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1.  Isolated Clonal Cytogenetic Abnormalities after High-Dose Therapy 
Therapy-related myeloid neoplasms (t-MN) are well-recognized complications of high-dose cytotoxic therapy (HDT), such as autologous stem cell transplantation (ASCT). Clonal marrow cytogenetic abnormalities (CMCA) in the setting of normal bone marrow pathology have also been reported after HDT, but their significance remains unclear. We retrospectively evaluated occurrences of CMCA and t-MN in 785 patients treated with HDT at Johns Hopkins University between 1997 and 2007. Most patients received ASCT, but 106 patients who received high-dose cyclophosphamide without ASCT were also included in this study, as this is our institutional standard for malignant and nonmalignant lymphoproliferative disorders in need of HDT. Twenty-two patients developed t-MN, with an estimated cumulative incidence of 3.5% at 4 years. Eleven patients developed isolated CMCA, either transient or persistent without pathologic evidence of t-MN. Altogether, only 20 of the patients with reported CMCA subsequently developed t-MN during the follow-up period. Therefore, in the absence of pathologic evidence of t-MN, CMCA should not be considered diagnostic of t-MN.
doi:10.1016/j.bbmt.2014.03.033
PMCID: PMC4149219  PMID: 24732780
Treatment-related myeloid neoplasms; Clonal cytogenetic abnormalities; Chromosomal alterations in myeloid neoplasms
2.  Sarcomere Mutation-Specific Expression Patterns in Human Hypertrophic Cardiomyopathy 
Background
Heterozygous mutations in sarcomere genes in hypertrophic cardiomyopathy (HCM) are proposed to exert their effect through gain-of-function for missense mutations or loss-of-function for truncating mutations. However, allelic expression from individual mutations has not been sufficiently characterized to support this exclusive distinction in human HCM.
Methods and Results
Sarcomere transcript and protein levels were analyzed in septal myectomy and transplant specimens from 46 genotyped HCM patients with or without sarcomere gene mutations and 10 control hearts. For truncating mutations in MYBPC3, the average ratio of mutant:wild-type transcripts was ~1:5, in contrast to ~1:1 for all sarcomere missense mutations, confirming that nonsense transcripts are uniquely unstable. However, total MYBPC3 mRNA was significantly increased by ~9 fold in HCM samples with MYBPC3 mutations compared to control hearts and to HCM samples without sarcomere gene mutations. Full-length MYBPC3 protein content was not different between MYBPC3 mutant HCM and control samples and no truncated proteins were detected. By absolute quantification of abundance (AQUA) with multiple reaction monitoring, stoichiometric ratios of mutant sarcomere proteins relative to wild-type were strikingly variable in a mutation-specific manner, with the fraction of mutant protein ranging from 30–84%.
Conclusions
These results challenge the concept that haploinsufficiency is a unifying mechanism for HCM caused by MYBPC3 truncating mutations. The range of allelic imbalance for several missense sarcomere mutations suggests that certain mutant proteins may be more or less stable, or incorporate more or less efficiently into the sarcomere than wild-type proteins. These mutation-specific properties may distinctly influence disease phenotypes.
doi:10.1161/CIRCGENETICS.113.000448
PMCID: PMC4254656  PMID: 25031304
hypertrophic cardiomyopathy; sarcomere; gene expression; human; proteomics; expression/regulation
3.  Increased Dietary Intake of Saturated Fatty Acid Heptadecanoic Acid (C17:0) Associated with Decreasing Ferritin and Alleviated Metabolic Syndrome in Dolphins 
PLoS ONE  2015;10(7):e0132117.
Similar to humans, bottlenose dolphins (Tursiops truncatus) can develop metabolic syndrome and associated high ferritin. While fish and fish-based fatty acids may protect against metabolic syndrome in humans, findings have been inconsistent. To assess potential protective factors against metabolic syndrome related to fish diets, fatty acids were compared between two dolphin populations with higher (n = 30, Group A) and lower (n = 19, Group B) mean insulin (11 ± 12 and 2 ± 5 μIU/ml, respectively; P < 0.0001) and their dietary fish. In addition to higher insulin, triglycerides, and ferritin, Group A had lower percent serum heptadecanoic acid (C17:0) compared to Group B (0.3 ± 0.1 and 1.3 ± 0.4%, respectively; P < 0.0001). Using multivariate stepwise regression, higher percent serum C17:0, a saturated fat found in dairy fat, rye, and some fish, was an independent predictor of lower insulin in dolphins. Capelin, a common dietary fish for Group A, had no detectable C17:0, while pinfish and mullet, common in Group B’s diet, had C17:0 (41 and 67 mg/100g, respectively). When a modified diet adding 25% pinfish and/or mullet was fed to six Group A dolphins over 24 weeks (increasing the average daily dietary C17:0 intake from 400 to 1700 mg), C17:0 serum levels increased, high ferritin decreased, and blood-based metabolic syndrome indices normalized toward reference levels. These effects were not found in four reference dolphins. Further, higher total serum C17:0 was an independent and linear predictor of lower ferritin in dolphins in Group B dolphins. Among off the shelf dairy products tested, butter had the highest C17:0 (423mg/100g); nonfat dairy products had no detectable C17:0. We hypothesize that humans’ movement away from diets with potentially beneficial saturated fatty acid C17:0, including whole fat dairy products, could be a contributor to widespread low C17:0 levels, higher ferritin, and metabolic syndrome.
