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1.  Facilitation by intracellular carbonic anhydrase of Na+–HCO3− co-transport but not Na+/H+ exchange activity in the mammalian ventricular myocyte 
The Journal of Physiology  2014;592(5):991-1007.
Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO2 to H+ and HCO3− ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein–protein binding (a ‘transport metabolon’) or local functional interaction. Flux facilitation has previously been investigated by heterologous co-expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO3− or H+ transport via the native acid-extruding proteins, Na+–HCO3− cotransport (NBC) and Na+/H+ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CAi and CAe) are distinguished using membrane-permeant and –impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na+ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CAi activity alone. Results are quantitatively consistent with a model where CAi catalyses local H+ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO3− ions. In well-superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO2/HCO3− buffer is clamped at equilibrium. The CAi insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer-shuttles supply sufficient intracellular H+ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO3− transport, and hence pHi regulation in the heart.
doi:10.1113/jphysiol.2013.265439
PMCID: PMC3948559  PMID: 24297849
2.  Molecular weight dependent vertical composition profiles of PCDTBT:PC71BM blends for organic photovoltaics 
Scientific Reports  2014;4:5286.
We have used Soxhlet solvent purification to fractionate a broad molecular weight distribution of the polycarbazole polymer PCDTBT into three lower polydispersity molecular weight fractions. Organic photovoltaic devices were made using a blend of the fullerene acceptor PC71BM with the molecular weight fractions. An average power conversion efficiency of 5.89% (peak efficiency of 6.15%) was measured for PCDTBT blend devices with a number average molecular weight of Mn = 25.5 kDa. There was significant variation between the molecular weight fractions with low (Mn = 15.0 kDa) and high (Mn = 34.9 kDa) fractions producing devices with average efficiencies of 5.02% and 3.70% respectively. Neutron reflectivity measurements on these polymer:PC71BM blend layers showed that larger molecular weights leads to an increase in the polymer enrichment layer thickness at the anode interface, this improves efficiency up to a limiting point where the polymer solubility causes a reduction of the PCDTBT concentration in the active layer.
doi:10.1038/srep05286
PMCID: PMC4055896  PMID: 24924096
3.  Ten-Year Effects of the ACTIVE Cognitive Training Trial on Cognition and Everyday Functioning in Older Adults 
Objective
To determine the effects of cognitive training on cognitive abilities and everyday function over 10 years.
Design, Setting, and Participants
Ten-year follow-up of a randomized, controlled single-blind trial with 3 intervention groups and a no-contact control group. A volunteer sample of 2832 persons (mean baseline age, 73.6 years; 26% African American) living independently in 6 US cities.
Interventions
Ten-session training for memory, reasoning, or speed-of-processing.; 4-session booster training at 11 and at 35 months after training.
Measurements
Objectively measured cognitive abilities and self-reported and performance-based measures of everyday function.
Results
Participants in each intervention group reported less difficulty with instrumental activities of daily living (IADL) (memory: effect size, 0.48 [99% CI, 0.12-0.84]; reasoning: effect size, 0.38 [99% CI, 0.02-0.74]; speed-of-processing: effect size, 0.36 [99% CI, 0.01-0.72]). At mean age of 82 years, about 60% of trained participants compared to 50% of controls (p<.05) were at or above their baseline level of self-reported IADL function at 10 years. The reasoning and speed-of-processing interventions maintained their effects on their targeted cognitive abilities at 10 years (reasoning: effect size, 0.23 [99% CI, 0.09-0.38]; speed-of-processing: effect size, 0.66 [99% CI, 0.43-0.88]). Memory training effects were no longer maintained for memory performance. Booster training produced additional and durable improvement for the reasoning intervention for reasoning performance (effect size, 0.21 [99% CI, 0.01-0.41]) and the speed-of-processing intervention for speed-of-processing performance (effect size, 0.62 [99% CI, 0.31-0.93]).
Conclusions
Each ACTIVE cognitive intervention resulted in less decline in self-reported IADL compared with the control group. Reasoning and speed, but not memory, training resulted in improved targeted cognitive abilities for 10 years.
doi:10.1111/jgs.12607
PMCID: PMC4055506  PMID: 24417410
cognitive training; elderly; cognitive abilities; everyday function; training maintenance
4.  Modifying Measures Based on Differential Item Functioning (DIF) Impact Analyses 
Journal of aging and health  2012;24(6):1044-1076.
Objectives
Measure modification can impact comparability of scores across groups and settings. Changes in items can affect the percent admitting to a symptom.
Methods
Using item response theory (IRT) methods, well-calibrated items can be used interchangeably, and the exact same item does not have to be administered to each respondent, theoretically permitting wider latitude in terms of modification.
