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Find video protocols related to scientific articles indexed in Pubmed.
Inhibition of interleukin-1? decreases aneurysm formation and progression in a novel model of thoracic aortic aneurysms.
Circulation
PUBLISHED: 09-10-2014
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Thoracic aortic aneurysms (TAAs) are common, but experimental TAA models are limited and the role of interleukin-1? (IL-1?) is undetermined.
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Planned Cardiac Reexploration in the Intensive Care Unit Is a Safe Procedure.
Ann. Thorac. Surg.
PUBLISHED: 08-27-2014
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Cardiac surgical reexploration is necessary in approximately 5% of all patients. However, the impact of routine, planned reexploration performed in the intensive care unit (ICU) remains poorly defined. This study evaluated postoperative outcomes after cardiac reexplorations to determine the safety and efficacy of a planned approach in the ICU.
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Cryptic and complex chromosomal aberrations in early-onset neuropsychiatric disorders.
Am. J. Hum. Genet.
PUBLISHED: 08-08-2014
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Structural variation (SV) is a significant component of the genetic etiology of both neurodevelopmental and psychiatric disorders; however, routine guidelines for clinical genetic screening have been established only in the former category. Genome-wide chromosomal microarray (CMA) can detect genomic imbalances such as copy-number variants (CNVs), but balanced chromosomal abnormalities (BCAs) still require karyotyping for clinical detection. Moreover, submicroscopic BCAs and subarray threshold CNVs are intractable, or cryptic, to both CMA and karyotyping. Here, we performed whole-genome sequencing using large-insert jumping libraries to delineate both cytogenetically visible and cryptic SVs in a single test among 30 clinically referred youth representing a range of severe neuropsychiatric conditions. We detected 96 SVs per person on average that passed filtering criteria above our highest-confidence resolution (6,305 bp) and an additional 111 SVs per genome below this resolution. These SVs rearranged 3.8 Mb of genomic sequence and resulted in 42 putative loss-of-function (LoF) or gain-of-function mutations per person. We estimate that 80% of the LoF variants were cryptic to clinical CMA. We found myriad complex and cryptic rearrangements, including a "paired" duplication (360 kb, 169 kb) that flanks a 5.25 Mb inversion that appears in 7 additional cases from clinical CNV data among 47,562 individuals. Following convergent genomic profiling of these independent clinical CNV data, we interpreted three SVs to be of potential clinical significance. These data indicate that sequence-based delineation of the full SV mutational spectrum warrants exploration in youth referred for neuropsychiatric evaluation and clinical diagnostic SV screening more broadly.
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Separating proteins with activated carbon.
Langmuir
PUBLISHED: 06-30-2014
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Activated carbon is applied to separate proteins based on differences in their size and effective charge. Three guidelines are suggested for the efficient separation of proteins with activated carbon. (1) Activated carbon can be used to efficiently remove smaller proteinaceous impurities from larger proteins. (2) Smaller proteinaceous impurities are most efficiently removed at a solution pH close to the impurity's isoelectric point, where they have a minimal effective charge. (3) The most efficient recovery of a small protein from activated carbon occurs at a solution pH further away from the protein's isoelectric point, where it is strongly charged. Studies measuring the binding capacities of individual polymers and proteins were used to develop these three guidelines, and they were then applied to the separation of several different protein mixtures. The ability of activated carbon to separate proteins was demonstrated to be broadly applicable with three different types of activated carbon by both static treatment and by flowing through a packed column of activated carbon.
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Far-red organic fluorophores contain a fluorescent impurity.
Chemphyschem
PUBLISHED: 02-03-2014
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Far-red organic fluorophores commonly used in traditional and super-resolution localization microscopy are found to contain a fluorescent impurity with green excitation and near-red emission. This near-red fluorescent impurity can interfere with some multicolor stochastic optical reconstruction microscopy/photoactivated localization microscopy measurements in live cells and produce subtle artifacts in chemically fixed cells. We additionally describe alternatives to avoid artifacts in super-resolution localization microscopy.