doi:10.1371/journal.pone.0132117
PMCID: PMC4511797  PMID: 26200116
4.  Enhanced Prediction of Src Homology 2 (SH2) Domain Binding Potentials Using a Fluorescence Polarization-derived c-Met, c-Kit, ErbB, and Androgen Receptor Interactome* 
Many human diseases are associated with aberrant regulation of phosphoprotein signaling networks. Src homology 2 (SH2) domains represent the major class of protein domains in metazoans that interact with proteins phosphorylated on the amino acid residue tyrosine. Although current SH2 domain prediction algorithms perform well at predicting the sequences of phosphorylated peptides that are likely to result in the highest possible interaction affinity in the context of random peptide library screens, these algorithms do poorly at predicting the interaction potential of SH2 domains with physiologically derived protein sequences. We employed a high throughput interaction assay system to empirically determine the affinity between 93 human SH2 domains and phosphopeptides abstracted from several receptor tyrosine kinases and signaling proteins. The resulting interaction experiments revealed over 1000 novel peptide-protein interactions and provided a glimpse into the common and specific interaction potentials of c-Met, c-Kit, GAB1, and the human androgen receptor. We used these data to build a permutation-based logistic regression classifier that performed considerably better than existing algorithms for predicting the interaction potential of several SH2 domains.
doi:10.1074/mcp.M113.034876
PMCID: PMC4083110  PMID: 24728074
5.  Lateral Wedge Insoles as a Conservative Treatment for Pain in Patients With Medial Knee Osteoarthritis 
JAMA  2013;310(7):722-730.
IMPORTANCE
There is no consensus regarding the efficacy of lateral wedge insoles as a treatment for pain in medial knee osteoarthritis.
OBJECTIVE
To evaluate whether lateral wedge insoles reduce pain in patients with medial knee osteoarthritis compared with an appropriate control.
DATA SOURCES
Databases searched include the Cochrane Central Register of Controlled Trials, EMBASE, AMED, MEDLINE, CINAHL Plus, ScienceDirect, SCOPUS, Web of Science, and BIOSIS from inception to May 2013, with no limits on study date or language. The metaRegister of Controlled Trials and the NHS Evidence website were also searched.
STUDY SELECTION
Included were randomized trials comparing shoe-based treatments (lateral heel wedge insoles or shoes with variable stiffness soles) aimed at reducing medial knee load, with a neutral or no wedge control condition in patients with painful medial knee osteoarthritis. Studies must have included patient-reported pain as an outcome.
DATA EXTRACTION AND SYNTHESIS
Trial data were extracted independently by 2 researchers using a standardized form. Risk of bias was assessed using the Cochrane Risk of Bias tool by 2 observers. Eligible studies were pooled using a random-effects approach.
MAIN OUTCOME AND MEASURES
Change in self-reported knee pain at follow-up.
RESULTS
Twelve trials met inclusion criteria with a total of 885 participants of whom 502 received lateral wedge treatment. The pooled standardized mean difference (SMD) suggested a favorable association with lateral wedges compared with control (SMD, −0.47; 95% CI, −0.80 to −0.14); however, substantial heterogeneity was present (I2 = 82.7%). This effect size represents an effect of −2.12 points on the 20-point Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain scale. Larger trials with a lower risk of bias suggested a null association. Meta-regression analyses showed that higher effect sizes (unstandardized β, 1.07 [95% CI, 0.28 to 1.87] for trials using a no treatment control) were seen in trials using a no wedge treatment control group (n = 4 trials; SMD, −1.20 [95% CI, −2.09 to −0.30]) and lower effect sizes (unstandardized β, 0.26 [95% CI, 0.002 to 0.52] for each bias category deemed low risk) when the study method was deemed at low risk of bias. Among trials in which the control treatment was a neutral insole (n = 7), lateral wedges showed no association (SMD, −0.03 [95% CI, −0.18 to 0.12] on WOMAC; this represents an effect of −0.12 points), and results showed little heterogeneity (I2 = 7.1%).
CONCLUSIONS AND RELEVANCE
Although meta-analytic pooling of all studies showed a statistically significant association between use of lateral wedges and lower pain in medial knee osteoarthritis, restriction of studies to those using a neutral insole comparator did not show a significant or clinically important association. These findings do not support the use of lateral wedges for this indication.
doi:10.1001/jama.2013.243229
PMCID: PMC4458141  PMID: 23989797
6.  All-Trans Retinoic Acid Activity in Acute Myeloid Leukemia: Role of Cytochrome P450 Enzyme Expression by the Microenvironment 
PLoS ONE  2015;10(6):e0127790.
Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved outcome in acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of resistance have been previously reported, yet the majority of AML blasts are sensitive to atRA in vitro. Even in APL, single agent atRA induces remission without cure. The microenvironment expression of cytochrome P450 (CYP)26, a retinoid-metabolizing enzyme was shown to determine normal hematopoietic stem cell fate. Accordingly, we hypothesized that the bone marrow (BM) microenvironment is responsible for difference between in vitro sensitivity and in vivo resistance of AML to atRA-induced differentiation. We observed that the pro-differentiation effects of atRA on APL and non-APL AML cells as well as on leukemia stem cells from clinical specimens were blocked by BM stroma. In addition, BM stroma produced a precipitous drop in atRA levels. Inhibition of CYP26 rescued atRA levels and AML cell sensitivity in the presence of stroma. Our data suggest that stromal CYP26 activity creates retinoid low sanctuaries in the BM that protect AML cells from systemic atRA therapy. Inhibition of CYP26 provides new opportunities to expand the clinical activity of atRA in both APL and non-APL AML.
doi:10.1371/journal.pone.0127790
PMCID: PMC4457893  PMID: 26047326
7.  Epithelial–mesenchymal transition-associated secretory phenotype predicts survival in lung cancer patients 
Carcinogenesis  2014;35(6):1292-1300.
Summary
A gene signature derived from EASP strongly correlated with nodal metastasis, advanced tumor stage and poor differentiation and predicted survival in three independent lung cancer data sets, including early-stage patients. It may serve as a prognostic signature to identify high-risk early-stage patients.
In cancer cells, the process of epithelial–mesenchymal transition (EMT) confers migratory and invasive capacity, resistance to apoptosis, drug resistance, evasion of host immune surveillance and tumor stem cell traits. Cells undergoing EMT may represent tumor cells with metastatic potential. Characterizing the EMT secretome may identify biomarkers to monitor EMT in tumor progression and provide a prognostic signature to predict patient survival. Utilizing a transforming growth factor-β-induced cell culture model of EMT, we quantitatively profiled differentially secreted proteins, by GeLC-tandem mass spectrometry. Integrating with the corresponding transcriptome, we derived an EMT-associated secretory phenotype (EASP) comprising of proteins that were differentially upregulated both at protein and mRNA levels. Four independent primary tumor-derived gene expression data sets of lung cancers were used for survival analysis by the random survival forests (RSF) method. Analysis of 97-gene EASP expression in human lung adenocarcinoma tumors revealed strong positive correlations with lymph node metastasis, advanced tumor stage and histological grade. RSF analysis built on a training set (n = 442), including age, sex and stage as variables, stratified three independent lung cancer data sets into low-, medium- and high-risk groups with significant differences in overall survival. We further refined EASP to a 20 gene signature (rEASP) based on variable importance scores from RSF analysis. Similar to EASP, rEASP predicted survival of both adenocarcinoma and squamous carcinoma patients. More importantly, it predicted survival in the early-stage cancers. These results demonstrate that integrative analysis of the critical biological process of EMT provides mechanism-based and clinically relevant biomarkers with significant prognostic value.
doi:10.1093/carcin/bgu041
PMCID: PMC4043235  PMID: 24510113
8.  Influence of Uncomplicated Phacoemulsification on Central Macular Thickness in Diabetic Patients: A Meta-Analysis 
PLoS ONE  2015;10(5):e0126343.
Objective
To evaluate the effect of uncomplicated phacoemulsification on central macular thickness (CMT) and best corrected visual acuity (BCVA) in both diabetic patients without diabetic retinopathy (DR) and diabetic patients with mild to moderate non-proliferative diabetic retinopathy (NPDR).
Methods
Potential prospective observational studies were searched through PubMed and EMBASE. Standardized mean difference (SMD) and 95% confidence interval (CI) for changes in CMT and BCVA were evaluated at postoperative 1, 3 and 6 months. The pooled effect estimates were calculated in the use of a random-effects model.
Results
A total of 10 studies involving 190 eyes of diabetic patients without diabetic retinopathy and 143 eyes of diabetic patients with NPDR were identified. CMT values demonstrated a statistically significant increase after uncomplicated phacoemulsification at 1 month (SMD, -0.814; 95%CI, -1.230 to -0.399), 3 months (SMD, -0.565; 95%CI, -0.927 to -0.202) and 6 months (SMD, -0.458; 95%CI, -0.739 to -0.177) in diabetic patients with NPDR. There was no statistical difference in CMT values at postoperative 1 month (SMD, -1.206; 95%CI, -2.433 to 0.021)and no statistically significant increase in CMT values at postoperative3 months (SMD, -0.535; 95%CI, -1.252 to 0.182) and 6 months (SMD, -1.181; 95%CI, -2.625 to 0.263) in diabetic patients without DR.BCVA was significantly increased at postoperative 1 month (SMD, 1.149; 95%CI, 0.251 to 2.047; and SMD,1.349; 95%CI, 0.264 to 2.434, respectively) and 6 months (SMD, 1.295; 95%CI, 0.494 to 2.096; and SMD, 2.146; 95%CI, 0.172 to 4.120, respectively) in both diabetic patients without DR and diabetic patients with NPDR. Sensitivity analysis showed that the results were relatively stable and reliable.
