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Find video protocols related to scientific articles indexed in Pubmed.
Preoperative factors and early complications associated with hemiarthroplasty and total hip arthroplasty for displaced femoral neck fractures.
Geriatr Orthop Surg Rehabil
PUBLISHED: 11-01-2014
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Displaced femoral neck fractures are common injuries in the elderly individuals. There is controversy about the best treatment with regard to total hip arthroplasty (THA) versus hemiarthroplasty. This study uses the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) database to evaluate the preoperative risk factors associated with the decision to perform THA over hemiarthroplasty. We also evaluate the risk factors associated with postoperative complications after each procedure. Patients older than 50 years undergoing hemiarthroplasty or THA after fracture in the NSQIP database from 2007 to 2010 were compared to each other in terms of preoperative medical conditions, postoperative complications, and length of stay. Multivariate logistic regression models were used to adjust for preoperative risk factors for undergoing a THA versus a hemiarthroplasty and for complications after each procedure. In all, 783 patients underwent hemiarthroplasty and 419 underwent THA for fracture. Hemiarthroplasty patients had longer hospital stays. On multivariate logistic regression, the only significant predictor for having a THA after fracture over hemiarthroplasty was being aged 50 to 64 years. The patient characteristics/comorbidities that favored having a hemiarthroplasty were age >80 years, hemiplegia, being underweight, having a dependent functional status, being on dialysis, and having an early surgery. High body mass index, American Society of Anesthesiologists (ASA) class, gender, and other comorbidities were not predictors of having one procedure over another. Disseminated cancer and diabetes were predictive of complications after THA while being overweight, obese I, or a smoker were protective. High ASA class and do-not-resuscitate status were significant predictors of complications after a hemiarthroplasty. This study identified clinical factors influencing surgeons toward performing either THA or hemiarthroplasty for elderly patients after femoral neck fractures. Younger, healthier patients were more likely to receive THA. Patients particularly at higher risks of complications after hemiarthroplasty should be monitored closely.
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Paleoindian settlement of the high-altitude Peruvian Andes.
Science
PUBLISHED: 10-25-2014
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Study of human adaptation to extreme environments is important for understanding our cultural and genetic capacity for survival. The Pucuncho Basin in the southern Peruvian Andes contains the highest-altitude Pleistocene archaeological sites yet identified in the world, about 900 meters above confidently dated contemporary sites. The Pucuncho workshop site [4355 meters above sea level (masl)] includes two fishtail projectile points, which date to about 12.8 to 11.5 thousand years ago (ka). Cuncaicha rock shelter (4480 masl) has a robust, well-preserved, and well-dated occupation sequence spanning the past 12.4 thousand years (ky), with 21 dates older than 11.5 ka. Our results demonstrate that despite cold temperatures and low-oxygen conditions, hunter-gatherers colonized extreme high-altitude Andean environments in the Terminal Pleistocene, within about 2 ky of the initial entry of humans to South America.
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Effects of Diagnostic Inclusion Criteria on Prevalence and Population Characteristics in Database Research.
Psychiatr Serv
PUBLISHED: 10-17-2014
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Studies of serious mental illnesses that use administrative databases have employed various criteria to establish diagnoses of interest. Several studies have assessed the validity of diagnostic inclusion criteria against research diagnoses. However, no studies have examined the effect of diagnostic inclusion criteria on prevalence and population characteristics across such groups.
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Genetic suppression of a phosphomimic myosin II identifies system-level factors that promote myosin II cleavage furrow accumulation.
Mol. Biol. Cell
PUBLISHED: 10-17-2014
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How myosin II localizes to the cleavage furrow in Dictyostelium and metazoan cells remains largely unknown despite significant advances in understanding its regulation. We designed a genetic selection using cDNA library suppression of 3xAsp myosin II to identify factors involved in myosin cleavage furrow accumulation. The 3xAsp mutant is deficient in bipolar thick filament assembly, fails to accumulate at the cleavage furrow, cannot rescue myoII-null cytokinesis, and has impaired mechanosensitive accumulation. Eleven genes suppressed this dominant cytokinesis deficiency when 3xAsp was expressed in WT cells. 3xAsp myosin II's localization to the cleavage furrow was rescued by constructs encoding rcdBB, mmsdh, RMD1, actin, one novel protein, and a 14-3-3 hairpin. Further characterization showed that RMD1 is required for myosin II cleavage furrow accumulation, acting parallel with mechanical stress. Analysis of several mutant strains revealed that different thresholds of myosin II activity are required for daughter cell symmetry than for furrow ingression dynamics. Finally, an engineered myosin II with a longer lever arm (2xELC), producing a highly mechanosensitive motor, could also partially suppress the intragenic 3xAsp. Overall, myosin II accumulation is the result of multiple parallel and partially redundant pathways that comprise a cellular contractility control system.
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Meningitis and a safe dexamethasone-eluting intracochlear electrode array.
Cochlear Implants Int
PUBLISHED: 10-16-2014
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Objectives To evaluate the potential risk of pneumococcal meningitis associated with the use of a dexamethasone-eluting intracochlear electrode array as compared with a control array. Methods In two phases, adult Hooded-Wistar rats were implanted via the middle ear with an intracochlear array and were inoculated with Streptococcus pneumoniae 5 days post-surgery. Phase I created a dosing curve by implanting five groups (n = 6) with a control array, then inoculating 5 days later with different numbers of S. pneumoniae: 0 CFU, 10(3) CFU, 10(4) CFU, 10(4) CFU repeated, or 10(5) CFU (colony forming units). A target infection rate of 20% was aimed for and 10(4) CFU was the closest to this target with 33% infection rate. In phase II, we implanted two groups (n = 10), one with a dexamethasone-eluting array, the other a control array, and both groups were inoculated with 10(4) CFU of S. pneumoniae 5 days post-surgery. Results The dexamethasone-eluting array group had a 40% infection rate; the control array group had a 60% infection rate. This difference was not statistically significant with a P value of ?0.5. Conclusion The use of a dexamethasone-eluting intracochlear electrode array did not increase the risk of meningitis in rats when inoculated with S. pneumoniae via the middle ear 5 days following implantation.
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Enhanced fidelity to treatment for bipolar disorder: results from a randomized controlled implementation trial.
Psychiatr Serv
PUBLISHED: 09-25-2014
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The authors compared fidelity to bipolar disorder treatment at community practices that received a standard or enhanced version of a novel implementation intervention called Replicating Effective Programs (REP).
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Driving Reaction Times in Patients With Foot and Ankle Pathology Before and After Image-Guided Injection: Pain Relief Without Improved Function.
Foot Ankle Spec
PUBLISHED: 09-12-2014
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Foot and ankle pathology is common in the driving population. Local anesthetic steroid injections are frequent ambulatory treatments. Brake reaction time (BRT) has validated importance in motor vehicle safety. There are no prior studies examining the effect of foot and ankle pathology and injection treatment on the safe operation of motor vehicles. We studied BRT in patients with foot and ankle musculoskeletal disease before and after image-guided injection treatment.
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Excess mortality in bipolar disorders.