Results
Recommendations regarding modifications vary, depending on the use of the measure. In the context of research, adjustments can be made at the analytic level by freeing and fixing parameters based on findings of differential item functioning (DIF). The consequences of DIF for clinical decision making depend on whether or not the patient’s performance level approaches the scale decision cutpoint. High-stakes testing may require item removal or separate calibrations to ensure accurate assessment.
Discussion
Guidelines for modification based on DIF analyses and illustrations of the impact of adjustments are presented.
doi:10.1177/0898264312436877
PMCID: PMC4030595  PMID: 22422759
differential item functioning; DIF; factorial invariance; impact of DIF; ethnicity; measure modification
5.  Outcomes of Related Donor HLA-Identical or HLA-Haploidentical Allogeneic Blood or Marrow Transplantation for Peripheral T Cell Lymphoma 
The role of allogeneic blood or marrow transplantation (alloBMT) for peripheral T cell lymphoma (PTCL) remains to be defined. There is growing interest in reduced-intensity conditioning (RIC) regimens and/or utilization of human leukocyte antigen haploidentical (haplo) grafts given concerns about treatment-associated toxicities and donor availability. We reviewed the outcomes of 44 consecutive, related donor alloBMTs for PTCL performed at Johns Hopkins Hospital from 1994 to 2011, including 18 RIC/haplo alloBMTs. Patients receiving RIC (n = 24) were older, with median age of 59 years (range, 24 to 70), than patients receiving myeloablative conditioning (MAC, n = 20), with median age of 46 years (range, 18 to 64), P =.01. The median age at RIC/haplo alloBMT was 60 years. The estimated 2-year progression-free survival (PFS) was 40% (95% confidence interval [CI], 26% to 55%) and overall survival (OS) was 43% (95% CI, 28% to 59%). In older patients (≥60, n = 14), the estimated 2-year PFS and OS were 38% (95% CI, 18% to 79%) and 45% (95% CI, 24% to 86%), respectively. On unadjusted analysis, there was a tendency toward superior outcomes for alloBMT in first remission versus beyond first remission, with an estimated 2-year PFS of 53% (95% CI, 33% to 77%) versus 29% (95% CI, 9% to 45%), P = .08. On competing risk analysis, the 1-year cumulative incidence of relapse was 38% for MAC/HLA-identical alloBMTs and 34% for RIC/haplo alloBMTs. Estimated 1-year nonrelapse mortality was 10% for MAC and 8% for RIC (11% for RIC/haplo alloBMT). On unadjusted landmark analysis, patients with acute grade II-IV or chronic graft-versus-host disease (GVHD) had a 17% probability of relapse (95% CI, 0% to 39%), compared with 66% (95% CI, 48% to 84%) in patients without GVHD, P = .04. Utilization of RIC and alternative donors expands treatment options in PTCL to those who are older and unable to tolerate high-dose conditioning, with outcomes comparable with approaches using myeloablative regimens and HLA-matched donors. AlloBMT may be appropriate in first remission in select high-risk cases.
doi:10.1016/j.bbmt.2013.01.006
PMCID: PMC4020434  PMID: 23370119
Peripheral T-cell lymphoma; Allogeneic blood or marrow; transplantation; Haploidentical; Reduced-intensity conditioning; Survival outcomes
6.  Phase II Study of Risk-Adapted Therapy of Newly Diagnosed, Aggressive Non-Hodgkin Lymphoma Based on Midtreatment FDG-PET Scanning 
In newly diagnosed aggressive non-Hodgkin lymphoma (NHL), a positive midtreatment fluorine-18 fluorodeoxyglucose positron emission tomography (PET) scan often carries a poor prognosis, with reported 2-year event-free survival (EFS) rates of 0% to 30% after standard therapy. To determine the outcome of early treatment intensification for midtreatment PET-positive disease, a phase II trial of risk-adapted therapy was conducted. Fifty-nine newly diagnosed patients, 98% with B cell lymphoma, had PET/CT performed after 2 or 3 cycles of first-line chemotherapy. Those with negative PET on semiquantitative visual interpretation completed standard therapy. Those with positive PET received platinum-based salvage chemotherapy, high-dose therapy, and autologous stem cell transplantation (ASCT). Midtreatment PET was positive in 33 (56%); 28 received ASCT with an actuarial 2-year EFS of 75% (95% confidence interval, 60%–93%). On intention-to-treat analysis, 2-year EFS was 67% (53%–86%) in all PET-positive patients and 89% (77%–100%) in PET-negative patients. No association was found between the International Prognostic Index category and the midtreatment PET result. The favorable outcome achieved here in historically poor-risk patients warrants further, more definitive investigation of treatment modification based on early PET scanning.
doi:10.1016/j.bbmt.2008.11.026
PMCID: PMC4020440  PMID: 19167684
Lymphoma; Positron emission tomography (PET); Prognosis; Autologous stem cell transplantation
7.  H3K36me3 key to Polycomb-mediated gene silencing in lineage specification 
Nature structural & molecular biology  2012;19(12):1214-1215.