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Elevated peptides in lung lavage fluid associated with bronchiolitis obliterans syndrome.
PLoS ONE
PUBLISHED: 01-01-2014
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The objective of this discovery-level investigation was to use mass spectrometry to identify low mass compounds in bronchoalveolar lavage fluid from lung transplant recipients that associate with bronchiolitis obliterans syndrome.
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Ezrin tunes the magnitude of humoral immunity.
J. Immunol.
PUBLISHED: 09-16-2013
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Ezrin is a member of the ezrin-radixin-moesin family of membrane-actin cytoskeleton cross-linkers that participate in a variety of cellular processes. In B cells, phosphorylation of ezrin at different sites regulates multiple processes, such as lipid raft coalescence, BCR diffusion, microclustering, and endosomal JNK activation. In this study, we generated mice with conditional deletion of ezrin in the B cell lineage to investigate the physiological significance of ezrins function in Ag receptor-mediated B cell activation and humoral immunity. B cell development, as well as the proportion and numbers of major B cell subsets in peripheral lymphoid organs, was unaffected by the loss of ezrin. Using superresolution imaging methods, we show that, in the absence of ezrin, BCRs respond to Ag binding by accumulating into larger and more stable signaling microclusters. Loss of ezrin led to delayed BCR capping and accelerated lipid raft coalescence. Although proximal signaling proteins showed stronger activation in the absence of ezrin, components of the distal BCR signaling pathways displayed distinct effects. Ezrin deficiency resulted in increased B cell proliferation and differentiation into Ab-secreting cells ex vivo and stronger T cell-independent and -dependent responses to Ag in vivo. Overall, our data demonstrate that ezrin regulates amplification of BCR signals and tunes the strength of B cell activation and humoral immunity.
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Association of race and socioeconomic status with the use of endovascular repair to treat thoracic aortic diseases.
J. Vasc. Surg.
PUBLISHED: 03-11-2013
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Descending thoracic aortic diseases may be treated with either open thoracic aortic repair or thoracic endovascular aortic repair (TEVAR). Previous studies have demonstrated that race and socioeconomic status (SES) affect access to care and treatment allocation in vascular surgery. We hypothesized that racial minorities and lower SES patients have decreased propensity to have their thoracic aortic disease treated with TEVAR.
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Surgical outcomes analysis of pediatric peritoneal dialysis catheter function in a rural region.
J. Pediatr. Surg.
PUBLISHED: 02-05-2013
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The purpose of this study was to analyze the experience with peritoneal dialysis (PD) at a high-volume, single center institution that supports a rural population.
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Rapamycin blocks fibrocyte migration and attenuates bronchiolitis obliterans in a murine model.
Ann. Thorac. Surg.
PUBLISHED: 01-30-2013
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Fibrocytes are integral in the development of fibroproliferative disease. The CXCL12/CXCR4 chemokine axis has been shown to play a central role in fibrocyte migration and the development of bronchiolitis obliterans (BO) after lung transplantation. Inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin has been shown to decrease expression of both CXCR4 and its receptor agonist CXCL12. Thus, we hypothesized that rapamycin treatment would decrease fibrocyte trafficking into tracheal allografts and prevent BO.
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The effect of race and gender on pediatric surgical outcomes within the United States.
J. Pediatr. Surg.
PUBLISHED: 01-28-2013
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The purpose of this study was to examine risk-adjusted associations between race and gender on postoperative morbidity, mortality, and resource utilization in pediatric surgical patients within the United States.
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Primary payer status is significantly associated with postoperative mortality, morbidity, and hospital resource utilization in pediatric surgical patients within the United States.
J. Pediatr. Surg.
PUBLISHED: 01-22-2013
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Current healthcare reform efforts have highlighted the potential impact of insurance status on patient outcomes. The influence of primary payer status (PPS) within the pediatric surgical patient population remains unknown. The purpose of this study was to examine risk-adjusted associations between PPS and postoperative mortality, morbidity, and resource utilization in pediatric surgical patients within the United States.