Curr Psychiatry Rep
PUBLISHED: 09-08-2014
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Bipolar disorder is associated with high mortality, and people with this disorder on average may die 10-20 years earlier than the general population. This excess and premature mortality continues to occur despite a large and expanding selection of treatment options dating back to lithium and now including anticonvulsants, antipsychotics, and evidence-based psychotherapies. This review summarizes recent findings on mortality in bipolar disorder, with an emphasis on the role of suicide (accounting for about 15% of deaths in this population) and cardiovascular disease (accounting for about 35-40% of deaths). Recent care models and treatments incorporating active outreach, integrated mental and physical health care, and an emphasis on patient self-management have shown promise in reducing excess mortality in this population.
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A discrete-time survival model with random effects for designing and analyzing repeated low-dose challenge experiments.
Biostatistics
PUBLISHED: 09-03-2014
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Repeated low-dose (RLD) challenge designs are important in HIV vaccine research. Current methods for RLD designs rely heavily on an assumption of homogeneous risk of infection among animals, which, upon violation, can lead to invalid inferences and underpowered study designs. We propose to fit a discrete-time survival model with random effects that allows for heterogeneity in the risk of infection among animals and allows for predetermined challenge dose changes over time. Based on this model, we derive likelihood ratio tests and estimators for vaccine efficacy. A two-stage approach is proposed for optimizing the RLD design under cost constraints. Simulation studies demonstrate good finite sample properties of the proposed method and its superior performance compared to existing methods. We illustrate the application of the heterogeneous infection risk model on data from a real simian immunodeficiency virus vaccine study using Rhesus Macaques. The results of our study provide useful guidance for future RLD experimental design.
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Building a temperature-sensitive ion channel.
Cell
PUBLISHED: 08-30-2014
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The biophysical basis of temperature-sensitive ion channel gating has been a tough nut to crack. Chowdhury, et al. use a protein engineering approach to render a temperature-insensitive voltage-gated channel cold- or heat-responsive to reveal principles for temperature-gating and a plausible model for molecularly enabling this mode of environmental responsiveness.
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Design principles for transposition flaps: the rhombic (single-lobed), bilobed, and trilobed flaps.
Dermatol Surg
PUBLISHED: 08-28-2014
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When tension at a cutaneous defect is too great for primary closure or causes distortion of surrounding anatomy, transposition flaps provide a useful reconstruction option.
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Bacterial fluoride resistance, Fluc channels, and the weak acid accumulation effect.
J. Gen. Physiol.
PUBLISHED: 08-27-2014
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Fluoride ion (F(-)) is a ubiquitous environmental threat to microorganisms, which have evolved a family of highly selective "Fluc" F(-) channels that export this inhibitory anion from their cytoplasm. It is unclear, however, how a thermodynamically passive mechanism like an ion channel can protect against high concentrations of external F(-). We monitored external F(-) concentrations in Escherichia coli suspensions and showed that, in bacteria lacking Fluc, F(-) accumulates when the external medium is acidified, as a predicted function of the transmembrane pH gradient. This weak acid accumulation effect, which results from the high pKa (3.4) and membrane permeability of HF, is abolished by Fluc channels. We also found that, although bacterial growth is inhibited by high concentrations of F(-), bacteria can withstand cytoplasmic F(-) at levels a hundred times higher than those that inhibit proliferation, resuming growth when the F(-) challenge is removed.
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SciClone: inferring clonal architecture and tracking the spatial and temporal patterns of tumor evolution.
PLoS Comput. Biol.
PUBLISHED: 08-07-2014
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The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.
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Molecular alterations of PIK3CA in uterine carcinosarcoma, clear cell, and serous tumors.
Int. J. Gynecol. Cancer
PUBLISHED: 08-01-2014
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Type II endometrial carcinomas-uterine carcinosarcomas or uterine malignant mesodermal mixed tumors (UMMMTs), clear cell carcinomas (UCCs), and uterine serous carcinomas (USCs)-are aggressive malignancies that present with advanced disease and have high mortality rates. PIK3CA mutations are commonly found in endometrial cancers. The objective of the study was to characterize molecular alterations in the PIK3CA gene in these tumors.
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Local fasciocutaneous sliding flaps for soft-tissue defects of the dorsum of the hand.
JAMA Dermatol
PUBLISHED: 07-31-2014
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Appropriate coverage of defects that expose tendon, joints, and/or neurovascular structures is necessary to preserve optimal hand function. Local, random-pattern flaps and skin grafts may be inadequate because of the hand's finite skin reservoir or the presence of a poorly vascularized and mobile wound bed. Described herein is a novel method of dorsal hand reconstruction.
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F-/Cl- selectivity in CLCF-type F-/H+ antiporters.
J. Gen. Physiol.
PUBLISHED: 07-30-2014
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Many bacterial species protect themselves against environmental F(-) toxicity by exporting this anion from the cytoplasm via CLC(F) F(-)/H(+) antiporters, a subclass of CLC superfamily anion transporters. Strong F(-) over Cl(-) selectivity is biologically essential for these membrane proteins because Cl(-) is orders of magnitude more abundant in the biosphere than F(-). Sequence comparisons reveal differences between CLC(F)s and canonical Cl(-)-transporting CLCs within regions that, in the canonical CLCs, coordinate Cl(-) ion and govern anion transport. A phylogenetic split within the CLC(F) clade, manifested in sequence divergence in the vicinity of this ion-binding center, raises the possibility that these two CLC(F) subclades might exhibit differences in anion selectivity. Several CLC(F) homologues from each subclade were examined for F(-)/Cl(-) selectivity of anion transport and equilibrium binding. Differences in both of these anion-selectivity metrics correlate with sequence divergence among CLC(F)s. Chimeric constructs identify two residues in this region that largely account for the subclade differences in selectivity. In addition, these experiments serendipitously uncovered an unusually steep, Cl(-)-specific voltage dependence of transport that greatly enhances F(-) selectivity at low voltage.
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Multiparametric cardiovascular magnetic resonance surveillance of acute cardiac allograft rejection and characterisation of transplantation-associated myocardial injury: a pilot study.
J Cardiovasc Magn Reson
PUBLISHED: 07-20-2014
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Serial surveillance endomyocardial biopsies are performed in patients who have recently undergone heart transplantation in order to detect acute cardiac allograft rejection (ACAR) before symptoms occur, however the biopsy process is associated with a number of limitations. This study aimed to prospectively and longitudinally evaluate the performance of multiparametric cardiovascular magnetic resonance (CMR) for detecting and monitoring ACAR in the early phase post-transplant, and characterize graft recovery following transplantation.
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Self-efficacy and quality of life among people with bipolar disorder.
J. Nerv. Ment. Dis.
PUBLISHED: 07-11-2014
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People with bipolar disorders report a lower quality of life than the general population does, and few mutable factors associated with health-related quality of life (HRQoL) among people with bipolar disorders have been identified. Using a cross-sectional design, these analyses examined whether self-efficacy was associated with mental and physical HRQoL in a sample of 141 patients with bipolar disorder who completed baseline assessments for two randomized controlled trials. Multiple linear regression analyses indicated that higher levels of self-efficacy were associated with higher mental and physical HRQoL, after controlling for demographic factors and clinical factors (including mood symptoms, comorbid medical conditions, and substance use). Future research should examine whether targeted treatments that aim to improve self-efficacy (such as self-management interventions) lead to improvements in HRQoL among people with bipolar disorder and other serious mental illnesses.
