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1.  Trends in drug resistance codons in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Kenyan parasites from 2008 to 2012 
Malaria Journal  2014;13:250.
Background
Sulphadoxine-pyrimethamine (SP), an antifolate, was replaced by artemether-lumefantrine as the first-line malaria drug treatment in Kenya in 2004 due to the wide spread of resistance. However, SP still remains the recommended drug for intermittent preventive treatment in pregnant women and infants (IPTP/I) owing to its safety profile. This study assessed the prevalence of mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes associated with SP resistance in samples collected in Kenya between 2008 and 2012.
Methods
Field isolates collected from Kisumu, Kisii, Kericho and Malindi district hospitals were assessed for genetic polymorphism at various loci within Pfdhfr and Pfdhps genes by sequencing.
Results
Among the Pfdhfr mutations, codons N51I, C59R, S108N showed highest prevalence in all the field sites at 95.5%, 84.1% and 98.6% respectively. Pfdhfr S108N prevalence was highest in Kisii at 100%. A temporal trend analysis showed steady prevalence of mutations over time except for codon Pfdhps 581 which showed an increase in mixed genotypes. Triple Pfdhfr N51I/C59R/S108N and double Pfdhps A437G/ K540E had high prevalence rates of 86.6% and 87.9% respectively. The Pfdhfr/Pfdhps quintuple, N51I/C59R/S108N/A437G/K540E mutant which has been shown to be the most clinically relevant marker for SP resistance was observed in 75.7% of the samples.
Conclusion
SP resistance is still persistently high in western Kenya, which is likely due to fixation of key mutations in the Pfdhfr and Pfdhps genes as well as drug pressure from other antifolate drugs being used for the treatment of malaria and other infections. In addition, there is emergence and increasing prevalence of new mutations in Kenyan parasite population. Since SP is used for IPTP/I, molecular surveillance and in vitro susceptibility assays must be sustained to provide information on the emergence and spread of SP resistance.
doi:10.1186/1475-2875-13-250
PMCID: PMC4094641  PMID: 24989984
Malaria; Plasmodium falciparum dihydrofolate reductase; Plasmodium falciparum dihydropteroate synthase
2.  Polymorphisms in Pfmdr1, Pfcrt, and Pfnhe1 Genes Are Associated with Reduced In Vitro Activities of Quinine in Plasmodium falciparum Isolates from Western Kenya 
In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, an alternative first-line treatment for severe malaria, and for treatment of malaria in the first trimester of pregnancy. Quinine has been shown to have frequent clinical failures, and yet the mechanisms of action and resistance have not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes, including multidrug resistance 1 (Pfmdr1), the chloroquine resistance transporter (Pfcrt), and the sodium/hydrogen exchanger gene (Pfnhe1). Here, we investigated the association between in vitro quinine susceptibility and genetic polymorphisms in Pfmdr1codons 86 and 184, Pfcrt codon 76, and Pfnhe1 ms4760 in 88 field isolates from western Kenya. In vitro activity was assessed based on the drug concentration that inhibited 50% of parasite growth (the IC50), and parasite genetic polymorphisms were determined from DNA sequencing. Data revealed there were significant associations between polymorphism in Pfmdr1-86Y, Pfmdr1-184F, or Pfcrt-76T and quinine susceptibility (P < 0.0001 for all three associations). Eighty-two percent of parasites resistant to quinine carried mutant alleles at these codons (Pfmdr1-86Y, Pfmdr1-184F, and Pfcrt-76T), whereas 74% of parasites susceptible to quinine carried the wild-type allele (Pfmdr1-N86, Pfmdr1-Y184, and Pfcrt-K76, respectively). In addition, quinine IC50 values for parasites with Pfnhe1 ms4760 3 DNNND repeats were significantly higher than for those with 1 or 2 repeats (P = 0.033 and P = 0.0043, respectively). Clinical efficacy studies are now required to confirm the validity of these markers and the importance of parasite genetic background.