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Genetic and pharmacologic disruption of interleukin-1? signaling inhibits experimental aortic aneurysm formation.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 01-03-2013
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Abdominal aortic aneurysms (AAAs) are common, but their exact pathogenesis remains unknown and no specific medical therapies are available. We sought to evaluate interleukin-1? (IL-1?) and interleukin-1 receptor (IL-1R) in an experimental AAA model to identify novel therapeutic targets for AAA treatment.
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Unconventional temperature enhanced magnetism in Fe1.1Te.
Phys. Rev. Lett.
PUBLISHED: 05-26-2011
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Our inelastic neutron scattering study of spin excitations in iron telluride reveals remarkable thermal evolution of the collective magnetism. In the temperature range relevant for the superconductivity in FeTe(1-x)Se(x) materials, where the local-moment behavior is dominated by liquidlike correlations of emergent spin plaquettes, we observe unusual, marked increase of magnetic fluctuations upon heating. The effective spin per Fe at T ? 10??K, in the phase with weak antiferromagnetic order, corresponds to S ? 1, consistent with the recent analyses that emphasize importance of Hunds coupling [K. Haule and G. Kotliar, New J. Phys. 11, 025021 (2009).]. However, it grows to S ? 3/2 in the high-T disordered phase, suggestive of the Kondo-type behavior, where local magnetic moments are entangled with the itinerant electrons.
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Spin waves and magnetic exchange interactions in insulating Rb(0.89)Fe(1.58)Se(2).
Nat Commun
PUBLISHED: 05-24-2011
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The parent compounds of iron pnictide superconductors are bad metals with a collinear antiferromagnetic structure and Néel temperatures below 220?K. Although alkaline iron selenide A(y)Fe(1.6+x)Se(2) (A=K, Rb, Cs) superconductors are isostructural with iron pnictides, in the vicinity of the undoped limit they are insulators, forming a block antiferromagnetic order and having Néel temperatures of roughly 500?K. Here we show that the spin waves of the insulating antiferromagnet Rb(0.89)Fe(1.58)Se(2) can be accurately described by a local moment Heisenberg Hamiltonian. A fitting analysis of the spin wave spectra reveals that the next-nearest neighbour couplings in Rb(0.89)Fe(1.58)Se(2), (Ba,Ca,Sr)Fe(2)As(2), and Fe(1.05)Te are of similar magnitude. Our results suggest a common origin for the magnetism of all the Fe-based superconductors, despite having different ground states and antiferromagnetic orderings.
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An improved Larock synthesis of quinolines via a Heck reaction of 2-bromoanilines and allylic alcohols.
Org. Lett.
PUBLISHED: 04-06-2011
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A modified Larock method has been developed for the one-pot synthesis of substituted quinolines via a Heck reaction of 2-bromoanilines and allylic alcohols followed by dehydrogenation with diisopropyl azodicarboxylate (DIAD).
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Large-scale phosphoproteomics analysis of whole saliva reveals a distinct phosphorylation pattern.
J. Proteome Res.
PUBLISHED: 03-01-2011
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In-depth knowledge of bodily fluid phosphoproteomes, such as whole saliva, is limited. To better understand the whole saliva phosphoproteome, we generated a large-scale catalog of phosphorylated proteins. To circumvent the wide dynamic range of phosphoprotein abundance in whole saliva, we combined dynamic range compression using hexapeptide beads, strong cation exchange HPLC peptide fractionation, and immobilized metal affinity chromatography prior to mass spectrometry. In total, 217 unique phosphopeptides sites were identified representing 85 distinct phosphoproteins at 2.3% global FDR. From these peptides, 129 distinct phosphorylation sites were identified of which 57 were previously known, but only 11 of which had been previously identified in whole saliva. Cellular localization analysis revealed salivary phosphoproteins had a distribution similar to all known salivary proteins, but with less relative representation in "extracellular" and "plasma membrane" categories compared to salivary glycoproteins. Sequence alignment showed that phosphorylation occurred at acidic-directed kinase, proline-directed, and basophilic motifs. This differs from plasma phosphoproteins, which predominantly occur at Golgi casein kinase recognized sequences. Collectively, these results suggest diverse functions for salivary phosphoproteins and multiple kinases involved in their processing and secretion. In all, this study should lay groundwork for future elucidation of the functions of salivary protein phosphorylation.