Conclusion
Uncomplicated phacoemulsification in diabetic patients with mild to moderate NPDR seemed to influence significantly the subclinical thickening of the macular zones at postoperative 1, 3 and 6 months compared with diabetic patients without DR. BCVA was significantly improved in both diabetic patients without DR and diabetic patients with mild to moderate NPDR.
doi:10.1371/journal.pone.0126343
PMCID: PMC4428827  PMID: 25965404
9.  Lanthanide nano-drums: a new class of molecular nanoparticles for potential biomedical applications† 
Faraday discussions  2014;175:241-255.
We are developing a new class of lanthanide-based self-assembling molecular nanoparticles as potential reporter molecules for imaging, and as multi-functional nanoprobes or nanosensors in diagnostic systems. These lanthanide “nano-drums” are homogeneous 4d–4f clusters approximately 25 to 30 Å in diameter that can emit from the visible to near-infrared (NIR) wavelengths. Here, we present syntheses, crystal structures, photophysical properties, and comparative cytotoxicity data for six nano-drums containing either Eu, Tb, Lu, Er, Yb or Ho. Imaging capabilities of these nano-drums are demonstrated using epifluorescence, total internal reflection fluorescence (TIRF), and two-photon microscopy. We discuss how these molecular nanoparticles can to be adapted for a range of assays, particularly by taking advantage of functionalization strategies with chemical moieties to enable conjugation to protein or nucleic acids.
doi:10.1039/c4fd00117f
PMCID: PMC4407017  PMID: 25284181
10.  The CAM-S: Development and Validation of a New Scoring System for Delirium Severity in 2 Cohorts 
Annals of internal medicine  2014;160(8):526-533.
Background
Quantifying the severity of delirium is essential to advance clinical care through improved understanding of delirium impact, prognosis, pathophysiology, and response to treatment.
Objectives
To develop and validate a new delirium severity measure (CAM-S) based on the Confusion Assessment Method
Design
Validation analysis in two independent cohorts.
Setting
Three academic medical centers
Patients
First cohort included 300 patients age ≥ 70 years scheduled for major surgery; second included 919 medicine admissions age ≥ 70 years.
Measurements
A 4-item short form and 10-item long-form were developed. The association of the maximal CAM-S score during hospitalization with hospital and post-hospital outcomes related to delirium was evaluated.
Results
CAM-S scores demonstrated strong associations with all clinical outcomes examined, with significant gradients across severity categories in adjusted analyses, adding substantively to delirium diagnoses alone. Representative results included adjusted mean length of stay (LOS), which increased across levels of CAM-S short form severity from 6.5 (95% confidence interval, CI, 6.2-6.9) to 12.7 days (95% CI, 11.2-14.3)(Ptrend < 0.001). Comparable results across increasing levels of the CAM-S long form severity were 5.6 (95% CI, 5.1-6.1) to 11.9 days (95% CI, 10.8-12.9) (Ptrend < 0.001). Representative results for the composite outcome of adjusted relative risk of death or nursing home residence at 90 days increased across levels of CAM-S short form severity from 1.0 (referent) to 2.5 (95% CI, 1.9-3.3)(Ptrend < 0.001). Comparable results for the CAM-S long form severity were 1.0 (referent) to 2.5 (95% CI, 1.6-3.7) (Ptrend < 0.001).
Limitations
Data on clinical outcomes were drawn from an older dataset involving patients age 70 years and older.
Conclusions
CAM-S provides a new delirium severity measure with strong psychometric properties and strong associations with important clinical outcomes.
doi:10.7326/M13-1927
PMCID: PMC4038434  PMID: 24733193
Delirium; Confusion Assessment Method; elderly; validation; predictive validity
11.  High-Dose Cyclophosphamide Without Stem Cell Rescue for the Treatment of Refractory Behcet’s Disease 
Objective
Behcet’s disease (BD) is a systemic inflammatory disorder characterized by recurrent mucocutaneous ulcerations, ocular inflammation, and numerous severe systemic inflammatory manifestations. While most patients respond to standard immunosuppressive therapies, a subset will develop refractory disease. In this report, the use of a novel therapy for the treatment of BD is described.
Methods
Two patients with severe refractory BD who were treated with nonmyeloablative high-dose cyclophosphamide therapy without stem cell rescue.
Results
After treatment, both patients were completely weaned off immunosuppressive therapy and entered disease-free remissions of 18 and 24 months, respectively.
Conclusions
These data suggest high-dose cyclophosphamide therapy without stem cell rescue as an alternative for the treatment of refractory BD.
doi:10.1016/j.semarthrit.2010.12.002
PMCID: PMC4390035  PMID: 21295330
Behcet’s disease; vasculitis; cyclophosphamide
12.  The Relationship Between Reductions in Knee Loading and Immediate Pain Response Whilst Wearing Lateral Wedged Insoles in Knee Osteoarthritis 
Studies of lateral wedge insoles (LWIs) in medial knee osteoarthritis (OA) have shown reductions in the average external knee adduction moment (EKAM) but no lessening of knee pain. Some treated patients actually experience increases in the EKAM which could explain the overall absence of pain response. We examined whether, in patients with painful medial OA, reductions in the EKAM were associated with lessening of knee pain. Each patient underwent gait analysis whilst walking in a control shoe and two LWI’s. We evaluated the relationship between change in EKAM and change in knee pain using Spearman Rank Correlation coefficients and tested whether dichotomizing patients into biomechanical responders (decreased EKAM) and non-responders (increased EKAM) would identify those with reductions in knee pain. In 70 patients studied, the EKAM was reduced in both LWIs versus control shoe (−5.21% and −6.29% for typical and supported wedges, respectively). The change in EKAM using LWIs was not significantly associated with the direction of knee pain change. Further, 54% were biomechanical responders, but these persons did not have more knee pain reduction than non-responders. Whilst LWIs reduce EKAM, there is no clearcut relationship between change in medial load when wearing LWIs and corresponding change in knee pain.