A newly uncovered activity of a family of Polycomb-group proteins provides insight into the mechanisms by which active genes become repressed during the transition from pluripotency to restricted cell fates as stem cells undergo lineage specification.
doi:10.1038/nsmb.2458
PMCID: PMC4019213  PMID: 23211767
8.  Bottom-Up Mass Spectrometry–Based Proteomics as an Investigative Analytical Tool for Discovery and Quantification of Proteins in Biological Samples 
Advances in Wound Care  2013;2(9):549-557.
Objective
The objective of this overview is to introduce bottom-up mass spectrometry (MS)–based proteomics approaches and strategies, widely used in other biomedical research fields, to the wound-healing research community.
Approaches
Two major proteomics workflows are discussed: gel-based and gel-free chromatographic separation to reduce the complexity of the sample at protein and peptide level, respectively, prior to nano–liquid chromatography–tandem mass spectrometry analysis. Other strategies to discover less abundant proteins present in the sample, are also briefly discussed along with label-free and label-incorporated methods for protein quantification. Overall, the experimental workflows are designed and continually improved to increase the number of proteins identifiable and quantifiable.
Discussion
Recent advances and improvements in all areas of proteomics workflow from sample preparation, to acquisition of massive amounts of data, to bioinformatics analysis have made this technology an indispensable tool for in-depth large-scale characterization of complex proteomes. This technology has been successfully applied in studies focusing on biomarker discovery, differential protein expression, protein–protein interactions, and post-translational modifications in complex biological samples such as cerebrospinal fluid, serum and plasma, and urine from patients. The publications from these studies have reported greater number of identified proteins, novel biomarker candidates, and post-translational modifications previously unknown.
Conclusions
The qualitative and quantitative protein analysis of the protein population of wound tissues or fluids at different stages is important in wound healing research. Given the complexities and analytical challenges of these samples, MS-based proteomic workflows further improved with recent advances offer a powerful and attractive technology for this purpose.
doi:10.1089/wound.2012.0384
PMCID: PMC3842888  PMID: 24761338
9.  Impaired Assembly and Post-Translational Regulation of 26S Proteasome in Human End Stage Heart Failure 
Circulation. Heart failure  2013;6(3):10.1161/CIRCHEARTFAILURE.112.000119.
Background
This study examined the hypothesis that 26S proteasome dysfunction in human end stage heart failure is associated with decreased docking of the 19S regulatory particle to the 20S proteasome. Previous studies have demonstrated that 26S proteasome activity is diminished in human end stage heart failure associated with oxidation of the 19S regulatory particle Rpt5 subunit. Docking of the 19S regulatory particle to the 20S proteasome requires functional Rpt subunit ATPase activity and phosphorylation of the α-type subunits.
Methods and Results
An enriched proteasome fraction was prepared from 7 human non-failing and 10 failing heart explants. Native gel electrophoresis assessed docking of 19S- to 20S proteasome revealing three proteasome populations (20S-, 26S-, and 30S proteasomes). In failing hearts, 30S proteasomes were significantly lower (P=0.048) by 37% suggesting diminished docking. Mass spectrometry-based phosphopeptide analysis demonstrated that the relative ratio of phosphorylated:non-phosphorylated α7 subunit (serine250) of the 20S proteasome was significantly less (P=0.011) by almost 80% in failing hearts. Rpt ATPase activity was determined in the enriched fraction and following immunoprecipitation with an Rpt6 antibody. ATPase activity (ρmol PO4/μg protein/h) of the total fraction was lowered from 291 ± 97 to 194 ± 27 and in the immunoprecipitated fraction from 42 ± 12 to 3 ± 2 (P=0.005) in failing hearts.