doi:10.1128/AAC.02472-14
PMCID: PMC4068580  PMID: 24752268
3.  Dose–Response Cocaine Pharmacokinetics and Metabolite Profile Following Intravenous Administration and Arterial Sampling in Unanesthetized, Freely Moving Male Rats 
Despite the wealth of experimental data on cocaine abuse, there are no published dose–response pharmacokinetic studies with bolus IV cocaine injection in the male rat. The present study examined the pharmacokinetics of arterial plasma concentrations of cocaine and metabolite profile [benzoylecgonine (BE), ecgonine methyl ester (EME), norcocaine (NC)] following a single IV injection of 0.5, 1.0, or 3.0 mg/kg cocaine. Male Sprague–Dawley rats (N = 25) were anesthetized and surgically instrumented with both jugular vein (drug administration) and carotid artery (blood withdrawal) catheters and allowed to recover for at least 24 h. Arterial plasma samples (200 µl) were obtained at eight time points (0.5, 1, 1.5, 2, 5, 10, 20, 30 min) following IV bolus injection (15-s injection, 15-s flush) and analyzed by single ion monitoring using GC/MS. Nonlinear regression and noncompartmental pharmacokinetic analysis were employed. Mean ± SEM peak plasma concentrations of cocaine occurred at 30 s in a dose–response manner (370 ± 14, 755 ± 119, 2553 ± 898 ng/ml for 0.5,1.0, and 3.0 mg/kg groups, respectively). T1/2∝ was < 1 min for all groups, but inversely related to dose. T1/2β was independent of dose (13.3 ± 1.6,13.0 ± 1.5, and 12.0 ± 2.0 min for 0.5,1.0, and 3.0 mg/ kg groups, respectively). MRT (16.0,15.9,14.5 min), VdSS (3.3, 3.2, and 2.8 1/kg), and ClTOT (204, 201, and 195 ml/min/kg) also provided little evidence of dose-dependent effects. Although the metabolic profile of IV cocaine was similarly ordered for all dose groups (BE > EME > NC), a quantitative shift in metabolite profile was evident as a function of increasing dose. This metabolic shift, perhaps attributable to saturation of plasma and liver esterases, suggests that the recently reported pharmacodynamic effects positively correlated with IV cocaine dose are unlikely attributable to NC, a minor but pharmacologically active metabolite. In sum, the IV pharmacokinetic profile in rats is distinct from that observed via the SC, IP, and PO routes of administration and offers the potential to provide a reasonable clinically relevant rodent model.
PMCID: PMC4041984  PMID: 9088006
Cocaine; Drug administration routes; Pharmacokinetics; Rats, Sprague–Dawley; Benzoylecgonine; Ecgonine methyl ester; Norcocaine
4.  Breast Cancer in Systemic Lupus Erythematosus 
Oncology  2013;85(2):117-121.
Objective
Evidence points to a decreased breast cancer risk in systemic lupus erythematosus (SLE). We analyzed data from a large multisite SLE cohort, linked to cancer registries.
Methods
Information on age, SLE duration, cancer date, and histology was available. We analyzed information on histological type and performed multivariate logistic regression analyses of histological types according to age, SLE duration, and calendar year.
Results
We studied 180 breast cancers in the SLE cohort. Of the 155 cases with histology information, 11 were referred to simply as ‘carcinoma not otherwise specified’. In the remaining 144 breast cancers, the most common histological type was ductal carcinoma (n = 95; 66%) followed by lobular adenocarcinoma (n = 11; 8%), 15 cancers were of mixed histology, and the remaining ones were special types. In our regression analyses, the independent risk factors for lobular versus ductal carcinoma was age [odds ratio (OR) 1.07, 95% confidence interval (CI) 1.01–1.14] and for the ‘special’ subtypes it was age (OR 1.06, 95% CI 1.01–1.10) and SLE duration (OR 1.05, 95% CI 1.00–1.11).
Conclusions
Generally, up to 80% of breast cancers are ductal carcinomas. Though our results are not definitive, in the breast cancers that occur in SLE, there may be a slight decrease in the ductal histological type. In our analyses, age and SLE duration were independent predictors of histological status.
doi:10.1159/000353138
PMCID: PMC3934367  PMID: 23887245
Breast cancer; Systemic lupus erythematosus; Histopathology; Epidemiology
5.  Ecosystem Services in Agricultural Landscapes: A Spatially Explicit Approach to Support Sustainable Soil Management 
The Scientific World Journal  2014;2014:483298.