doi:10.1002/jor.22666
PMCID: PMC4372252  PMID: 24903067
osteoarthritis; knee; pain; adduction moment; lateral wedge
13.  The chemistry, physiology and pathology of pH in cancer 
Cell survival is conditional on the maintenance of a favourable acid–base balance (pH). Owing to intensive respiratory CO2 and lactic acid production, cancer cells are exposed continuously to large acid–base fluxes, which would disturb pH if uncorrected. The large cellular reservoir of H+-binding sites can buffer pH changes but, on its own, is inadequate to regulate intracellular pH. To stabilize intracellular pH at a favourable level, cells control trans-membrane traffic of H+-ions (or their chemical equivalents, e.g. ) using specialized transporter proteins sensitive to pH. In poorly perfused tumours, additional diffusion-reaction mechanisms, involving carbonic anhydrase (CA) enzymes, fine-tune control extracellular pH. The ability of H+-ions to change the ionization state of proteins underlies the exquisite pH sensitivity of cellular behaviour, including key processes in cancer formation and metastasis (proliferation, cell cycle, transformation, migration). Elevated metabolism, weakened cell-to-capillary diffusive coupling, and adaptations involving H+/H+-equivalent transporters and extracellular-facing CAs give cancer cells the means to manipulate micro-environmental acidity, a cancer hallmark. Through genetic instability, the cellular apparatus for regulating and sensing pH is able to adapt to extracellular acidity, driving disease progression. The therapeutic potential of disturbing this sequence by targeting H+/H+-equivalent transporters, buffering or CAs is being investigated, using monoclonal antibodies and small-molecule inhibitors.
doi:10.1098/rstb.2013.0099
PMCID: PMC3917353  PMID: 24493747
pH regulation; pH sensing; buffering; carbonic anhydrase; diffusion
14.  Polybrominated diphenyl ether serum concentrations in a Californian population of children, their parents, and older adults: an exposure assessment study 
Environmental Health  2015;14:23.
Background
Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in many household items. Given concerns over their potential adverse health effects, we identified predictors and evaluated temporal changes of PBDE serum concentrations.
Methods
PBDE serum concentrations were measured in young children (2-8 years old; N = 67), parents of young children (<55 years old; N = 90), and older adults (≥55 years old; N = 59) in California, with concurrent floor wipe samples collected in participants’ homes in 2008-2009. We also measured serum concentrations one year later in a subset of children (N = 19) and parents (N = 42).
Results
PBDE serum concentrations in children were significantly higher than in adults. Floor wipe concentration is a significant predictor of serum BDE-47, 99, 100 and 154. Positive associations were observed between the intake frequency of canned meat and serum concentrations of BDE-47, 99 and 154, between canned meat entrees and BDE-154 and 209, as well as between tuna and white fish and BDE-153. The model with the floor wipe concentration and food intake frequencies explained up to 40% of the mean square prediction error of some congeners. Lower home values and renting (vs. owning) a home were associated with higher serum concentrations of BDE-47, 99 and 100. Serum concentrations measured one year apart were strongly correlated as expected (r = 0.70-0.97) with a slight decreasing trend.
Conclusions
Floor wipe concentration, food intake frequency, and housing characteristics can explain 12-40% of the prediction error of PBDE serum concentrations. Decreasing temporal trends should be considered when characterizing long-term exposure.
Electronic supplementary material
The online version of this article (doi:10.1186/s12940-015-0002-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12940-015-0002-2
PMCID: PMC4381357  PMID: 25884939
Children; Flame retardants; PBDEs; Serum concentration; Temporal variability
15.  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(Pt 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
16.  A Tale of Two Methods: Chart and Interview Methods for Identifying Delirium 
Background
Interview and chart-based methods for identifying delirium have been validated. However, relative strengths and limitations of each method have not been described, nor has a combined approach (using both interviews and chart), been systematically examined.
Objectives
To compare chart and interview-based methods for identification of delirium.
Design, Setting and Participants
Participants were 300 patients aged 70+ undergoing major elective surgery (majority were orthopedic surgery) interviewed daily during hospitalization for delirium using the Confusion Assessment Method (CAM; interview-based method) and whose medical charts were reviewed for delirium using a validated chart-review method (chart-based method). We examined rate of agreement on the two methods and patient characteristics of those identified using each approach. Predictive validity for clinical outcomes (length of stay, postoperative complications, discharge disposition) was compared. In the absence of a gold-standard, predictive value could not be calculated.