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Clonal architecture of secondary acute myeloid leukemia defined by single-cell sequencing.
PLoS Genet.
PUBLISHED: 07-01-2014
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Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions-the population frequency of individual clones, their genetic composition, and their evolutionary relationships-which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.
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Cardiovascular magnetic resonance validation of fractional changes in annulo-apical angles and tricuspid annular plane systolic excursion for rapid assessment of right ventricular systolic function.
J Magn Reson Imaging
PUBLISHED: 06-14-2014
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To evaluate the use of right ventricular (RV) annulo-apical angle (AA) changes acquired by magnetic resonance imaging (MRI), alongside tricuspid annular plane systolic excursion (TAPSE), for its association with RV systolic function.
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Proof of dual-topology architecture of Fluc F- channels with monobody blockers.
Nat Commun
PUBLISHED: 06-05-2014
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Fluc-type F(-) channels--used by microorganisms for resisting fluoride toxicity--are unusual in their quaternary architecture: they are thought to associate as dimers with the two subunits in antiparallel transmembrane orientation. Here, we subject this unusual structural feature to a direct test. Single purified Fluc channels recorded in planar lipid bilayers are constitutively open, with rare, short-lived closings. Using combinatorial libraries, we generated synthetic binding proteins, 'monobodies,' that specifically bind to Fluc homologues with nanomolar affinity. Reversible binding of monobodies to two different Fluc channel homologues is seen in single-channel recordings as long-lived nonconducting events that follow bimolecular kinetics. By applying monobodies sequentially to the two sides of the bilayer in a double-sided perfusion manoeuvre, we show that Fluc channels present monobody-binding epitopes to both sides of the membrane. The result establishes that Fluc subunits are arranged in dimeric antiparallel orientation.
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Age-related mutations associated with clonal hematopoietic expansion and malignancies.
Nat. Med.
PUBLISHED: 05-29-2014
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Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. The Cancer Genome Atlas (TCGA) provides a unique resource for comprehensive discovery of mutations and genes in blood that may contribute to the clonal expansion of hematopoietic stem/progenitor cells. Here, we analyzed blood-derived sequence data from 2,728 individuals from TCGA and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia and/or lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5-6% of people older than 70 years) contain mutations that may represent premalignant events that cause clonal hematopoietic expansion.
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Morbidity and Readmission After Open Reduction and Internal Fixation of Ankle Fractures Are Associated With Preoperative Patient Characteristics.
Clin. Orthop. Relat. Res.
PUBLISHED: 05-23-2014
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Ankle fractures are common and can be associated with severe morbidity. Risk factors for short-term adverse events and readmission after open reduction and internal fixation (ORIF) of ankle fractures have not been fully characterized.
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Enhanced In Vitro Transcytosis of Simian Immunodeficiency Virus Mediated by Vaccine-Induced Antibody Predicts Transmitted/Founder Strain Number After Rectal Challenge.
J. Infect. Dis.
PUBLISHED: 05-21-2014
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?The time to acquisition of simian immunodeficiency virus (SIV) infection following low-dose repeated rectal challenge correlated inversely with the number of transmitted/founder strains among macaques vaccinated with ALVAC-SIV/gp120 or gp120 alone. We determined if the ability of postvaccination, prechallenge sera to enhance SIVmac251 transcytosis across epithelial cells was associated with transmitted/founder strain number.
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Effect of solution and solid-phase conditions on the Fe(II)-accelerated transformation of ferrihydrite to lepidocrocite and goethite.
Environ. Sci. Technol.
PUBLISHED: 04-30-2014
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Aqueous ferrous iron (Fe(II)) accelerates the transformation of ferrihydrite into secondary, more crystalline minerals however the factors controlling the rate and, indeed, the underlying mechanism of this transformation process remain unclear. Here, we present the first detailed study of the kinetics of the Fe(II)-accelerated transformation of ferrihydrite to goethite, via lepidocrocite, for a range of pH and Fe(II) concentrations and, from the results obtained, provide insight into the factors controlling the transformation rate and the processes responsible for transformation. A reaction scheme for the Fe(II)-accelerated secondary mineralization of ferrihydrite is developed in which an Fe(II) atom attaches to the ferrihydrite surface where it is immediately oxidized to Fe(III) with the resultant electron transferred, sequentially, to other iron oxyhydroxide Fe(III) atoms before release to solution as Fe(II). This freshly precipitated Fe(III) forms the nuclei for the formation of secondary minerals and also facilitates the ongoing uptake of Fe(II) from solution by creation of fresh surface sites. The concentration of solid-associated Fe(II) and the rate of transport of Fe(II) to the oxyhydroxide surface appear to determine which particular secondary minerals form and their rates of formation. Lepidocrocite growth is enhanced at lower solid-associated Fe(II) concentrations while conditions leading to more rapid uptake of Fe(II) from solution lead to higher goethite growth rates.
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Bone marrow stromal cell adhesion and morphology on micro- and sub-micropatterned titanium.
J Biomed Nanotechnol
PUBLISHED: 04-17-2014
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The objective of this study was to investigate the adhesion and morphology of bone marrow derived stromal cells (BMSCs) on bulk titanium (Ti) substrates with precisely-patterned surfaces consisting of groove-based gratings with groove widths ranging from 50 micro m down to 0.5 micro m (500 nm). Although it is well known that certain surface patterning enhances osteoblast (bone-forming cell) functions, past studies on cell-pattern interactions reported in the literature have heavily relied on surface patterning on materials with limited clinical relevance for orthopedic applications, such as polymeric substrates. The clinical need for improving osseointegration and juxtaposed bone formation around load-bearing Ti implants motivated this in vitro study. BMSCs were selected as model cells due to their important role in bone regeneration. The results showed significantly greater BMSC adhesion density and more favorable cell morphology on sub-micropatterned gratings when compared with larger micropatterned gratings and non-patterned control surfaces after both 24 hr and 72 hr cultures. We observed increasing cellular alignment and elongation with decreasing feature size. We also identified two distinctive cellular morphologies: Type I-Attached and spread cells that elongated along the pattern axes; and Type II-Superficially adhered round cells. Sub-micropatterned gratings demonstrated significantly greater Type I cell density than the non-patterned control, and lower Type II cell density than the larger micropatterned gratings. Collectively, these results suggest potential for rationally designing nano-scale surface topography on Ti implants to improve osseointegration.
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Kinetics and mechanism of auto- and copper-catalyzed oxidation of 1,4-naphthohydroquinone.
Free Radic. Biol. Med.