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Hexapeptide libraries for enhanced protein PTM identification and relative abundance profiling in whole human saliva.
J. Proteome Res.
PUBLISHED: 01-14-2011
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Dynamic range compression (DRC) by hexapeptide libraries increases MS/MS-based identification of lower-abundance proteins in complex mixtures. However, two unanswered questions impede fully realizing DRCs potential in shotgun proteomics. First, does DRC enhance identification of post-translationally modified proteins? Second, can DRC be incorporated into a workflow enabling relative protein abundance profiling? We sought to answer both questions analyzing human whole saliva. Addressing question one, we coupled DRC with covalent glycopeptide enrichment and MS/MS. With DRC we identified ?2 times more N-linked glycoproteins and their glycosylation sites than without DRC, dramatically increasing the known salivary glycoprotein catalog. Addressing question two, we compared differentially stable isotope-labeled saliva samples pooled from healthy and metastatic breast cancer women using a multidimensional peptide fractionation-based workflow, analyzing in parallel one sample portion with DRC and one portion without. Our workflow categorizes proteins with higher absolute abundance, whose relative abundance ratios are altered by DRC, from proteins of lower absolute abundance detected only after DRC. Within each of these salivary protein categories, we identified novel abundance changes putatively associated with breast cancer, demonstrating feasibility and benefits of DRC for relative abundance profiling. Collectively, our results bring us closer to realizing the full potential of DRC for proteomic studies.
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Non-ATG-initiated translation directed by microsatellite expansions.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-20-2010
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Trinucleotide expansions cause disease by both protein- and RNA-mediated mechanisms. Unexpectedly, we discovered that CAG expansion constructs express homopolymeric polyglutamine, polyalanine, and polyserine proteins in the absence of an ATG start codon. This repeat-associated non-ATG translation (RAN translation) occurs across long, hairpin-forming repeats in transfected cells or when expansion constructs are integrated into the genome in lentiviral-transduced cells and brains. Additionally, we show that RAN translation across human spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1) CAG expansion transcripts results in the accumulation of SCA8 polyalanine and DM1 polyglutamine expansion proteins in previously established SCA8 and DM1 mouse models and human tissue. These results have implications for understanding fundamental mechanisms of gene expression. Moreover, these toxic, unexpected, homopolymeric proteins now should be considered in pathogenic models of microsatellite disorders.
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Mass spectrometric identification of phosphorylation sites in guanylyl cyclase A and B.
Biochemistry
PUBLISHED: 11-08-2010
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Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologues. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487), and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529), and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild-type, S487A, and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.
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LTQ-iQuant: A freely available software pipeline for automated and accurate protein quantification of isobaric tagged peptide data from LTQ instruments.
Proteomics
PUBLISHED: 09-08-2010
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Pulsed Q dissociation enables combining LTQ ion trap instruments with isobaric peptide tagging. Unfortunately, this combination lacks a technique which accurately reports protein abundance ratios and is implemented in a freely available, flexible software pipeline. We developed and implemented a technique assigning collective reporter ion intensity-based weights to each peptide abundance ratio and calculating a proteins weighted average abundance ratio and p-value. Using an iTRAQ-labeled standard mixture, we compared our techniques performance to the commercial software MASCOT, finding that it performed better than MASCOTs nonweighted averaging and median peptide ratio techniques, and equal to its weighted averaging technique. We also compared performance of the LTQ-Orbitrap plus our technique to 4800 MALDI TOF/TOF plus Protein Pilot, by analyzing an iTRAQ-labeled stem cell lysate. We found highly correlated protein abundance ratios, indicating that the LTQ-Orbitrap plus our technique yields results comparable to the current standard. We implemented our technique in a freely available, automated software pipeline, called LTQ-iQuant, which is mzXML-compatible; supports iTRAQ 4-plex and 8-plex LTQ data; and can be modified for and have weights trained to a users LTQ and other isobaric peptide tagging methods. LTQ-iQuant should make LTQ instruments and isobaric peptide tagging accessible to more proteomic researchers.