Soil degradation has been associated with a lack of adequate consideration of soil ecosystem services. We demonstrate a broadly applicable method for mapping changes in the supply of two priority soil ecosystem services to support decisions about sustainable land-use configurations. We used a landscape-scale study area of 302 km2 in northern Victoria, south-eastern Australia, which has been cleared for intensive agriculture. Indicators representing priority soil services (soil carbon sequestration and soil water storage) were quantified and mapped under both a current and a future 25-year land-use scenario (the latter including a greater diversity of land uses and increased perennial crops and irrigation). We combined diverse methods, including soil analysis using mid-infrared spectroscopy, soil biophysical modelling, and geostatistical interpolation. Our analysis suggests that the future land-use scenario would increase the landscape-level supply of both services over 25 years. Soil organic carbon content and water storage to 30 cm depth were predicted to increase by about 11% and 22%, respectively. Our service maps revealed the locations of hotspots, as well as potential trade-offs in service supply under new land-use configurations. The study highlights the need to consider diverse land uses in sustainable management of soil services in changing agricultural landscapes.
doi:10.1155/2014/483298
PMCID: PMC3925562  PMID: 24616632
6.  Mitochondria, Bioenergetics, and the Epigenome in Eukaryotic and Human Evolution 
Studies on the origin of species have focused largely on anatomy, yet animal populations are generally limited by energy. Animals can adapt to available energy resources at three levels: (1) evolution of different anatomical forms between groups of animals through nuclear DNA (nDNA) mutations, permitting exploitation of alternative energy reservoirs and resulting in new species with novel niches, (2) evolution of different physiologies within intraspecific populations through mutations in mitochondrial DNA (mtDNA) and nDNA bioenergetic genes, permitting adjustment to energetic variation within a species’ niche, and (3) epigenomic regulation of dispersed bioenergetic genes within an individual via mitochondrially generated high-energy intermediates, permitting individual adjustment to environmental fluctuations. Because medicine focuses on changes within our species, clinically relevant variation is more likely to involve changes in bioenergetics than anatomy. This may explain why mitochondrial diseases and epigenomic diseases frequently have similar phenotypes and why epigenomic diseases are being found to involve mitochondrial dysfunction. Therefore, common complex diseases may be the result of changes in any of a large number of mtDNA and nDNA bioenergetic genes or to altered epigenomic regulation of these bioenergetic genes. All of these changes result in similar bioenergetic failure and consequently related phenotypes.
doi:10.1101/sqb.2009.74.031
PMCID: PMC3905750  PMID: 19955254
7.  SF-36 summary and subscale scores are reliable outcomes of neuropsychiatric events in systemic lupus erythematosus 
Annals of the rheumatic diseases  2011;70(6):961-967.
Objective
To examine change in health-related quality of life (HRQoL) in association with clinical outcomes of neuropsychiatric (NP) events in SLE.
Methods
An international study evaluated newly diagnosed SLE patients for NP events attributed to SLE and non-SLE causes. Outcome of events was determined by physician-completed 7-point scale and compared to patient-completed SF-36 questionnaires. Statistical analysis used linear mixed-effects regression models with patient specific random effects.
Results
274 patients (92% female; 68% Caucasian), from a cohort of 1400, had ≥ 1 NP event where the interval between assessments was 12.3 ± 2 months. The overall difference in change between visits in mental component summary (MCS) scores of the SF-36 was significant (p<0.0001) following adjustments for gender, ethnicity, center and previous score. A consistent improvement in NP status (N=295) was associated with an increase in the mean(SD) adjusted MCS score of 3.66(0.89) in SF-36 scores. Between paired visits where NP status consistently deteriorated (N=30), the adjusted MCS score decreased by 4.00(1.96). For the physical component summary (PCS) scores the corresponding changes were +1.73(0.71) and −0.62(1.58) (p<0.05) respectively. Changes in SF-36 subscales were in the same direction (p<0.05; with the exception of role physical). Sensitivity analyses confirmed these findings. Adjustment for age, education, medications, SLE disease activity, organ damage, disease duration, attribution and characteristics of NP events did not substantially alter the results.