Conclusions
These studies suggest that diminished 26S activity in failing human hearts may be related to impaired docking of the 19S to the 20S as a result of decreased Rpt subunit ATPase activity and α7 subunit phosphorylation.
doi:10.1161/CIRCHEARTFAILURE.112.000119
PMCID: PMC3864674  PMID: 23515276
ubiquitin proteasome system; heart failure; human; phosphorylation
10.  INTRODUCTION TO PATIENT-REPORTED OUTCOME ITEM BANKS: ISSUES IN MINORITY AGING RESEARCH 
In 2004 NIH awarded contracts to initiate the development of high quality psychological and neuropsychological outcome measures for improved assessment of health-related outcomes. The workshop introduced these measurement development initiatives, the measures created, and the NIH supported resource (Assessment Center) for internet or tablet-based test administration and scoring. Presentation covered: (a) item response theory (IRT) and assessment of test bias, (b) construction of item banks and computerized adaptive testing, and (c) the different ways in which qualitative analyses contribute to the definition of construct domains and the refinement of outcome constructs. The panel discussion included questions about representativeness of samples, and assessment of cultural bias.
doi:10.1586/erp.13.10
PMCID: PMC4004066  PMID: 23570428
11.  Coevolution of brain structures in amnestic mild cognitive impairment 
NeuroImage  2012;0:10.1016/j.neuroimage.2012.10.029.
Network accounts of the progression of Alzheimer’s disease (AD), based on cross-sectional brain imaging observations, postulate that the biological course of the disease is characterized by coordinated spatial patterns of brain change to distributed cognitive networks. This study tests this conjecture by quantifying inter-regional covariance in cortical gray matter atrophy rates in 317 Alzheimer’s Disease Neuroimaging Initiative participants who were clinically diagnosed with amnestic mild cognitive impairment at baseline and underwent serial MRI at 6-month intervals over the course of 2 years. A factor analysis model identified five factors (i.e. groupings of regions) that exhibited highly correlated rates of atrophy. Four groupings approximately corresponded to coordinated change within the posterior default mode network, prefrontal cortex, medial temporal lobe, and regions largely spared by the early pathological course of AD (i.e., sensorimotor and occipital cortex), while the fifth grouping represented diffuse, global atrophy. The data-driven observation of “frontal aging” superimposed upon medial temporal atrophy typical of early AD and default mode network changes supports the view that in individuals at high risk of eventual clinical AD, multiple patterns of distributed neuronal death corresponding to multiple biological substrates may be active.
doi:10.1016/j.neuroimage.2012.10.029
PMCID: PMC3593811  PMID: 23103689
MRI parcellation; longitudinal cortical change; Alzheimer’s disease; exploratory factor analysis; distributed networks
12.  Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy 
PLoS Genetics  2014;10(4):e1004192.
Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms.
Author Summary
The central dogma of biology explains that DNA is transcribed to mRNA that is further translated into protein. Many genome-wide studies have implicated genetic variation that influences gene expression and that ultimately affect downstream complex traits including response to drugs. However, because of technical limitations, few studies have evaluated the contribution of genetic variation on protein expression and ensuing effects on downstream phenotypes. To overcome this challenge, we used a novel technology to simultaneously measure the baseline expression of 441 proteins in lymphoblastoid cell lines and compared them with publicly available genetic data. To further illustrate the utility of this approach, we compared protein-level measurements with chemotherapeutic induced apoptosis and cell-growth inhibition data. This study demonstrates the importance of using protein information to understand the functional consequences of genetic variants identified in genome-wide association studies. This protein data set will also have broad utility for understanding the relationship between other genome-wide studies of complex traits.
doi:10.1371/journal.pgen.1004192
PMCID: PMC3974641  PMID: 24699359
13.  Hyaluronidase expression and biofilm involvement in Staphylococcus aureus UAMS-1 and its sarA, agr and sarA agr regulatory mutants 
Microbiology  2013;159(Pt 4):782-791.
In a previous study, two proteins identified as hyaluronidases were detected in spent media by MS and found to be in greater quantity in the sarA and sarA agr mutant strains when compared with the parent and agr mutant strains of Staphylococcus aureus UAMS-1. In the present study, spent media and total RNA were isolated from UAMS-1 and its regulatory mutants and analysed for hyaluronidase activity and steady-state hyaluronidase (hysA) RNA message levels. Hyaluronidase activity was observed throughout all time points examined regardless of the regulatory effects of sarA and agr but activity was always substantially higher in the sarA and sarA agr mutant strains than in the UAMS-1 parent and agr mutant strains. Northern analysis did not detect hysA message for either the UAMS-1 parent or the agr mutant strains at any time point examined, while steady-state hysA message levels were detected throughout growth for the sarA mutant strain, but only at exponential and early post-exponential growth for the sarA agr mutant strain. An in vitro biofilm plate assay, pre-coated with human plasma as a source of hyaluronic acid, demonstrated no significant increase in biofilm for a sarA mutant strain of S. aureus UAMS-1 defective in hyaluronidase activity when compared with the sarA mutant strain. These data indicate that, while hysA message levels and hyaluronidase activity are elevated in the sarA mutant strains of S. aureus UAMS-1, the increase in activity did not contribute to the biofilm-negative phenotype observed in the sarA mutant strain of S. aureus UAMS-1.