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Novel In Situ Collection of Tumor Interstitial Fluid from a Head and Neck Squamous Carcinoma Reveals a Unique Proteome with Diagnostic Potential.
Clin Proteomics
PUBLISHED: 07-25-2010
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INTRODUCTION: Tumors lack normal drainage of secreted fluids and consequently build up tumor interstitial fluid (TIF). Unlike other bodily fluids, TIF likely contains a high proportion of tumor-specific proteins with potential as biomarkers. METHODS: Here, we evaluated a novel technique using a unique ultrafiltration catheter for in situ collection of TIF and used it to generate the first catalog of TIF proteins from a head and neck squamous cell carcinoma (HNSCC). To maximize proteomic coverage, TIF was immunodepleted for high abundance proteins and digested with trypsin, and peptides were fractionated in three dimensions prior to mass spectrometry. RESULTS: We identified 525 proteins with high confidence. The HNSCC TIF proteome was distinct compared to proteomes of other bodily fluids. It contained a relatively high proportion of proteins annotated by Gene Ontology as "extracellular" compared to other secreted fluid and cellular proteomes, indicating minimal cell lysis from our in situ collection technique. Several proteins identified are putative biomarkers of HNSCC, supporting our catalogs value as a source of potential biomarkers. CONCLUSIONS: In all, we demonstrate a reliable new technique for in situ TIF collection and provide the first HNSCC TIF protein catalog with value as a guide for others seeking to develop tumor biomarkers. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12014-010-9050-3) contains supplementary material, which is available to authorized users.
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Minimal peroxide exposure of neuronal cells induces multifaceted adaptive responses.
PLoS ONE
PUBLISHED: 06-14-2010
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Oxidative exposure of cells occurs naturally and may be associated with cellular damage and dysfunction. Protracted low level oxidative exposure can induce accumulated cell disruption, affecting multiple cellular functions. Accumulated oxidative exposure has also been proposed as one of the potential hallmarks of the physiological/pathophysiological aging process. We investigated the multifactorial effects of long-term minimal peroxide exposure upon SH-SY5Y neural cells to understand how they respond to the continued presence of oxidative stressors. We show that minimal protracted oxidative stresses induce complex molecular and physiological alterations in cell functionality. Upon chronic exposure to minimal doses of hydrogen peroxide, SH-SY5Y cells displayed a multifactorial response to the stressor. To fully appreciate the peroxide-mediated cellular effects, we assessed these adaptive effects at the genomic, proteomic and cellular signal processing level. Combined analyses of these multiple levels of investigation revealed a complex cellular adaptive response to the protracted peroxide exposure. This adaptive response involved changes in cytoskeletal structure, energy metabolic shifts towards glycolysis and selective alterations in transmembrane receptor activity. Our analyses of the global responses to chronic stressor exposure, at multiple biological levels, revealed a viable neural phenotype in-part reminiscent of aged or damaged neural tissue. Our paradigm indicates how cellular physiology can subtly change in different contexts and potentially aid the appreciation of stress response adaptations.
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Cardiac catheterization within 24 hours of valve surgery is significantly associated with acute renal failure.
J. Thorac. Cardiovasc. Surg.
PUBLISHED: 04-29-2010
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Acute renal failure after valve surgery carries significant morbidity and mortality. Preoperative cardiac catheterization is the standard of care. For convenience, catheterization just before surgery is simplest for patients. However, it is not known if this timing of radiocontrast administration significantly affects renal function. We hypothesized that preoperative cardiac catheterization within 24 hours of valve surgery is associated with the development of acute renal failure.