Conclusion
Changes in SF-36 summary and subscale scores, in particular those related to mental health, are strongly associated with the clinical outcome of NP events in SLE patients.
doi:10.1136/ard.2010.138792
PMCID: PMC3795436  PMID: 21342917
Systemic lupus erythematosus; Neuropsychiatric; Inception cohort; Health related quality of life; SF-36
8.  Toxicologic Assessment of a Commercial Decolorized Whole Leaf Aloe Vera Juice, Lily of the Desert Filtered Whole Leaf Juice with Aloesorb 
Journal of Toxicology  2013;2013:802453.
Aloe vera, a common ingredient in cosmetics, is increasingly being consumed as a beverage supplement. Although consumer interest in aloe likely stems from its association with several health benefits, a concern has also been raised by a National Toxicology Program Report that a nondecolorized whole leaf aloe vera extract taken internally by rats was associated with intestinal mucosal hyperplasia and ultimately malignancy. We tested a decolorized whole leaf (DCWL) aloe vera, treated with activated charcoal to remove the latex portion of the plant, for genotoxicity in bacteria, acute/subacute toxicity in B6C3F1 mice, and subchronic toxicity in F344 rats. We found this DCWL aloe vera juice to be nongenotoxic in histidine reversion and DNA repair assays. Following acute administration, mice exhibited no adverse signs at 3- or 14-day evaluation periods. When fed to male and female F344 rats over 13 weeks, DCWL aloe led to no toxicity as assessed by behavior, stools, weight gain, feed consumption, organ weights, and hematologic or clinical chemistry profiles. These rats had intestinal mucosal morphologies—examined grossly and microscopically—that were similar to controls. Our studies show that oral administration of this DCWL aloe juice has a different toxicology profile than that of the untreated aloe juice at exposures up to 13 weeks.
doi:10.1155/2013/802453
PMCID: PMC3608129  PMID: 23554812
10.  Efficacy of phototherapy devices and outcomes among extremely low birth weight infants: multi-center observational study 
Objective
Evaluate the efficacy of phototherapy (PT) devices and the outcomes of extremely premature infants treated with those devices.
Study Design
This substudy of the National Institute of Child Health and Human Development Neonatal Research Network PT trial included 1404 infants treated with a single type of PT device during the first 24±12 h of treatment. The absolute (primary outcome) and relative decrease in total serum bilirubin (TSB) and other measures were evaluated. For infants treated with one PT type during the 2-week intervention period (n =1223), adjusted outcomes at discharge and 18 to 22 months corrected age were determined.
Result
In the first 24 h, the adjusted absolute (mean (±s.d.)) and relative (%) decrease in TSB (mg dl−1) were: light-emitting diodes (LEDs) −2.2 (±3), −22%; Spotlights −1.7 (±2), −19%; Banks −1.3 (±3), −8%; Blankets −0.8 (±3), −1%; (P<0.0002). Some findings at 18 to 22 months differed between groups.
Conclusion
LEDs achieved the greatest initial absolute reduction in TSB but were similar to Spots in the other performance measures. Long-term effects of PT devices in extremely premature infants deserve rigorous evaluation.
doi:10.1038/jp.2012.39
PMCID: PMC3570170  PMID: 22499082
extremely low birth weight; neonatal jaundice; neurodevelopmental outcome; phototherapy
13.  Sleep-Related Falling Out of Bed in Parkinson's Disease 
Background and Purpose
Sleep-related falling out of bed (SFOB), with its potential for significant injury, has not been a strong focus of investigation in Parkinson's disease (PD) to date. We describe the demographic and clinical characteristics of PD patients with and without SFOB.
Methods
We performed a retrospective analysis of 50 consecutive PD patients, who completed an REM sleep behavior disorder screening questionnaire (RBDSQ), questionnaires to assess for RBD clinical mimickers and questions about SFOB and resulting injuries. Determination of high risk for RBD was based on an RBDSQ score of 5 or greater.