Results
The cumulative incidence of delirium was 23% (n= 68) by the interview-based method, 12% (n=35) by the chart-based method and 27% (n=82) by the combined approach. Overall agreement was 80%; kappa was 0.30. The methods differed in detection of psychomotor features and time of onset. The chart-based method missed delirium in CAM-identified patients laacking features of psychomotor agitation or inappropriate behavior. The CAM-based method missed chart-identified cases occurring during the night shift. The combined method had high predictive validity for all clinical outcomes.
Conclusions
Interview and chart-based methods have specific strengths for identification of delirium. A combined approach captures the largest number and the broadest range of delirium cases.
doi:10.1111/jgs.12684
PMCID: PMC3959564  PMID: 24512042
Delirium; Methodology; Epidemiology
17.  HLA-haploidentical donor lymphocyte infusions for patients with relapsed hematologic malignancies after related HLA-haploidentical bone marrow transplantation 
Background
Treatment of relapse after related HLA-haploidentical T-cell replete bone marrow transplantation (haploBMT) with post-transplantation cyclophosphamide (PTCy) using haploidentical donor lymphocyte infusion (haploDLI) is not documented.
Methods
All patients who received haploDLI after haploBMT with PTCy between 6/2003 and 10/2012 were identified and assessed for graft-versus-host disease (GvHD) and outcomes.
Results
40 patients received 52 haploDLI doses. Sixteen patients had acute myeloid leukemia, 11 had lymphomas, and 34 had nonmyeloablative conditioning prior to haploBMT. The median time from haploBMT to relapse was 183 (range, 0–1,399) days. The median age at haploDLI was 48 (range, 3–70) years. The first haploDLI doses were 1×105 CD3+ cells/kg with subsequent escalation. The most commonly used first haploDLI dose was 1×106 CD3+ cells/kg. The median follow-up after haploDLI was 7 (mean, 15.4, range, 0.5–96) months for the entire cohort, and 17.5 (mean, 28 range, 2.4–96) months for the responders. Acute GvHD developed in 10 patients (25%), 6 patients had grade 3–4, and 3 developed chronic GvHD. Twelve (30%) patients achieved a complete response (CR) with a median duration of 11.8 (mean, 22.5, range, 0.4–94) months. At last follow-up, 8 responders were alive in CR; 6 for over a year.
Conclusions
HaploDLI for relapse after haploBMT is associated with acceptable toxicities and can result in durable responses.
doi:10.1016/j.bbmt.2013.11.020
PMCID: PMC4010132  PMID: 24296490
Donor lymphocyte infusion (DLI); bone marrow transplantation (BMT); stem cell transplantation; haploidentical; Human leukocyte antigen (HLA)
18.  Targeting SQSTM1/p62 Induces Cargo Loading Failure and Converts Autophagy to Apoptosis via NBK/Bik 
Molecular and Cellular Biology  2014;34(18):3435-3449.
In selective autophagy, the adaptor protein SQSTM1/p62 plays a critical role in recognizing/loading cargo (e.g., malfolded proteins) into autophagosomes for lysosomal degradation. Here we report that whereas SQSTM1/p62 levels fluctuated in a time-dependent manner during autophagy, inhibition or knockdown of Cdk9/cyclin T1 transcriptionally downregulated SQSTM1/p62 but did not affect autophagic flux. These interventions, or short hairpin RNA (shRNA) directly targeting SQSTM1/p62, resulted in cargo loading failure and inefficient autophagy, phenomena recently described for Huntington's disease neurons. These events led to the accumulation of the BH3-only protein NBK/Bik on endoplasmic reticulum (ER) membranes, most likely by blocking loading and autophagic degradation of NBK/Bik, culminating in apoptosis. Whereas NBK/Bik upregulation was further enhanced by disruption of distal autophagic events (e.g., autophagosome maturation) by chloroquine (CQ) or Lamp2 shRNA, it was substantially diminished by inhibition of autophagy initiation (e.g., genetically by shRNA targeting Ulk1, beclin-1, or Atg5 or pharmacologically by 3-methyladenine [3-MA] or spautin-1), arguing that NBK/Bik accumulation stems from inefficient autophagy. Finally, NBK/Bik knockdown markedly attenuated apoptosis in vitro and in vivo. Together, these findings identify novel cross talk between autophagy and apoptosis, wherein targeting SQSTM1/p62 converts cytoprotective autophagy to an inefficient form due to cargo loading failure, leading to NBK/Bik accumulation, which triggers apoptosis.
doi:10.1128/MCB.01383-13
PMCID: PMC4135623  PMID: 25002530
19.  The evolution of treatment strategies for patients with chronic myeloid leukemia relapsing after allogeneic bone marrow transplantation: Can tyrosine kinase inhibitors replace donor lymphocyte infusions? 
Leukemia & lymphoma  2014;56(1):128-134.