PUBLISHED: 02-19-2014
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Although quinones represent a class of organic compounds that may exert toxic effects both in vitro and in vivo, the molecular mechanisms involved in quinone species toxicity are still largely unknown, especially in the presence of transition metals, which may both induce the transformation of the various quinone species and result in generation of harmful reactive oxygen species. In this study, the oxidation of 1,4-naphthohydroquinone (NH2Q) in the absence and presence of nanomolar concentrations of Cu(II) in 10 mM NaCl solution over a pH range of 6.5-7.5 has been investigated, with detailed kinetic models developed to describe the predominant mechanisms operative in these systems. In the absence of copper, the apparent oxidation rate of NH2Q increased with increasing pH and initial NH2Q concentration, with concomitant oxygen consumption and peroxide generation. The doubly dissociated species, NQ(2-), has been shown to be the reactive species with regard to the one-electron oxidation by O2 and comproportionation with the quinone species, both generating the semiquinone radical (NSQ(·-)). The oxidation of NSQ(·-) by O2 is shown to be the most important pathway for superoxide (O2(·-)) generation with a high intrinsic rate constant of 1.0×10(8)M(-1)s(-1). Both NSQ(·-) and O2(·-) served as chain-propagating species in the autoxidation of NH2Q. Cu(II) is capable of catalyzing the oxidation of NH2Q in the presence of O2 with the oxidation also accelerated by increasing the pH. Both the uncharged (NH2Q(0)) and the mono-anionic (NHQ(-)) species were found to be the kinetically active forms, reducing Cu(II) with an intrinsic rate constant of 4.0×10(4) and 1.2×10(7)M(-1)s(-1), respectively. The presence of O2 facilitated the catalytic role of Cu(II) by rapidly regenerating Cu(II) via continuous oxidation of Cu(I) and also by efficient removal of NSQ(·-) resulting in the generation of O2(·-). The half-cell reduction potentials of various redox couples at neutral pH indicated good agreement between thermodynamic and kinetic considerations for various key reactions involved, further validating the proposed mechanisms involved in both the autoxidation and the copper-catalyzed oxidation of NH2Q in circumneutral pH solutions.
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High-throughput miniaturized bioreactors for cell culture process development: reproducibility, scalability, and control.
Biotechnol. Prog.
PUBLISHED: 02-05-2014
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Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr™) is an automated micro-bioreactor system with miniature single-use bioreactors with a 10-15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in-line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr™ was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr™ system as a high throughput system for cell culture process development.
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The spectrum of eye disease in hospitalized adults living with HIV, 1995-2010.
AIDS Patient Care STDS
PUBLISHED: 02-03-2014
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Eye disease is a well-documented complication of HIV infection. Opportunistic infections generally comprised the majority of pre-antiretroviral therapy (ART) eye complications. With the introduction of ART, opportunistic infections diminished. However, early ART regimens were cumbersome regarding side effects and pill burden, making patient compliance difficult. Newer ART regimens are better tolerated and consist of fewer pills, theoretically making compliance easier and therapy more effective. The aim of this chart review study is to examine eye disease epidemiology in HIV patients as ART has evolved. We reviewed 222 admissions at Thomas Jefferson University Hospitals for 188 patients. These cases were divided into two groups. The first group was comprised of patients admitted from 1995 through 2003, while the second group consisted of patients admitted from 2003 to 2010. Eye disease epidemiology was compared between the two groups. Our study did note a significant decrease in eye diseases caused by opportunistic infections in the 2003-2010 patient group. Noninfectious eye disease is a significant complication in this group.
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Functional heterogeneity of genetically defined subclones in acute myeloid leukemia.
Cancer Cell
PUBLISHED: 01-31-2014
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The relationships between clonal architecture and functional heterogeneity in acute myeloid leukemia (AML) samples are not yet clear. We used targeted sequencing to track AML subclones identified by whole-genome sequencing using a variety of experimental approaches. We found that virtually all AML subclones trafficked from the marrow to the peripheral blood, but some were enriched in specific cell populations. Subclones showed variable engraftment potential in immunodeficient mice. Xenografts were predominantly comprised of a single genetically defined subclone, but there was no predictable relationship between the engrafting subclone and the evolutionary hierarchy of the leukemia. These data demonstrate the importance of integrating genetic and functional data in studies of primary cancer samples, both in xenograft models and in patients.
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Integrated analysis of germline and somatic variants in ovarian cancer.
Nat Commun
PUBLISHED: 01-23-2014
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We report the first large-scale exome-wide analysis of the combined germline-somatic landscape in ovarian cancer. Here we analyse germline and somatic alterations in 429 ovarian carcinoma cases and 557 controls. We identify 3,635 high confidence, rare truncation and 22,953 missense variants with predicted functional impact. We find germline truncation variants and large deletions across Fanconi pathway genes in 20% of cases. Enrichment of rare truncations is shown in BRCA1, BRCA2 and PALB2. In addition, we observe germline truncation variants in genes not previously associated with ovarian cancer susceptibility (NF1, MAP3K4, CDKN2B and MLL3). Evidence for loss of heterozygosity was found in 100 and 76% of cases with germline BRCA1 and BRCA2 truncations, respectively. Germline-somatic interaction analysis combined with extensive bioinformatics annotation identifies 222 candidate functional germline truncation and missense variants, including two pathogenic BRCA1 and 1 TP53 deleterious variants. Finally, integrated analyses of germline and somatic variants identify significantly altered pathways, including the Fanconi, MAPK and MLL pathways.
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Voxel-wise quantification of myocardial blood flow with cardiovascular magnetic resonance: effect of variations in methodology and validation with positron emission tomography.
J Cardiovasc Magn Reson
PUBLISHED: 01-13-2014
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Quantitative assessment of myocardial blood flow (MBF) from cardiovascular magnetic resonance (CMR) perfusion images appears to offer advantages over qualitative assessment. Currently however, clinical translation is lacking, at least in part due to considerable disparity in quantification methodology. The aim of this study was to evaluate the effect of common methodological differences in CMR voxel-wise measurement of MBF, using position emission tomography (PET) as external validation.
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The R882H DNMT3A mutation associated with AML dominantly inhibits wild-type DNMT3A by blocking its ability to form active tetramers.
Cancer Cell
PUBLISHED: 01-08-2014
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Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in ?30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 within the catalytic domain (most commonly R882H), suggesting the possibility of dominant-negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by ?80% compared with the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity, but coexpression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes.
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Development of a safe dexamethasone-eluting electrode array for cochlear implantation.
Cochlear Implants Int
PUBLISHED: 01-03-2014
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Cochlear implantation can result in trauma leading to increased tissue response and loss of residual hearing. A single intratympanic application of the corticosteroid dexamethasone is sometimes used clinically during surgery to combat the potential effect of trauma on residual hearing. This project looked at the safety and efficacy of dexamethasone eluted from an intracochlear array in vivo.
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Clonal architectures and driver mutations in metastatic melanomas.
PLoS ONE
PUBLISHED: 01-01-2014
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To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS) and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1), protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT), as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2). Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3' base of dipyrimidine sequences while one patient (MEL9) with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.
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Satsurblia: New Insights of Human Response and Survival across the Last Glacial Maximum in the Southern Caucasus.