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Quantitative proteomics reveals myosin and actin as promising saliva biomarkers for distinguishing pre-malignant and malignant oral lesions.
PLoS ONE
PUBLISHED: 04-12-2010
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Oral cancer survival rates increase significantly when it is detected and treated early. Unfortunately, clinicians now lack tests which easily and reliably distinguish pre-malignant oral lesions from those already transitioned to malignancy. A test for proteins, ones found in non-invasively-collected whole saliva and whose abundances distinguish these lesion types, would meet this critical need.
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A dynamic range compression and three-dimensional peptide fractionation analysis platform expands proteome coverage and the diagnostic potential of whole saliva.
J. Proteome Res.
PUBLISHED: 10-10-2009
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Comprehensive identification of proteins in whole human saliva is critical for appreciating its full diagnostic potential. However, this is challenged by the large dynamic range of protein abundance within the fluid. To address this problem, we used an analysis platform that coupled hexapeptide libraries for dynamic range compression (DRC) with three-dimensional (3D) peptide fractionation. Our approach identified 2340 proteins in whole saliva and represents the largest saliva proteomic dataset generated using a single analysis platform. Three-dimensional peptide fractionation involving sequential steps of preparative isoelectric focusing (IEF), strong cation exchange, and capillary reversed-phase liquid chromatography was essential for maximizing gains from DRC. Compared to saliva not treated with hexapeptide libraries, DRC substantially increased identified proteins across physicochemical and functional categories. Approximately 20% of total salivary proteins are also seen in plasma, and proteins in both fluids show comparable functional diversity and disease-linkage. However, for a subset of diseases, saliva has higher apparent diagnostic potential. These results expand the potential for whole saliva in health monitoring/diagnostics and provide a general platform for improving proteomic coverage of complex biological samples.
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Public speaking attitudes: does curriculum make a difference?
J Voice
PUBLISHED: 05-28-2009
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In light of infamous levels of fear associated with public speaking, businesses are training staff in communication effectiveness and universities are requiring courses in public speaking. A variety of approaches to individual training are available, but few studies have assessed effectiveness of group instruction, as in academic curricula. The specific purpose of this study was to compare changes in scores on measures of self-perceived confidence, competence, and apprehension associated with public speaking after two types of courses: one focused on knowledge of the vocal mechanism and mastering vocal characteristics (pitch, volume, rate, quality), and one addressing general communication theory and public speaking. Seventy-one undergraduate students enrolled in "Voice and Diction" at George Washington University (GWU) and 68 enrolled in "Fundamental Speech" at Florida State University completed questionnaires before and after the courses. Scores on Self-Perceived Communication Competence Scale, Personal Report of Confidence as a Speaker, and Personal Report of Communication Apprehension-24, were compared within subjects (ie, prepost course) and between courses. Significant differences (p<0.05) were found on all measures: students reported less apprehension and more confidence and competence after the courses. No differences were found between the two courses when comparing the mean changes from pre- to postscore. Traditional public speaking curriculum of how to design and deliver a speech and curriculum tailored to the voice and speech mechanism succeeded in reducing public speaking apprehension and increasing feelings of confidence and competency for these undergraduate students.
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Orthotopic heart transplant versus left ventricular assist device: a national comparison of cost and survival.
J. Thorac. Cardiovasc. Surg.
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Orthotopic heart transplantation is the standard of care for end-stage heart disease. Left ventricular assist device implantation offers an alternative treatment approach. Left ventricular assist device practice has changed dramatically since the 2008 Food and Drug Administration approval of the HeartMate II (Thoratec, Pleasanton, Calif), but at what societal cost? The present study examined the cost and efficacy of both treatments over time.
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Identification and origin of N-linked ?-D-N-acetylglucosamine monosaccharide modifications on Arabidopsis proteins.
Plant Physiol.