doi:10.1099/mic.0.065367-0
PMCID: PMC3709827  PMID: 23393148
14.  Partially Mismatched Transplantation and Human Leukocyte Antigen Donor-Specific Antibodies 
The presence of donor human leukocyte antigen (HLA)-specific antibodies (DSA) increases engraftment failure risk in partially HLA-mismatched, or HLA-haploidentical, allogeneic marrow(alloBMT) transplantation. As preexisting sensitization to HLA antigens is not well characterized among candidates for HLA-haploidentical alloBMT, we retrospectively evaluated both the incidence and relative strength of DSA in this patient population. Based on correlations of solid-phase antibody assays on the Luminex (Luminex, Austin, TX) platform with actual crossmatch tests, DSA were characterized as weak for results that were consistent with negative flow cytometric crossmatch results or as moderate-to-strong for results consistent with positive flow cytometric or cytotoxicity crossmatches. We evaluated 296 alloBMT candidates; 111 (37.5%) were female. DSA were detected in 43 (14.5%) candidates, mostly among female candidates (42.9% female versus 12.5% male). Moderate-to-strong DSA strength was more frequently encountered when directed against haploidentical donors as compared with mismatched unrelated donors. DSAwere most commonly detected in female patients directed against their children. Because the presence of DSA has been considered prohibitive for HLA-mismatched alloBMT, we additionally report a desensitization methodology used to reduce DSA to negative or weak levels, ie, levels well below those detectable in a flow cytometric crossmatch. Nine patients without other available donors underwent desensitization. Eight who reduced their DSA to negative or weak levels proceeded to alloBMT and achieved full donor engraftment. These data support routine DSA evaluation in all patients considered for mismatched alloBMT; however, for patients with no other viable options, desensitization to weak or negative DSA levels may afford the opportunity for successful transplantation.
doi:10.1016/j.bbmt.2013.01.016
PMCID: PMC3768172  PMID: 23353119
Donor specific antibodies; Haploidentical allogeneic bone marrow transplantation; Desensitization
15.  Implications of Lifecourse Epidemiology for Research on Determinants of Adult Disease 
Public health reviews  2010;32(2):489-511.
Many diseases commonly associated with aging are now thought to have social and physiologic antecedents in early life. Understanding how the timing of exposure to early life risk factors influences later-life health may illuminate mechanisms driving adult health inequalities and identify possible points for effective interventions. Recognizing chronic diseases as developing across the lifecourse also has implications for the conduct of research on adult risk factors for disease. We review alternative conceptual models that describe how the timing of risk factor exposure relates to the development of disease. We propose some expansions of lifecourse models to improve their relevance for research on adult chronic disease, using the relationship between education and adult cognitive decline and dementia as an example. We discuss the important implications each of the lifecourse conceptual models has on study design, analysis, and interpretation of research on aging and chronic diseases. We summarize several research considerations implied by the lifecourse framework, including: advantages of analyzing change in function rather than onset of impairment; the pervasive challenge of survivor bias; the importance of controlling for possible confounding by early life conditions; and the likely heterogeneity in responses of adults to treatment.
PMCID: PMC3955391  PMID: 24639598
Lifecourse epidemiology; aging; chronic disease; models; dementia
16.  Evidence for Steric Regulation of Fibrinogen Binding to Staphylococcus aureus Fibronectin-binding Protein A (FnBPA)* 
The Journal of Biological Chemistry  2014;289(18):12842-12851.
Background: Staphylococcus aureus fibronectin-binding protein A (FnBPA) binds fibronectin and fibrinogen at adjacent sites.
Results: The fibrinogen-binding mechanism is similar but not identical to homologous bacterial proteins. Ternary complex formation by intact fibronectin and fibrinogen on adjacent FnBPA sites could not be demonstrated.
Conclusion: Fibrinogen binding is sterically regulated by fibronectin binding.
Significance: Steric regulation might result in targeting of S. aureus to fibrin clots.
