significant main effect of viscosity on plasma acetaminophen concentration (p<0.05). However, there was no significant difference in food intake at the subsequent test meal. Blood samples will be assayed for CCK and ghrelin. This study indicates that increasing the viscosity of a liquid meal can reduce postprandial feelings of hunger. This may be due, in part, to a reduced rate of gastric emptying. However, food viscosity has no effect on food intake at the next meal.This work was supported by a USDA/Iowa State University NWRC grant
Increased colonic iNOS gene expression from sustained Western‐style diet exposure
Authors: Zimmerly Ella M , Jang Hyeran , Sauer Julia , Choi Sang‐Woon 
Published: June 2021

Risk of colorectal cancer has been associated with inflammation. However, the proinflammatory effects of diet on colorectal carcinogenesis have not been elucidated. Overexpression of inducible nitric oxide synthase (iNOS) and cycloxygenase‐2 (COX‐2) are common to inflammation and colon cancer; their inhibition is chemopreventative. Mechanistically, nitric oxide (NO) has been implicated as a signaling molecule to promote tumorigenesis, and intermediates of NO have been shown to be mutagenic through direct DNA damage or decline in DNA repair. iNOS and COX‐2 have been linked through the stimulatory effect of nitric oxide and peroxynitrite on COX‐2 activity. In this study we used a dietary model of sporadic colon cancer to assess diet‐induced changes in inflammation. For six months C57BL/6 mice were fed either a control (AIN‐76A) or Western‐style diet, which is high in fat and calories and low in vitamin D, calcium, methionine and fiber. In the colonic mucosa we used real time RT‐PCR to examine relative gene expression of enzymes related to inflammation, including iNOS and COX‐2. The western‐style diet increased iNOS expression in the colonic mucosa (p<0.05) but not COX‐2 expression. These results indicate a Western‐style diet may exert a proinflammatory effect on colorectal carcinogenesis through increased iNOS expression. Supported by the NIH Grant R01 AG25834Grant Funding Source: NIH
Relationships Between Pathophysiology Course Performance and Primary Care Didactic and Clinical Courses and NCCPA Board Scores
Authors: McCleary Vikki L , Hosford Charles C , Kuntz Susan J , Larson Annette C , Laxen Mary Ann J , McHugo Jeanie M 
Published: June 2021

In 2006, University of North Dakota School of Medicine and Health Sciences (UNDSMHS) Physician Assistant (PA) Program initiated a study to determine if relationships exist between the pathophysiology course performance and the clinical primary and didactic course performance and also on national board scores in a masters level Physician Assistant Program.Academic student achievement data were collected in a retrospective document review of students admitted into the UNDSMHS PA Pilot Project in 2006 following IRB approval (# 200804‐303). Data were analyzed using PASW version 17.0. Pearson correlation coefficients were produced and interpreted.Records of twenty‐nine students (12males/17 females) were reviewed for this study. Mean age and mean years of clinical experience upon matriculation were 36.9 years and 10.0 years respectively. Mean baccalaureate science GPA was 3.0, and overall mean GPA was 3.2. Results of the data analysis revealed significant and positive relationships between the pathophysiology performance and primary care didactic and clinical courses and also on the National Commission on Certification of Physician Assistants.Results of this study may cultivate discussions centering on PA Program prerequisites and admissions criteria. Additional data will be collected to verify these findings.
Myogenin expression is independent of agrin treatment in C2C12 cell culture
Authors: Grow Wade A. , Henley Jessica B. 
Published: June 2021