PLoS ONE
PUBLISHED: 01-01-2014
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The region of western Georgia (Imereti) has been a major geographic corridor for human migrations during the Middle and Upper Palaeolithic (MP/UP). Knowledge of the MP and UP in this region, however, stems mostly from a small number of recent excavations at the sites of Ortvale Klde, Dzudzuana, Bondi, and Kotias Klde. These provide an absolute chronology for the Late MP and MP-UP transition, but only a partial perspective on the nature and timing of UP occupations, and limited data on how human groups in this region responded to the harsh climatic oscillations between 37,000-11,500 years before present. Here we report new UP archaeological sequences from fieldwork in Satsurblia cavein the same region. A series of living surfaces with combustion features, faunal remains, stone and bone tools, and ornaments provide new information about human occupations in this region (a) prior to the Last Glacial Maximum (LGM) at 25.5-24.4 ka cal. BP and (b) after the LGM at 17.9-16.2 ka cal. BP. The latter provides new evidence in the southern Caucasus for human occupation immediately after the LGM. The results of the campaigns in Satsurblia and Dzudzuana suggest that at present the most plausible scenario is one of a hiatus in the occupation of this region during the LGM (between 24.4-17.9 ka cal. BP). Analysis of the living surfaces at Satsurblia offers information about human activities such as the production and utilisation of lithics and bone tools, butchering, cooking and consumption of meat and wild cereals, the utilisation of fibers, and the use of certain woods. Microfaunal and palynological analyses point to fluctuations in the climate with consequent shifts in vegetation and the faunal spectrum not only before and after the LGM, but also during the two millennia following the end of the LGM.
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Infection with host-range mutant adenovirus 5 suppresses innate immunity and induces systemic CD4+ T cell activation in rhesus macaques.
PLoS ONE
PUBLISHED: 01-01-2014
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Ad5 is a common cause of respiratory disease and an occasional cause of gastroenteritis and conjunctivitis, and seroconversion before adolescence is common in humans. To gain some insight into how Ad5 infection affects the immune system of rhesus macaques (RM) 18 RM were infected with a host-range mutant Ad5 (Ad5hr) by 3 mucosal inoculations. There was a delay of 2 to 6 weeks after the first inoculation before plasmacytoid dendritic cell (pDC) frequency and function increased in peripheral blood. Primary Ad5hr infection suppressed IFN-? mRNA expression, but the second Ad5hr exposure induced a rapid increase in IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC). Primary Ad5hr infection suppressed CCL20, TNF and IL-1 mRNA expression in PBMC, and subsequent virus exposures further dampened expression of these pro-inflammatory cytokines. Primary, but not secondary, Ad5hr inoculation increased the frequency of CXCR3+ CD4+ T cells in blood, while secondary, but not primary, Ad5hr infection transiently increased the frequencies of Ki67+, HLADR+ and CD95+/CCR5+ CD4+ T cells in blood. Ad5hr infection induced polyfunctional CD4 and CD8+ T cells specific for the Ad5 hexon protein in all of the animals. Thus, infection with Ad5hr induced a complex pattern of innate and adaptive immunity in RM that included transient systemic CD4+ T cell activation and suppressed innate immunity on re-exposure to the virus. The complex effects of adenovirus infection on the immune system may help to explain the unexpected results of testing Ad5 vector expressing HIV antigens in Ad5 seropositive people.
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Adjudicated morbidity and mortality outcomes by age among individuals with HIV infection on suppressive antiretroviral therapy.
PLoS ONE
PUBLISHED: 01-01-2014
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Non-AIDS conditions such as cardiovascular disease and non-AIDS defining cancers dominate causes of morbidity and mortality among persons with HIV on suppressive combination antiretroviral therapy. Accurate estimates of disease incidence and of risk factors for these conditions are important in planning preventative efforts.
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Transient Increase of Interferon-Stimulated Genes and No Clinical Benefit by Chloroquine Treatment During Acute Simian Immunodeficiency Virus Infection of Macaques.
AIDS Res. Hum. Retroviruses
PUBLISHED: 12-24-2013
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Abstract Simian immunodeficiency virus (SIV) infection leads to AIDS in experimentally infected Rhesus macaques similarly to HIV-infected humans. In contrast, SIV infection of natural hosts is characterized by a down-regulation of innate acute responses to the virus within a few weeks of infection and results in limited pathology. Chloroquine (CQ) has been used in the treatment or prevention of malaria and has recently been shown to cause a decrease of immune activation and CD4 cell loss in HIV-infected individuals treated with antiretroviral therapy. Here, we treated Rhesus macaques with CQ during the acute phase of SIVmac251 infection with the intent to decrease viral-induced immune activation and possibly limit disease progression. Contrary to what was expected, CQ treatment resulted in a temporary increased expression of interferon (IFN)-stimulating genes and it worsened the recovery of CD4(+) T cells in the blood. Our findings confirm recent results observed in asymptomatic HIV-infected patients and suggest that CQ does not provide an obvious benefit in the absence of antiretroviral therapy.
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Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1.
J. Gen. Physiol.
PUBLISHED: 12-16-2013
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The leucine zipper, EF hand-containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca(2+) and K(+) homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca(2+) fluorophore and (45)Ca(2+)-based assays, we demonstrate directly that Letm1 is a Ca(2+) transporter, with apparent affinities of cations in the sequence of Ca(2+) ? Mn(2+) > Gd(3+) ? La(3+) > Sr(2+) > Ba(2+), Mg(2+), K(+), Na(+). Kinetic analysis yields a Letm1 turnover rate of 2 Ca(2+)/s and a Km of ?25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca(2+)/2 H(+) antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na(+)/Ca(2+) exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H(+)-dependent Ca(2+) transport mechanism identified in intact mitochondria.
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Efficacy of Influenza Vaccination of Elderly Rhesus Macaques Is Dramatically Improved by Addition of a Cationic Lipid/DNA Adjuvant.
J. Infect. Dis.
PUBLISHED: 10-17-2013
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Background.?The decreased immune response among elderly individuals results in reduced influenza vaccine efficacy. Strategies to improve vaccine efficacy in elderly individuals are needed. The goal of this study was to determine whether a cationic lipid/DNA complex (CLDC) can improve the efficacy of the trivalent inactivated influenza vaccine Fluzone in elderly nonhuman primates. Methods.?Elderly (age, >18 years) rhesus macaques were vaccinated with Fluzone, with or without CLDC, and challenged with a human seasonal influenza virus isolate, A/Memphis/7/2001(H1N1). Results.?We found that elderly macaques have significantly lower levels of circulating naive CD4(+) T cells, naive CD8(+) T cells, and B cells as compared to juvenile monkeys. Furthermore, on the day of challenge, recipients of Fluzone/CLDC had significantly higher plasma anti-influenza virus immunoglobulin G (P < .001) and immunoglobulin A (P < .001) titers than recipients of Fluzone alone. After virus challenge, only the Fluzone/CLDC-vaccinated animals had a significantly lower level of virus replication (P < .01) relative to the unvaccinated control animals. Conclusions.?These results demonstrate that CLDC can enhance the immunogenicity and efficacy of a licensed TIV in immunosenescent elderly monkeys.
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Accuracy and Completeness of Patient Information in Organic World-Wide Web Search for Mohs Surgery: A Prospective Cross-Sectional Multirater Study Using Consensus Criteria.