The adjacent fibrinogen (Fg)- and fibronectin (Fn)-binding sites on Fn-binding protein A (FnBPA), a cell surface protein from Staphylococcus aureus, are implicated in the initiation and persistence of infection. FnBPA contains a single Fg-binding site (that also binds elastin) and multiple Fn-binding sites. Here, we solved the structure of the N2N3 domains containing the Fg-binding site of FnBPA in the apo form and in complex with a Fg peptide. The Fg binding mechanism is similar to that of homologous bacterial proteins but without the requirement for “latch” strand residues. We show that the Fg-binding sites and the most N-terminal Fn-binding sites are nonoverlapping but in close proximity. Although Fg and a subdomain of Fn can form a ternary complex on an FnBPA protein construct containing a Fg-binding site and single Fn-binding site, binding of intact Fn appears to inhibit Fg binding, suggesting steric regulation. Given the concentrations of Fn and Fg in the plasma, this mechanism might result in targeting of S. aureus to fibrin-rich thrombi or elastin-rich tissues.
doi:10.1074/jbc.M113.543546
PMCID: PMC4007472  PMID: 24627488
Fibrinogen; Fibronectin; Isothermal Titration Calorimetry; Staphylococcus aureus; Surface Plasmon Resonance (SPR); X-ray Crystallography; FnBPA
17.  Combining Human and Rat Sequences in Her-2 DNA Vaccines Blunts Immune Tolerance and Drives Antitumor Immunity 
Cancer research  2010;70(1):119-128.
Immune tolerance to tumor-associated self-antigens poses a major challenge in the ability to mount an effective cancer vaccine response. To overcome immune tolerance to HER-2, we formulated DNA vaccines that express both human HER-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode HER-2/Neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/c × C57BL/6 F1 (F1), or C57BL/6 (B6) background, which exhibit decreasing immune responsiveness to HER-2. Analysis of various cocktails or hybrid vaccines defined a requirement for particular combination of HER/2/Neu sequences to effectively prime immune effector cells in HER-2 Tg mice. In B6 HER-2 Tg mice, rejection of HER-2–positive tumors protected mice from HER-2–negative tumors, providing evidence of epitope spreading. Our findings show that a strategy of combining heterologous antigen with self-antigens could produce a potent DNA vaccine that may be applicable to other tumor-associated antigens.
doi:10.1158/0008-5472.CAN-09-2554
PMCID: PMC3947604  PMID: 20048073
18.  Modeling Learning and Memory Using Verbal Learning Tests: Results From ACTIVE 
Objective.
To investigate the influence of memory training on initial recall and learning.
Method.
The Advanced Cognitive Training for Independent and Vital Elderly study of community-dwelling adults older than age 65 (n = 1,401). We decomposed trial-level recall in the Auditory Verbal Learning Test (AVLT) and Hopkins Verbal Learning Test (HVLT) into initial recall and learning across trials using latent growth models.
Results.
Trial-level increases in words recalled in the AVLT and HVLT at each follow-up visit followed an approximately logarithmic shape. Over the 5-year study period, memory training was associated with slower decline in Trial 1 AVLT recall (Cohen’s d = 0.35, p = .03) and steep pre- and posttraining acceleration in learning (d = 1.56, p < .001). Findings were replicated using the HVLT (decline in initial recall, d = 0.60, p = .01; pre- and posttraining acceleration in learning, d = 3.10, p < .001). Because of the immediate training boost, the memory-trained group had a higher level of recall than the control group through the end of the 5-year study period despite faster decline in learning.
Discussion.
This study contributes to the understanding of the mechanisms by which training benefits memory and expands current knowledge by reporting long-term changes in initial recall and learning, as measured from growth models and by characterization of the impact of memory training on these components. Results reveal that memory training delays the worsening of memory span and boosts learning.
doi:10.1093/geronb/gbs053
PMCID: PMC3693605  PMID: 22929389
AVLT; Growth modeling; HVLT; Memory training; Older adults
19.  Once-daily budesonide MMX in active, mild-to-moderate ulcerative colitis: results from the randomised CORE II study 
Gut  2013;63(3):433-441.
Objective
Budesonide MMX is a novel oral formulation of budesonide that uses Multi-Matrix System (MMX) technology to extend release to the colon. This study compared the efficacy of budesonide MMX with placebo in patients with active, mild-to-moderate ulcerative colitis (UC).
Design
Patients were randomised 1:1:1:1 to receive budesonide MMX 9 mg or 6 mg, or Entocort EC 9 mg (budesonide controlled ileal-release capsules; reference arm) or placebo once daily for 8 weeks. The primary endpoint was combined clinical and endoscopic remission, defined as UC Disease Activity Index score ≤1 with a score of 0 for rectal bleeding and stool frequency, no mucosal friability on colonoscopy, and a ≥1-point reduction in endoscopic index score from baseline.