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Many plant proteins are modified with N-linked oligosaccharides at asparagine-X-serine/threonine sites during transit through the endoplasmic reticulum and the Golgi. We have identified a number of Arabidopsis (Arabidopsis thaliana) proteins with modifications consisting of an N-linked N-acetyl-D-glucosamine monosaccharide (N-GlcNAc). Electron transfer dissociation mass spectrometry analysis of peptides bearing this modification mapped the modification to asparagine-X-serine/threonine sites on proteins that are predicted to transit through the endoplasmic reticulum and Golgi. A mass labeling method was developed and used to study N-GlcNAc modification of two thioglucoside glucohydrolases (myrosinases), TGG1 and TGG2 (for thioglucoside glucohydrolase). These myrosinases are also modified with high-mannose (Man)-type glycans. We found that N-GlcNAc and high-Man-type glycans can occur at the same site. It has been hypothesized that N-GlcNAc modifications are generated when endo-?-N-acetylglucosaminidase (ENGase) cleaves N-linked glycans. We examined the effects of mutations affecting the two known Arabidopsis ENGases on N-GlcNAc modification of myrosinase and found that modification of TGG2 was greatly reduced in one of the single mutants and absent in the double mutant. Surprisingly, N-GlcNAc modification of TGG1 was not affected in any of the mutants. These data support the hypothesis that ENGases hydrolyze high-Man glycans to produce some of the N-GlcNAc modifications but also suggest that some N-GlcNAc modifications are generated by another mechanism. Since N-GlcNAc modification was detected at only one site on each myrosinase, the production of the N-GlcNAc modification may be regulated.
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Fibroblast growth factor receptor 1 activation in mammary tumor cells promotes macrophage recruitment in a CX3CL1-dependent manner.
PLoS ONE
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Tumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for growth and progression of the primary tumor. Previous studies have shown that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. These studies also demonstrated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium where macrophages contributed to iFGFR1-mediated epithelial cell proliferation and angiogenesis. The studies presented here further utilize this model to identify the mechanisms that regulate FGFR1-induced macrophage recruitment. Results from this study elucidate a novel role for the inflammatory chemokine CX3CL1 in FGFR1-induced macrophage migration. Specifically, we illustrate that activation of both the inducible FGFR1 construct in mouse mammary epithelial cells and endogenous FGFR in the triple negative breast cancer cell line, HS578T, leads to expression of the chemokine CX3CL1. Furthermore, we demonstrate that FGFR-induced CX3CL1 is sufficient to recruit CX3CR1-expressing macrophages in vitro. Finally, blocking CX3CR1 in vivo leads to decreased iFGFR1-induced macrophage recruitment, which correlates with decreased angiogenesis. While CX3CL1 is a known target of FGF signaling in the wound healing environment, these studies demonstrate that FGFR activation also leads to induction of CX3CL1 in a tumor setting. Furthermore, these results define a novel role for CX3CL1 in promoting macrophage recruitment during mammary tumor formation, suggesting that the CX3CL1/CX3CR1 axis may represent a potential therapeutic approach for targeting breast cancers associated with high levels of tumor-associated macrophages.
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Primary payer status is associated with mortality and resource utilization for coronary artery bypass grafting.
Circulation
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Medicaid and uninsured populations are a significant focus of current healthcare reform. We hypothesized that outcomes after coronary artery bypass grafting (CABG) in the United States is dependent on primary payer status.
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Ex vivo rehabilitation of non-heart-beating donor lungs in preclinical porcine model: delayed perfusion results in superior lung function.
J. Thorac. Cardiovasc. Surg.
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Ex vivo lung perfusion (EVLP) is a promising modality for the evaluation and treatment of marginal donor lungs. The optimal timing of EVLP initiation and the potential for rehabilitation of donor lungs with extended warm ischemic times is unknown. The present study compared the efficacy of different treatment strategies for uncontrolled non-heart-beating donor lungs.
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Protein carbonylation and adipocyte mitochondrial function.
J. Biol. Chem.
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Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1? subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1? subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.
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Workflow for analysis of high mass accuracy salivary data set using MaxQuant and ProteinPilot search algorithm.