Dermatol Surg
PUBLISHED: 10-09-2013
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Many patients obtain medical information from the Internet. Inaccurate information affects patient care and perceptions.
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Vismodegib: a hedgehog pathway inhibitor for locally advanced and metastatic basal cell carcinomas.
J Drugs Dermatol
PUBLISHED: 10-03-2013
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Basal cell carcinomas (BCCs) are the most common cancer in the United States, and the overwhelming majority of BCCs are the result of hedgehog pathway activation. While locally advanced and metastatic BCC are rare, currently available treatments remain limited and are often unsuccessful. Vismodegib inhibits a key regulatory protein in the hedgehog pathway and was approved by the United States Food and Drug Administration in 2012. This orally-administered medication offers a novel approach for treating locally advanced and metastatic BCC. The following review will address vismodegibs mechanism of action, published clinical trial data, and the questions that still remain unanswered about this new medication.
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Community mental health provider modifications to cognitive therapy: implications for sustainability.
Psychiatr Serv
PUBLISHED: 10-02-2013
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This study identified modifications to an evidence-based psychosocial treatment (cognitive therapy) within a community mental health system after clinicians had received intensive training and consultation.
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Collaborative chronic care models for mental health conditions: cumulative meta-analysis and metaregression to guide future research and implementation.
Med Care
PUBLISHED: 08-14-2013
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Prior meta-analysis indicates that collaborative chronic care models (CCMs) improve mental and physical health outcomes for individuals with mental disorders. This study aimed to investigate the stability of evidence over time and identify patient and intervention factors associated with CCM effects to facilitate implementation and sustainability of CCMs in clinical practice.
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Neandertals made the first specialized bone tools in Europe.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-12-2013
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Modern humans replaced Neandertals ?40,000 y ago. Close to the time of replacement, Neandertals show behaviors similar to those of the modern humans arriving into Europe, including the use of specialized bone tools, body ornaments, and small blades. It is highly debated whether these modern behaviors developed before or as a result of contact with modern humans. Here we report the identification of a type of specialized bone tool, lissoir, previously only associated with modern humans. The microwear preserved on one of these lissoir is consistent with the use of lissoir in modern times to obtain supple, lustrous, and more impermeable hides. These tools are from a Neandertal context proceeding the replacement period and are the oldest specialized bone tools in Europe. As such, they are either a demonstration of independent invention by Neandertals or an indication that modern humans started influencing European Neandertals much earlier than previously believed. Because these finds clearly predate the oldest known age for the use of similar objects in Europe by anatomically modern humans, they could also be evidence for cultural diffusion from Neandertals to modern humans.
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Iron redox transformations in continuously photolyzed acidic solutions containing natural organic matter: kinetic and mechanistic insights.
Environ. Sci. Technol.
PUBLISHED: 08-02-2013
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In this work, the various pathways contributing to the formation and decay of Fe(II) in photolyzed acidic solutions containing Suwannee River fulvic acid (SRFA) are investigated. Results of experimental and computational studies suggest that ligand to metal charge transfer (LMCT), superoxide-mediated iron reduction and interaction with reduced organic species that are present intrinsically in SRFA each contribute to Fe(III) reduction with LMCT the most likely dominant pathway under these conditions. Fe(II) oxidation occurs as a result of its interaction with a variety of light-generated species including (i) short-lived organic species, (ii) relatively stable semiquinone-like organic species, and (iii) hydroperoxy radicals. While not definitive, a hypothesis that the short-lived organic species are similar to peroxyl radicals appears most consistent with our experimental and modeling results. The semiquinone-like organic species formed during photolysis by superoxide-mediated oxidation of reduced organic moieties are long-lived in the dark but prone to rapid oxidation by singlet oxygen ((1)O2) under irradiated conditions and thus play a minor role in Fe(II) oxidation in the light. A kinetic model is developed that adequately describes all aspects of the experimental data obtained and which is capable of predicting Fe(II) oxidation rates and Fe(III) reduction rates in the presence of natural organic matter and light.
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Cardiac MRI of patients with implanted electrical cardiac devices.
Heart
PUBLISHED: 07-19-2013
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Implantable pulse generators and defibrillators have traditionally been considered contraindications to MRI. However, recent data have challenged this paradigm and demonstrated that patients with newer generation devices can safely undergo MRI, including cardiac MRI, provided basic precautions are taken. Indeed, the introduction of MRI conditional systems has led to a conceptual shift in clinical decision making-can this patient undergo MRI safely? is being superseded by should this patient be implanted with an MRI conditional device?. This review outlines the risks associated with MRI in patients with implanted cardiac devices, and discusses practical measures to minimise risks and facilitate safe and diagnostic scanning.
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New directions for HIV vaccine development from animal models.
Curr Opin HIV AIDS
PUBLISHED: 07-10-2013
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The development of a preventive HIV vaccine remains an unresolved challenge. Animal models that can predict the results of HIV vaccine efficacy trials and identify the immune mechanisms responsible for vaccine protection would be most useful for HIV vaccine development. The purpose of the current review is to critique recent developments in the use of animal models of HIV infection in preclinical studies of AIDS vaccines and to describe how the use of improved animal models can inform the development of an HIV vaccine.
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Copper-catalyzed hydroquinone oxidation and associated redox cycling of copper under conditions typical of natural saline waters.
Environ. Sci. Technol.
PUBLISHED: 07-10-2013
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A detailed kinetic model has been developed to describe the oxidation of Cu(I) by O2 and the reduction of Cu(II) by 1,4-hydroquinone (H2Q) in the presence of O2 in 0.7 M NaCl solution over a pH range of 6.5-8.0. The reaction between Cu(I) and O2 is shown to be the most important pathway in the overall oxidation of Cu(I), with the rate constant for this oxidation process increasing with an increasing pH. In 0.7 M NaCl solutions, Cu(II) is capable of catalyzing the oxidation of H2Q in the presence of O2 with the monoanion, HQ(-), the kinetically active hydroquinone form, reducing Cu(II) with an intrinsic rate constant of (5.0 ± 0.4) × 10(7) M(-1) s(-1). Acting as a chain-propagating species, the deprotonated semiquinone radical (SQ(•)?(-)) generated from both the one-electron oxidation of H2Q and the one-electron reduction of 1,4-benzoquinone (BQ) also reacts rapidly with Cu(II) and Cu(I), with the same rate constant of (2.0 ± 0.5) × 10(7) M(-1) s(-1). In addition to its role in reformation of Cu(II) via continuous oxidation of Cu(I), O2 rapidly removes SQ(•)?(-), resulting in the generation of O2(•)?(-). Agreement between half-cell reduction potentials of different redox couples provides confirmation of the veracity of the proposed model describing the interactions of copper and quinone species in circumneutral pH saline solutions.
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[2+2+2] cycloadditions of siloxy alkynes with 1,2-diazines: from reaction discovery to identification of an antiglycolytic chemotype.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 07-02-2013
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Cycloaddition uncovered: The title reaction produces novel polycyclic compounds with high efficiency and excellent diastereoselectivity under mild reaction conditions. A small-molecule library, synthesized using this reaction, yielded a novel chemotype which inhibited glycolytic ATP production by blocking glucose uptake in CHO-K1 cells. DMF=N,N-dimethylformamide, Tf=trifluoromethanesulfonyl, TIPS=triisopropylsilyl.