Results
Thirteen patients reported history of SFOB (26%). Visual hallucinations, sleep-related injury, quetiapine and amantadine use were more common in those patients reporting SFOB. Twenty-two patients (44%) fulfilled criteria for high risk for RBD, 12 of which (55%) reported SFOB. Five patients reported injuries related to SFOB. SFOB patients had higher RBDSQ scores than non-SFOB patients (8.2±3.0 vs. 3.3±2.0, p<0.01). For every one unit increase in RBDSQ score, the likelihood of SFOB increased two-fold (OR 2.4, 95% CI 1.3-4.2, p<0.003).
Conclusions
SFOB may be a clinical marker of RBD in PD and should prompt confirmatory polysomnography and pharmacologic treatment to avoid imminent injury. Larger prospective studies are needed to identify risk factors for initial and recurrent SFOB in PD.
doi:10.3988/jcn.2012.8.1.51
PMCID: PMC3325432  PMID: 22523513
Parkinson's disease; REM sleep behavior disorder; sleep disturbance; falls; sleep-related injury
14.  Emerging Endoscopic and Photodynamic Techniques for Bladder Cancer Detection and Surveillance 
TheScientificWorldJournal  2011;11:2550-2558.
This review provides an overview of emerging techniques, namely, photodynamic diagnosis (PDD), narrow band imaging (NBI), Raman spectroscopy, optical coherence tomography, virtual cystoscopy, and endoscopic microscopy for its use in the diagnosis and surveillance of bladder cancer. The technology, clinical evidence and future applications of these approaches are discussed with particular emphasis on PDD and NBI. These approaches show promise to optimise cystoscopy and transurethral resection of bladder tumours.
doi:10.1100/2011/412739
PMCID: PMC3253550  PMID: 22235185
bladder; cancer; diagnosis; surveillance; fluorescence; cystoscopy; narrow; band; imaging; optical; coherence; tomography; photodynamic; diagnosis; Raman; spectroscopy; virtual; cystoscopy; endoscopic; microscope; urothelial; cell; carcinoma
15.  Mechanisms of recurrence of Ta/T1 bladder cancer 
INTRODUCTION
Bladder cancer recurrence occurs via four mechanisms - incomplete resection, tumour cell re-implantation, growth of microscopic tumours, and new tumour formation. The first two mechanisms are influenced by clinicians before and immediately after resection; the remaining mechanisms have the potential to be influenced by chemopreventive agents. However, the relative importance and timing of these mechanisms is currently unknown. Our objective was to postulate the incidence and timing of these mechanisms by investigating the location of bladder cancer recurrences over time.
PATIENTS AND METHODS
The topographical locations of tumours and their recurrences were analysed retrospectively for 169 patients newly-diagnosed with Ta/T1 bladder cancer, with median follow-up of 33.8 months. Tumours were assigned to one or more of six bladder sectors, and time to recurrence and location of recurrences were recorded.
RESULTS
Median time to first tumour recurrence was 40 months. Median times between subsequent recurrences were 6.6, 7.9, 8.0 and 6.6 months for recurrences 1 to 2, 2 to 3, 3 to 4, and 4 to 5, respectively. The risk of first tumour recurrence in any given bladder sector increased by nearly 4-fold if the primary tumour was resected from that sector (P < 0.001); this association was not significant for subsequent recurrences. The proportion of tumour recurrences in multiple bladder sectors increased from 13% for the first recurrence to 100% for recurrence seven onwards.