Proteomics
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LTQ Orbitrap data analyzed with ProteinPilot can be further improved by MaxQuant raw data processing, which utilizes precursor-level high mass accuracy data for peak processing and MGF creation. In particular, ProteinPilot results from MaxQuant-processed peaklists for Orbitrap data sets resulted in improved spectral utilization due to an improved peaklist quality with higher precision and high precursor mass accuracy (HPMA). The output and postsearch analysis tools of both workflows were utilized for previously unexplored features of a three-dimensional fractionated and hexapeptide library (ProteoMiner) treated whole saliva data set comprising 200 fractions. ProteinPilots ability to simultaneously predict multiple modifications showed an advantage from ProteoMiner treatment for modified peptide identification. We demonstrate that complementary approaches in the analysis pipeline provide comprehensive results for the whole saliva data set acquired on an LTQ Orbitrap. Overall our results establish a workflow for improved protein identification from high mass accuracy data.
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Concomitant tricuspid valve operations affect outcomes after mitral operations: a multiinstitutional, statewide analysis.
Ann. Thorac. Surg.
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Mitral valve (MV) disease is often accompanied by concomitant tricuspid valve (TV) disease. This study determined the influence of performing TV procedures in the setting of MV operations within a multiinstitutional patient population.
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A functional screen provides evidence for a conserved, regulatory, juxtamembrane phosphorylation site in guanylyl cyclase a and B.
PLoS ONE
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Kinase homology domain (KHD) phosphorylation is required for activation of guanylyl cyclase (GC)-A and -B. Phosphopeptide mapping identified multiple phosphorylation sites in GC-A and GC-B, but these approaches have difficulty identifying sites in poorly detected peptides. Here, a functional screen was conducted to identify novel sites. Conserved serines or threonines in the KHDs of phosphorylated receptor GCs were mutated to alanine and tested for reduced hormone to detergent activity ratios. Mutation of Ser-489 in GC-B to alanine but not glutamate reduced the activity ratio to 60% of wild type (WT) levels. Similar results were observed with Ser-473, the homologous site in GC-A. Receptors containing glutamates for previously identified phosphorylation sites (GC-A-6E and GC-B-6E) were activated to ~20% of WT levels but the additional glutamate substitution for S473 or S489 increased activity to near WT levels. Substrate-velocity assays indicated that GC-B-WT-S489E and GC-B-6E-S489E had lower Km values and that WT-GC-B-S489A, GC-B-6E and GC-B-6E-S489A had higher Km values than WT-GC-B. Homologous desensitization was enhanced when GC-A contained the S473E substitution, and GC-B-6E-S489E was resistant to inhibition by a calcium elevating treatment or protein kinase C activation--processes that dephosphorylate GC-B. Mass spectrometric detection of a synthetic phospho-Ser-473 containing peptide was 200-1300-fold less sensitive than other phosphorylated peptides and neither mass spectrometric nor (32)PO(4) co-migration studies detected phospho-Ser-473 or phospho-Ser-489 in cells. We conclude that Ser-473 and Ser-489 are Km-regulating phosphorylation sites that are difficult to detect using current methods.
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SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling.
Mol. Cell
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Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.
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Cryoablation during left ventricular assist device implantation reduces postoperative ventricular tachyarrhythmias.
J. Thorac. Cardiovasc. Surg.
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The number of patients undergoing implantation of a HeartMate II left ventricular assist device (LVAD; Thoratec Corporation, Pleasanton, Calif) is rising. Ventricular tachyarrhythmia (VA) after placement of the device is common, especially among patients with preoperative VA. We sought to determine whether intraoperative cryoablation in select patients reduces the incidence of postoperative VA.
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The Cohn felt plug: an effective HeartMate II® reimplantation technique.
J Card Surg
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We report the first documented case of HeartMate II® left ventricular assist device (LVAD) reimplantation following Cohn Teflon felt plug repair of the initial left ventricular apical cannulation site. This case highlights the current limitations of the predictability of myocardial recovery while describing an effective technique for possible future LVAD reimplantation.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.