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Mutational landscape and significance across 12 major cancer types.
Nature
PUBLISHED: 06-21-2013
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The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known (for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase, Wnt/?-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the number of driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment.
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Development of a framework and coding system for modifications and adaptations of evidence-based interventions.
Implement Sci
PUBLISHED: 05-29-2013
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Evidence-based interventions are frequently modified or adapted during the implementation process. Changes may be made to protocols to meet the needs of the target population or address differences between the context in which the intervention was originally designed and the one into which it is implemented [Addict Behav 2011, 36(6):630-635]. However, whether modification compromises or enhances the desired benefits of the intervention is not well understood. A challenge to understanding the impact of specific types of modifications is a lack of attention to characterizing the different types of changes that may occur. A system for classifying the types of modifications that are made when interventions and programs are implemented can facilitate efforts to understand the nature of modifications that are made in particular contexts as well as the impact of these modifications on outcomes of interest.
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Fluoride-dependent interruption of the transport cycle of a CLC Cl-/H+ antiporter.
Nat. Chem. Biol.
PUBLISHED: 05-11-2013
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Cl(-)/H(+) antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl(-), Br(-), I(-), NO3(-) and SCN(-), but they seem to be very selective against F(-). The recent discovery of a new CLC clade of F(-)/H(+) antiporters, which are highly selective for F(-) over Cl(-), led us to investigate the mechanism of Cl(-)-over-F(-) selectivity by a CLC Cl(-)/H(+) antiporter, CLC-ec1. By subjecting purified CLC-ec1 to anion transport measurements, electrophysiological recording, equilibrium ligand-binding studies and X-ray crystallography, we show that F(-) binds in the Cl(-) transport pathway with affinity similar to Cl(-) but stalls the transport cycle. Examination of various mutant antiporters implies a lock-down mechanism of F(-) inhibition, in which F(-), by virtue of its unique hydrogen-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.
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Oral serum-derived bovine immunoglobulin improves duodenal immune reconstitution and absorption function in patients with HIV enteropathy.
AIDS
PUBLISHED: 05-11-2013
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To examine the impact of serum-derived bovine immunoglobulin, an oral medical food known to neutralize bacterial antigen and reduce intestinal inflammation, on restoration of mucosal immunity and gastrointestinal function in individuals with HIV enteropathy.
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Endocrine-therapy-resistant ESR1 variants revealed by genomic characterization of breast-cancer-derived xenografts.
Cell Rep
PUBLISHED: 05-06-2013
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To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.
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DGIdb: mining the druggable genome.
Nat. Methods
PUBLISHED: 04-28-2013
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The Drug-Gene Interaction database (DGIdb) mines existing resources that generate hypotheses about how mutated genes might be targeted therapeutically or prioritized for drug development. It provides an interface for searching lists of genes against a compendium of drug-gene interactions and potentially druggable genes. DGIdb can be accessed at http://dgidb.org/.
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A kinome-wide siRNA screen identifies multiple roles for protein kinases in hypoxic stress adaptation, including roles for IRAK4 and GAK in protection against apoptosis in VHL-/- renal carcinoma cells, despite activation of the NF-?B pathway.
J Biomol Screen
PUBLISHED: 04-16-2013
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Hypoxia induces changes to cancer cells that make them more resistant to treatment. We have looked at signaling pathways that facilitate these changes by screening the human kinome for effects on hypoxic responses in SW480 colon cancer cells. Hits identified in the screen were examined for effects on multiple molecular responses to hypoxia, including the endoplasmic reticulum stress and DNA damage responses in colon, melanoma, and renal cancer lines. To validate the hits from the small interfering RNA studies, we developed cell lines expressing stable short hairpin RNAs (shRNAs) in the A498 renal carcinoma cell line. Several lines, including those expressing shRNAs against DYRK1B, GAK, IHPK2, IRAK4, and MATK, showed an inability to form spheroid cultures. In addition, shRNAs targeting IRAK4 and GAK were incapable of 2D growth under anoxia. In the GAK shRNA-expressing line, nuclear factor-?B (NF-?B) was localized to the nucleus, but in the IRAK4 shRNA line, NF-?B levels were increased but the extent of nuclear localization was unchanged. Dominant negative mutants of IRAK4 and GAK also showed strong apoptotic effects in A498 cells under anoxia, supporting a direct link between these kinases and survival of the VHL(-/-) RCC line, which is typically highly resistant to hypoxic stress as a result of high and constitutive levels of Hif-1?.
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Comprehensive validation of cardiovascular magnetic resonance techniques for the assessment of myocardial extracellular volume.
Circ Cardiovasc Imaging
PUBLISHED: 04-03-2013
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Extracellular matrix expansion is a key element of ventricular remodeling and a potential therapeutic target. Cardiovascular magnetic resonance (CMR) T1-mapping techniques are increasingly used to evaluate myocardial extracellular volume (ECV); however, the most widely applied methods are without histological validation. Our aim was to perform comprehensive validation of (1) dynamic-equilibrium CMR (DynEq-CMR), where ECV is quantified using hematocrit-adjusted myocardial and blood T1 values measured before and after gadolinium bolus; and (2) isolated measurement of myocardial T1, used as an ECV surrogate.
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Multiparametric Cardiovascular Magnetic Resonance Assessment of Cardiac Allograft Vasculopathy.
J. Am. Coll. Cardiol.
PUBLISHED: 04-02-2013
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To evaluate the diagnostic performance of multiparametric cardiovascular magnetic resonance (CMR) for detecting cardiac allograft vasculopathy (CAV) using contemporary invasive epicardial artery and microvascular assessment techniques as reference standards, and to compare the performance of CMR with that of angiography.
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Substrate selectivity in glutamate-dependent acid resistance in enteric bacteria.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-25-2013
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The bacterial antiporter GadC plays a central role in the glutamate (Glu)-dependent acid resistance system, which protects enteric bacteria against the extreme acidity of the human stomach. Upon acid shock, GadC imports Glu into the cytoplasm, where Glu decarboxylases consume a cytoplasmic proton, which ends up as a "virtual" proton in the decarboxylated product ?-aminobutyric acid (GABA) and is then exported via GadC. It was therefore proposed that GadC counters intracellular acidification by continually pumping out virtual protons. This scenario, however, is oversimplified. In gastric environments, GadC encounters substrates in multiple carboxyl protonation forms (outside: Glu(-), Glu(0), Glu(+); inside: GABA(0), GABA(+)). Of the six possible combinations of antiport partners, Glu(+)/GABA(0) results in proton influx, Glu(0)/GABA(0) and Glu(+)/GABA(+) are proton neutral, and Glu(-)/GABA(0), Glu(-)/GABA(+), or Glu(0)/GABA(+) lead to proton extrusion. Which of these exchanges does GadC catalyze? To attack this problem, we developed an oriented GadC liposome system holding a three-unit inward pH gradient to mimic the conditions facing bacteria in the stomach. By assessing the electrogenicity of substrate transport, we demonstrate that GadC selectively exchanges Glu(-) or Glu(0) with GABA(+), resulting in effective proton extrusion of >0.9 H(+) per turnover to counter proton invasion into acid-challenged bacteria. We further show that GadC selects among protonated substrates using a charge-based mechanism, rather than directly recognizing the protonation status of the carboxyl groups. This result paves the way for future work to identify the molecular basis of GadCs substrate selectivity.