The optimal treatment for chronic myeloid leukemia (CML) relapsing following allogeneic bone marrow transplantation (alloBMT) is unknown. We performed a single-center retrospective analysis of 71 consecutive patients undergoing alloBMT for CML from 1995–2008. A multi-state model was used to quantify the cumulative incidences of complete molecular response (CMR) and death following alloBMT. The primary analysis was the comparison of three treatment interventions (tyrosine kinase inhibitor: TKI, donor lymphocyte infusion: DLI, and TKI+DLI) for relapsed disease post-alloBMT. Forty-five (63%) patients relapsed post-alloBMT (molecular relapse: n=16, cytogenetic relapse: n=20, hematologic relapse: n=2, advanced phase relapse: n=7) and 40 patients underwent one of three treatments: TKI-only (n=13), DLI-only (n=11), or TKI+DLI (n=16). Although not statistically significant, the TKI-only group had the highest cumulative incidence of CMR and the lowest cumulative incidence of death compared to DLI and TKI+DLI. These data support the finding that TKI therapy is active in the post-alloBMT setting.
doi:10.3109/10428194.2014.910868
PMCID: PMC4268088  PMID: 24712979
chronic myeloid leukemia; relapse; allogeneic transplantation
20.  Calibration and validation of an innovative approach for estimating general cognitive performance 
Neuroepidemiology  2014;42(3):144-153.
Objective
To evaluate a new approach for creating a composite measure of cognitive function, we calibrated a measure of general cognitive performance from existing neuropsychological batteries.
Methods
We applied our approach in an epidemiologic study and scaled the composite to a nationally representative sample of older adults. Criterion validity was evaluated against standard clinical diagnoses. Convergent validity was evaluated against the Mini-Mental State Examination (MMSE).
Results
The general cognitive performance factor was scaled to have a mean=50 and SD=10 in a nationally representative sample of older adults. A cut-point of approximately 45, corresponding with an MMSE of 23/24, optimally discriminated participants with and without dementia (sensitivity=0.94; specificity=0.90; AUC=0.97). The general cognitive performance factor was internally consistent (Cronbach's alpha=0.91) and provided reliable measures of functional ability across a wide range of cognitive functioning. It demonstrated minimal floor and ceiling effects, which is an improvement over most individual cognitive tests.
Conclusions
The cognitive composite is a highly reliable measure, with minimal floor and ceiling effects. We calibrated it using a nationally representative sample of adults over age 70 in the US and established diagnostically relevant cut-points. Our methods can be used to harmonize neuropsychological test results across diverse settings and studies.
doi:10.1159/000357647
PMCID: PMC3988278  PMID: 24481241
dementia; cognitive function; measurement; factor analysis; harmonization; calibration
21.  Newborn Screening for X-linked Adrenoleukodystrophy: Further evidence high throughput screening is feasible 
X-linked adrenoleukodystrophy (ALD) is characterized by adrenal insufficiency and neurologic involvement with onset at variable ages. Plasma very long chain fatty acids are elevated in ALD; even in asymptomatic patients. We demonstrated previously that liquid chromatography tandem mass spectrometry measuring C26:0 lysophosphatidylcholine reliably identifies affected males. We prospectively applied this method to 4689 newborn blood spot samples; no false positives were observed. We show that high throughput neonatal screening for ALD is methodologically feasible.
doi:10.1016/j.ymgme.2013.10.019
PMCID: PMC3935823  PMID: 24268529
Newborn screening; Adrenoleukodystrophy; Adrenomyeloneuropathy; Adrenal insufficiency; Peroxisomal disorders; Tandem mass spectrometry
22.  Na+ ions as spatial intracellular messengers for co-ordinating Ca2+ signals during pH heterogeneity in cardiomyocytes 
Cardiovascular Research  2014;105(2):171-181.
Aims
Contraction of the heart is regulated by electrically evoked Ca2+ transients (CaTs). H+ ions, the end products of metabolism, modulate CaTs through direct interactions with Ca2+-handling proteins and via Na+-mediated coupling between acid-extruding proteins (e.g. Na+/H+ exchange, NHE1) and Na+/Ca2+ exchange. Restricted H+ diffusivity in cytoplasm predisposes pH-sensitive Ca2+ signalling to becoming non-uniform, but the involvement of readily diffusible intracellular Na+ ions may provide a means for combatting this.
Methods and results
CaTs were imaged in fluo3-loaded rat ventricular myocytes paced at 2 Hz. Cytoplasmic [Na+] ([Na+]i) was imaged using SBFI. Intracellular acidification by acetate exposure raised diastolic and systolic [Ca2+] (also observed with acid-loading by ammonium prepulse or CO2 exposure). The systolic [Ca2+] response correlated with a rise in [Na+]i and sarcoplasmic reticulum Ca2+ load, and was blocked by the NHE1 inhibitor cariporide (CO2/HCO3−-free media). Exposure of one half of a myocyte to acetate using dual microperfusion (CO2/HCO3−-free media) raised diastolic [Ca2+] locally in the acidified region. Systolic [Ca2+] and CaT amplitude increased more uniformly along the length of the cell, but only when NHE1 was functional. Cytoplasmic Na+ diffusivity (DNa) was measured in quiescent cells, with strophanthidin present to inhibit the Na+/K+ pump. With regional acetate exposure to activate a local NHE-driven Na+-influx, DNa was found to be sufficiently fast (680 µm2/s) for transmitting the pH–systolic Ca2+ interaction over long distances.