Results
410 patients were evaluated for efficacy. Combined clinical and endoscopic remission rates with budesonide MMX 9 mg or 6 mg, Entocort EC and placebo were 17.4%, 8.3%, 12.6% and 4.5%, respectively. The difference between budesonide MMX 9 mg and placebo was significant (OR 4.49; 95% CI 1.47 to 13.72; p=0.0047). Budesonide MMX 9 mg was associated with numerically higher rates of clinical (42.2% vs 33.7%) and endoscopic improvement (42.2% vs 31.5%) versus placebo. The rate of histological healing (16.5% vs 6.7%; p=0.0361) and proportion of patients with symptom resolution (23.9% vs 11.2%; p=0.0220) were significantly higher for budesonide MMX 9 mg than placebo. Adverse event profiles were similar across groups.
Conclusion
Budesonide MMX 9 mg was safe and more effective than placebo at inducing combined clinical and endoscopic remission in patients with active, mild-to-moderate UC.
doi:10.1136/gutjnl-2012-304258
PMCID: PMC3933176  PMID: 23436336
Inflammatory Bowel Disease; IBD; Ulcerative Colitis
20.  Evidence of a Role for CD44 and Cell Adhesion in Mediating Resistance to Lenalidomide in Multiple Myeloma: Therapeutic Implications 
Leukemia  2013;28(2):373-383.
Resistance of myeloma to lenalidomide is an emerging clinical problem, and though it has been associated in part with activation of Wnt/β-catenin signaling, the mediators of this phenotype remained undefined. Lenalidomide-resistant models were found to overexpress the hyaluronan (HA)-binding protein CD44, a downstream Wnt/β-catenin transcriptional target. Consistent with a role of CD44 in cell adhesion-mediated drug-resistance (CAM-DR), lenalidomide-resistant myeloma cells were more adhesive to bone marrow stroma and HA-coated plates. Blockade of CD44 with monoclonal antibodies, free HA, or CD44 knockdown reduced adhesion and sensitized to lenalidomide. Wnt/β-catenin suppression by FH535 enhanced the activity of lenalidomide, as did interleukin-6 neutralization with siltuximab. Notably, all-trans-retinoic acid (ATRA) down-regulated total β-catenin, cell-surface and total CD44, reduced adhesion of lenalidomide-resistant myeloma cells, and enhanced the activity of lenalidomide in a lenalidomide-resistant in vivo murine xenograft model. Finally, ATRA sensitized primary myeloma samples from patients that had relapsed and/or refractory disease after lenalidomide therapy to this immunomodulatory agent ex vivo. Taken together, our findings support the hypotheses that CD44 and CAM-DR contribute to lenalidomide-resistance in multiple myeloma, that CD44 should be evaluated as a putative biomarker of sensitivity to lenalidomide, and that ATRA or other approaches that target CD44 may overcome clinical lenalidomide resistance.
doi:10.1038/leu.2013.174
PMCID: PMC3874423  PMID: 23760401
CD44; ATRA; lenalidomide; drug-resistance
21.  Emergence of Enteric Viruses in Production Chickens Is a Concern for Avian Health 
The Scientific World Journal  2014;2014:450423.
Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. Chicken astrovirus (CAstV), rotavirus, reovirus, chicken parvovirus (ChPV), fowl adenovirus of subgroup I (FAdV-1), and avian nephritis virus (ANV) were investigated using the conventional polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). In addition, the infectious bronchitis virus (IBV), which may play a role in enteric disease, was included. The viruses most frequently detected, either alone or in concomitance with other viruses, were IBV, ANV, rotavirus, and CAstV followed by parvovirus, reovirus, and adenovirus. This study demonstrates the diversity of viruses in Brazilian chicken flocks presenting enteric problems characterized by diarrhea, growth retard, loss weight, and mortality, which reflects the multicausal etiology of this disease.
doi:10.1155/2014/450423
PMCID: PMC3919086  PMID: 24578633
22.  Memory Training in the ACTIVE study: How Much is Needed and Who Benefits? 
Journal of aging and health  2012;25(0):10.1177/0898264312461937.
Objectives and Methods
Data from the memory training arm (n = 629) of the Advanced Cognitive Training for Independent and Vital Elderly (ACTIVE) trial were examined to characterize change in memory performance through five years of follow-up as a function of memory training, booster training, adherence to training, and other characteristics.
Results
Latent growth model analyses revealed that memory training was associated with improved memory performance through year five, but that neither booster training nor training adherence significantly influenced this effect. Baseline age was associated with change in memory performance attributable to the passage of time alone (i.e., to aging). Higher education and better self-rated health were associated with greater change in memory performance after training.