Skeletal muscle differentiation is influenced by myogenic regulatory factors, including MyoD and myogenin. Previous studies have reported that MyoD is expressed prior to myogenin. Our objective was to investigate this process using C2C12 cell culture. When C2C12 myoblasts proliferate to 80% density, growth medium (GM) is replaced with differentiation medium (DM). Subsequently, myoblasts fuse into myotubes within 72 hours in DM. During myotube formation, acetylcholine receptors (AChRs) cluster spontaneously. Treatment with motor neuron derived agrin increases the frequency of AChR clusters through an agrin signaling pathway that also clusters other postsynaptic components of the neuromuscular synapse. Using immunofluorescence, we observed MyoD in myoblast nuclei while myoblasts were in GM, with elimination of MyoD within 72 hours in DM. In contrast, we did not observe myogenin while myoblasts were in GM, with or without agrin treatment, but myogenin was observed in myotube nuclei within 24 hours in DM without agrin treatment. We conclude that myogenin expression is independent of agrin treatment in C2C12 cell culture.Wade A. Grow was supported by Midwestern University intramural funds.
Effects of Particle Size and Protein Composition on Structural Stability and Functional Remodeling of Human High‐Density Lipoproteins
Authors: Gao Xuan , Yuan Shujun , Jayaraman Shobini , Gursky Olga 
Published: June 2021

High‐density lipoproteins (HDL) are protein‐lipid complexes that prevent atherosclerosis by removing cell cholesterol. During cholesterol transport, HDL undergo fusion and rupture upon remodeling by plasma factors, and form subclasses differing in size, composition and function. Thus, large HDL are more cardioprotective than small HDL, while HDL(A‐I/A‐II) containing proteins A‐I and A‐II are thought to be less cardioprotective than HDL(A‐I) containing only A‐I, since A‐II is thought to inhibit HDL remodeling. To provide the basis for these functional differences, we tested the effects of particle size and composition on HDL stability, fusion and rupture.Our spectroscopic, light scattering, calorimetric, electron microscopic, and biochemical studies show that HDL stability decreases with increasing particle size. This may enhance cholesterol uptake from large lipid‐loaded HDL. Surprisingly, HDL(A‐I/A‐II) are slightly less stable than HDL(A‐I). Thus, contrary to the existing notion, A‐II promotes HDL fusion and A‐I dissociation; interestingly, A‐II inhibits HDL rupture. This effect, which may be due to deep penetration of the hydrophobic A‐II into the lipid, may shift the population towards larger more cardioprotective HDL. These results shed new light on the role of particle size and protein composition in HDL remodeling, and help reconcile conflicting reports regarding the role of A‐II on HDL. NIH grants: GM067260 and HL026355
Design development and application of a fluorescent probe to study changes in hERG channel density and trafficking; a mechanistic basis for cardiac arrhythmia
Authors: Bergeron Zachary L , Collier Abby , Cummins Theodore , Bingham Jon‐Paul 
Published: June 2021

Using a novel probe, we will investigate the impact of specific drugs on the surface expression of the hERG potassium ion channel in relation to the cardiac arrhythmias, Torsade de Pointes (TdP) and Long‐QT Syndrome (LQTS) using in vitro models.Specific AimsUsing BeKm2‐NBDX, we aim to prove that over time, drugs causing aqLQTS by direct block, and drugs causing aqLQTS through inhibition of trafficking events are manifesting the same type of Density‐Related aqLQTS.Methods(1) Utilize Solid Phase Peptide Synthesis (SPPS) to bioengineer a fluorescent probe based on the framework of the scorpion toxin peptide BeKm‐1. (2A) Validate the stable expression of the HEK293/hERG over‐expressing cell line by western blot analysis of both transfected and wild type cells and their utility for hERG binding studies with the BeKm2‐NBDX probe. (2B) Demonstrate effective probe conjugation to hERG channel proteins in stably transfected cells using plate‐based assays for quantitation and confocal microscopy for visualization. Further, to optimize environmental variables for maximal binding and fluorescence as well as for extracellular fluorescence inhibition in order to functionalize BeKm2‐NBDX for hERG trafficking and internalization studies. (3) Investigate the impact of (A) channel blocking, and (B) traffic inhibiting drugs known to interact with the hERG ion channel protein. Source of funding, AHA – SDG #0530204N.