Conclusions
Na+ ions are rapidly diffusible messengers that expand the spatial scale of cytoplasmic pH–CaT interactions, helping to co-ordinate global Ca2+ signalling during conditions of intracellular pH non-uniformity.
doi:10.1093/cvr/cvu251
PMCID: PMC4297422  PMID: 25514933
Acidosis; Calcium; Diffusion; E–C coupling; Na+–H+ exchange
23.  Depression, Antidepressants, and Falls Among Community-Dwelling Elderly People: The MOBILIZE Boston Study 
Background.
The mechanisms linking falls and depression are still unknown. The aim of the study is to examine the association between depression and antidepressants, with indoor and outdoor falls, and to investigate how antidepressants mediate this relationship.
Methods.
The study included 763 men and women aged 70 and older with baseline measures for depression and antidepressant use are captured with prospective data on falls from the “Maintenance of Balance, Independent Living, Intellect and Zest in the Elderly” (MOBILIZE) Boston study, which is a population-based longitudinal study (from 2005 to 2009).
Results.
Overall, the rate of falls was 26 falls/100 person-years. Seventeen percent of participants had clinically significant depressive symptoms (CSDS), and 12% used antidepressants. CSDS increased the risk of indoor and outdoor falls (incidence rate ratio [IRR] = 1.6, 95% confidence interval [CI] = 1.2–2.3, p < .01; IRR = 1.6, 95% CI = 1.2–2.2, p < .01). Antidepressant use increased the risk of outdoor falls by 70% and partially mediated the association between CSDS and outdoor falls (IRR = 1.7, 95% CI = 1.2–2.5, p < .05). There was no relationship between antidepressant use and indoor falls. Similar results were observed when depression was considered as a continuous variable.
Conclusions.
Depression increased the risk of indoor and outdoor falls. Antidepressant use among older adults with CSDS increased the risk of outdoor, but not indoor falls. Clinicians should carefully consider the role of antidepressants among older adults with CSDS and their potential increase for the risk of outdoor falls.
doi:10.1093/gerona/glt084
PMCID: PMC3814236  PMID: 23817088
Falls; Depression; Medication.
24.  The ACTIVE Cognitive Training Interventions and Trajectories of Performance among Older Adults 
Journal of aging and health  2012;25(0):10.1177/0898264312461938.
Objectives
Salthouse (2006) illustrated that among Advanced Cognitive Training for Independent and Vital Elderly (ACTIVE) randomized controlled trial participants, the pace of cognitive change over time accelerated for persons who had participated in training. Our goal was to determine if the pace of cognitive aging, net of effects due to practice, training, and loss of training gains, differed for persons who received training.
Methods
We evaluated change in cognitive performance over five years following brief cognitive training among older adults (N=1,659, age 65-94) in ACTIVE using a latent growth curve model.
Results
Reasoning training, but not memory or speed, attenuated aging-related change. But this model modification produced instability and was not statistically significant. Memory gains were maintained throughout follow-up. About half of reasoning and speed gains were lost, however all trained groups performed better than controls at 5 years. Performance differences at the end of the follow-up were equivalent to about 6, 4, and 8 years of aging for memory, reasoning and speed training, respectively.
Discussion
Training can appear to accelerate age-related change, because change over time is coupled with loss of training gains. Of the three training interventions, only reasoning training appeared to attenuate the pace of normative decline. However, our analysis is limited by follow-up that is short for precisely characterizing aging-related change.
doi:10.1177/0898264312461938
PMCID: PMC3866224  PMID: 23103453
Cognitive training interventions; Advanced Cognitive Training for Independent and Vital Elderly; training outcomes; growth curve modeling; older adults
25.  Movement of the human foot in 100 pain free individuals aged 18–45: implications for understanding normal foot function 
Background
Understanding motion in the normal healthy foot is a prerequisite for understanding the effects of pathology and thereafter setting targets for interventions. Quality foot kinematic data from healthy feet will also assist the development of high quality and research based clinical models of foot biomechanics. To address gaps in the current literature we aimed to describe 3D foot kinematics using a 5 segment foot model in a population of 100 pain free individuals.
Methods
Kinematics of the leg, calcaneus, midfoot, medial and lateral forefoot and hallux were measured in 100 self reported healthy and pain free individuals during walking. Descriptive statistics were used to characterise foot movements. Contributions from different foot segments to the total motion in each plane were also derived to explore functional roles of different parts of the foot.
Results
Foot segments demonstrated greatest motion in the sagittal plane, but large ranges of movement in all planes. All foot segments demonstrated movement throughout gait, though least motion was observed between the midfoot and calcaneus. There was inconsistent evidence of movement coupling between joints. There were clear differences in motion data compared to foot segment models reported in the literature.
Conclusions
The data reveal the foot is a multiarticular structure, movements are complex, show incomplete evidence of coupling, and vary person to person. The data provide a useful reference data set against which future experimental data can be compared and may provide the basis for conceptual models of foot function based on data rather than anecdotal observations.
doi:10.1186/s13047-014-0051-8
PMCID: PMC4260241  PMID: 25493100
Normal foot kinematics; Foot motion; Foot segments; Foot model

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