Discussion
These findings confirm that memory training can aid in maintaining long-term improvements in memory performance. Booster training and adherence to training do not appear to attenuate rates of normal age-related memory decline.
doi:10.1177/0898264312461937
PMCID: PMC3825774  PMID: 23103452
ACTIVE trial; memory training; training adherence; cognitive training; aging; memory decline
23.  AD pathology and cerebral infarctions are associated with memory and executive functioning one and five-years before death 
We provide rigorous psychometric evidence for distinct patterns of cognitive impairment for Alzheimer's disease (AD) and cerebral infarctions using 440 participants from the Religious Order Study. Latent variable models were used to decompose the effects of AD pathology and cerebral infarctions assessed at autopsy on overall cognition and specific neuropsychological tests at one and five-years prior to death. Results support clinical and univariate psychometric analyses that memory impairment is more pronounced in AD, and executive impairment more pronounced in the presence of cerebral infarctions. These specific effects are subtle relative to the stronger associations of both AD neuropathology and cerebral infarctions with overall level of cognitive impairment.
doi:10.1080/13803395.2012.740001
PMCID: PMC3605227  PMID: 23205616
Neuropathology; Alzheimer's disease; cerebrovascular disease; neuropsychological measures; latent variable models
24.  Methodology for developing quality indicators for the care of older people in the Emergency Department 
Background
Compared with younger people, older people have a higher risk of adverse health outcomes when presenting to emergency departments. As the population ages, older people will make up an increasing proportion of the emergency department population. Therefore it is timely that consideration be given to the quality of care received by older persons in emergency departments, and to consideration of those older people with special needs. Particular attention will be focused on important groups of older people, such as patients with cognitive impairment, residents of long term care and patients with palliative care needs. This project will develop a suite of quality indicators focused on the care of older persons in the emergency department.
Methods/design
Following input from an expert panel, an initial set of structural, process, and outcome indicators will be developed based on thorough systematic search in the scientific literature. All initial indicators will be tested in eight emergency departments for their validity and feasibility. Results of the data from the field studies will be presented to the expert panel at a second meeting. A suite of Quality Indicators for the older emergency department population will be finalised following a formal voting process.
Discussion
The predicted burgeoning in the number of older persons presenting to emergency departments combined with the recognised quality deficiencies in emergency department care delivery to this population, highlight the need for a quality framework for the care of older persons in emergency departments. Additionally, high quality of care is associated with improved survival & health outcomes of elderly patients. The development of well-selected, validated and economical quality indicators will allow appropriate targeting of resources (financial, education or quality management) to improve quality in areas with maximum potential for improvement.
doi:10.1186/1471-227X-13-23
PMCID: PMC4029470  PMID: 24314126
Emergency service; Hospital; Quality indicators; Health care; Geriatrics; Health services for the aged; Dementia; Cognitive impairment; Residential facilities; Protocol
25.  Induction of pro-apoptotic antibodies to triple negative breast cancer by vaccination with TRAIL death receptor DR5 DNA 
TNF-Related Apoptosis Inducing Ligand Receptor 2 (TRAIL-R2 or DR5) is expressed at elevated levels in a broad range of solid tumors to mediate apoptotic signals from TRAIL or agonist antibodies. We tested the hypothesis that DR5 DNA vaccination will induce pro-apoptotic antibody to trigger apoptosis of tumor cells. BALB/c mice were electrovaccinated with DNA encoding wild type human DR5 (phDR5) or its derivatives. Resulting immune serum or purified immune IgG induced apoptosis in triple negative breast cancer (TNBC) cells, which were also TRAIL-sensitive. The pro-apoptotic activity of immune serum at dilutions of 0.5-2% was comparable to that of 1-2 μg/ml of TRAIL. Apoptotic activity of immune serum was enhanced by antibody cross-linking. Apoptotic cell death induced by anti-DR5 antibody was shown by the cleavage of PARP and caspase-3. In contrast, immune serum had no effect on the proliferation of activated human T cells, which expressed low levels of DR5. In vivo, hDR5 reactive immune serum prevented growth of SUM159 TNBC cells in SCID mice. DR5 specific IFN-γ secreting T cells were also induced by DNA vaccination. Furthermore, the feasibility to overcome immune tolerance to self DR5 was shown by the induction of mouse DR5 binding antibody after electrovaccination of BALB/c mice with pmDR5ectm-Td1 encoding a fusion protein of mouse DR5 and an immunogenic fragment of tetanus toxin. These findings support DR5 as a promising vaccine target for controlling TNBC and other DR5 positive cancers.
doi:10.1002/ijc.27534
PMCID: PMC3387508  PMID: 22419388
DR5 (TRAIL-R2); DNA vaccine; antibody; apoptosis; triple negative breast cancer

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