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Substrate selectivity in arginine-dependent acid resistance in enteric bacteria.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-25-2013
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To successfully colonize the human gut, enteric bacteria must activate acid resistance systems to survive the extreme acidity (pH 1.5-3.5) of the stomach. The antiporter AdiC is the master orchestrator of the arginine-dependent system. Upon acid shock, it imports extracellular arginine (Arg) into the cytoplasm, providing the substrate for arginine decarboxylases, which consume a cellular proton ending up in a C-H bond of the decarboxylated product agmatine (Agm(2+)). Agm(2+) and the "virtual" proton it carries are exported via AdiC subsequently. It is widely accepted that AdiC counters intracellular acidification by continuously pumping out virtual protons. However, in the gastric environment, Arg is present in two carboxyl-protonation forms, Arg(+) and Arg(2+). Virtual proton pumping can only be achieved by Arg(+)/Agm(2+) exchange, whereas Arg(2+)/Agm(2+) exchange would produce no net proton movement. This study experimentally asks which exchange AdiC catalyzes, an issue previously unapproachable due to the absence of a reconstituted system mimicking the situation of bacteria in the stomach. Here, using an oriented liposome system able to hold a three-unit pH gradient, we demonstrate that Arg/Agm exchange by AdiC is strongly electrogenic with positive charge moved outward, and thus that AdiC mainly mediates Arg(+)/Agm(2+) exchange to support effective virtual proton pumping. Further experiments reveal a mechanistic surprise--that AdiC selects Arg(+) against Arg(2+) on the basis of gross valence, rather than by local scrutiny of protonation states of the carboxyl group, as had been suggested by Arg-bound AdiC crystal structures.
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Differential diagnosis of acute pericarditis from normal variant early repolarization and left ventricular hypertrophy with early repolarization: an electrocardiographic study.
Am. J. Med. Sci.
PUBLISHED: 02-21-2013
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Differentiation of ST-segment elevation on electrocardiogram (ECG) from acute pericarditis (AP), normal variant early repolarization (ER) and early repolarization of left ventricular hypertrophy (ERLVH) can be problematic. Hence, the authors evaluated the accuracy of the ST/T ratio in ECG to more optimally differentiate between AP, ST-segment elevation, ER and ERLVH.
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Viral RNA Levels and env Variants in Semen and Tissues of Mature Male Rhesus Macaques Infected with SIV by Penile Inoculation.
PLoS ONE
PUBLISHED: 01-01-2013
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HIV is shed in semen but the anatomic site of virus entry into the genital secretions is unknown. We determined viral RNA (vRNA) levels and the envelope gene sequence in the SIVmac 251 viral populations in the genital tract and semen of 5 adult male rhesus monkeys (Macaca mulatta) that were infected after experimental penile SIV infection. Paired blood and semen samples were collected from 1-9 weeks after infection and the monkeys were necropsied eleven weeks after infection. The axillary lymph nodes, testes, epididymis, prostate, and seminal vesicles were collected and vRNA levels and single-genome analysis of the SIVmac251 env variants was performed. At the time of semen collection, blood vRNA levels were between 3.09 and 7.85 log10 vRNA copies/ml plasma. SIV RNA was found in the axillary lymph nodes of all five monkeys and in 3 of 5 monkeys, all tissues examined were vRNA positive. In these 3 monkeys, vRNA levels (log10 SIVgag copies/ug of total tissue RNA) in the axillary lymph node (6.48±0.50) were significantly higher than in the genital tract tissues: testis (3.67±2.16; p<0.05), epididymis (3.08±1.19; p<0.0001), prostate (3.36±1.30; p<0.01), and seminal vesicle (2.67±1.50; p<0.0001). Comparison of the SIVmac251 env viral populations in blood plasma, systemic lymph node, and genital tract tissues was performed in two of the macaques. Visual inspection of the Neighbor-Joining phylograms revealed that in both animals, all the sequences were generally distributed evenly among all tissue compartments. Importantly, viral populations in the genital tissues were not distinct from those in the systemic tissues. Our findings demonstrate striking similarity in the viral populations in the blood and male genital tract tissues within 3 months of penile SIV transmission.
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Myocarditis, disseminated infection, and early viral persistence following experimental coxsackievirus B infection of cynomolgus monkeys.
PLoS ONE
PUBLISHED: 01-01-2013
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Coxsackievirus B (CVB) infection is a common cause of acute viral myocarditis. The clinical presentation of myocarditis caused by this enterovirus is highly variable, ranging from mildly symptoms to complete hemodynamic collapse. These variations in initial symptoms and in the immediate and long term outcomes of this disease have impeded development of effective treatment strategies. Nine cynomolgus monkeys were inoculated with myocarditic strains of CVB. Virological studies performed up to 28 days post-inoculation demonstrated the development of neutralizing antibody in all animals, and the presence of CVB in plasma. High dose intravenous inoculation (n = 2) resulted in severe disseminated disease, while low dose intravenous (n = 6) or oral infection (1 animal) resulted in clinically unapparent infection. Transient, minor, echocardiographic abnormalities were noted in several animals, but no animals displayed signs of significant acute cardiac failure. Although viremia rapidly resolved, signs of myocardial inflammation and injury were observed in all animals at the time of necropsy, and CVB was detected in postmortem myocardial specimens up to 28 days PI. This non-human primate system replicates many features of illness in acute coxsackievirus myocarditis and demonstrates that myocardial involvement may be common in enteroviral infection; it may provide a model system for testing of treatment strategies for enteroviral infections and acute coxsackievirus myocarditis.
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A family of fluoride-specific ion channels with dual-topology architecture.
Elife
PUBLISHED: 01-01-2013
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Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F(-) exporter proteins to lower cytoplasmic F(-) levels to counteract the anions toxicity. We show here that these Fluc proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F(-) over Cl(-). The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins. DOI:http://dx.doi.org/10.7554/eLife.01084.001.
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Preimplant transthoracic echocardiographic assessment of continuous flow left ventricular assist device.
Echocardiography
PUBLISHED: 12-16-2011
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For many patients with end-stage heart failure, heart transplantation is the only remaining option to prolong survival and provide symptom relief. Transthoracic echo is the modality of choice in assessing a patient for potential left ventricular assist device (LVAD) insertion. There are currently no guidelines available, and assessing this specific patient population can prove extremely challenging. As such, an understanding of LVAD mechanism, the important physiological consequences of device implantation together with the related echocardiographic examination is vital to accurately and effectively gauge correct patient selection and also improve implantation success. This review aims to highlight the common devices implanted, how these devices affect cardiac physiology and hemodynamics, and therefore discuss the major echocardiographic variables that should be assessed predevice implantation. (Echocardiography 2012;29:52-58).
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