CONCLUSIONS
First tumour recurrence appears different to subsequent recurrences; incomplete resection and tumour cell re-implantation may dominate at this time-point. Only later does genuine new tumour formation appear to increase in importance. This has important implications for clinical trials, especially those involving chemopreventive agents.
doi:10.1308/003588410X12664192076935
PMCID: PMC3182798  PMID: 20522307
Bladder cancer; Mechanisms; Recurrence
16.  Purification, folding, and characterization of Rec12 (Spo11) meiotic recombinase of fission yeast 
Meiotic recombination is initiated by controlled dsDNA breaks (DSBs). Rec12 (Spo11) protein of fission yeast is essential for the formation of meiotic DSBs in vivo, for meiotic recombination, and for segregation of chromosomes during meiosis I. Rec12 is orthologous to Top6A topoisomerase of Archaea and is likely the catalytic subunit of a meiotic recombinase that introduces recombinogenic DSBs. However, despite intensive effort, it has not been possible to produce Rec12 protein in a soluble form required to permit biochemical analyses of function. To obtain purified Rec12 protein for in vitro studies, a rec12+ cDNA was generated, cloned into vector pET15b(+), and expressed in Escherichia coli. Rec12 protein was produced at moderate levels and it partitioned into insoluble fractions of whole-cell extracts. The protein was enriched based upon its differential solubility in two different denaturants and was further purified by column chromatography. A combinatorial, fractional, factorial approach was used to identify conditions under which Rec12 protein could be refolded. Four parameters were most important and, following optimization, soluble Rec12 protein was obtained. Gel filtration demonstrated that refolded Rec12 protein exists as a monomer in solution, suggesting that additional proteins may be required to assemble biologically-active Rec12 dimers, as inferred previously from genetic data [Cell Chromosome 1 (2002) 1]. The production of refolded Rec12 in a soluble form will allow for characterization in vitro of this key meiotic recombination enzyme.
doi:10.1016/j.pep.2004.07.012
PMCID: PMC3127416  PMID: 15477092
Meiosis; Homologous recombination; Topoisomerase; Endonuclease
17.  A DNA binding motif of meiotic recombinase Rec12 (Spo11) defined by essential glycine-202, and persistence of Rec12 protein after completion of recombination 
Gene  2005;356:77-84.
The Rec12 (Spo11) protein of the fission yeast Schizosaccharomyces pombe is a meiosis-specific ortholog of the catalytic subunit of type VI topoisomerases and is thought to catalyze double-strand DNA breaks that initiate recombination. We tested the hypothesis that the rec12-117 allele affects the choice of pathways by which recombination is resolved. DNA sequence analysis revealed a single missense mutation in the coding region (rec12-G202E). The corresponding glycine-202 residue of Rec12 protein is strictly conserved in proteins of the Rec12/Spo11/Top6A family. It maps to the base of the DNA binding pocket in the crystal structure of the archaeal ortholog, Top6A. The rec12-G202E mutants lacked crossover and non-crossover recombination, demonstrating that rec12-G202E does not affect choice of resolution pathway. Like rec12-D15 null mutants, the rec12-G202E mutants suffered chromosome segregation errors in meiosis I. The Rec12-G202E protein was as stable as wild-type Rec12, demonstrating that glycine-202 is essential for a biochemical activity of Rec12 protein, rather than for its stability. These findings suggest that Rec12 facilitates binding of the meiotic recombinase to its substrate, DNA. Interestingly, the bulk of Rec12 protein persisted until the time of anaphase I, and a portion of Rec12 protein persisted until the time of anaphase II, after which it was undetectable. This suggests that Rec12 protein has additional meiotic functions after completion of recombination in prophase, as inferred previously from genetic studies.
doi:10.1016/j.gene.2005.04.039
PMCID: PMC3119478  PMID: 16009511
Homologous recombination; Genetic recombination; Meiosis; Fission yeast; Schizosaccharomyces pombe; Topoisomerase
18.  Atherosclerotic Vascular Events in a Multinational Inception Cohort of Systemic Lupus Erythematosus (SLE) 
Arthritis care & research  2010;62(6):881-887.
Objective
To describe vascular events during an 8 year follow-up in a multicentre SLE inception cohort and their attribution to atherosclerosis.
Methods
Clinical data including co-morbidities are recorded yearly. Vascular events are recorded and attributed to atherosclerosis or not. All events met standard clinical criteria. Factors associated with atherosclerotic vascular events were analysed using descriptive statistics, t-tests and χ2. Stepwise multivariate logistic regression was used to assess the association of factors with vascular events attributed to atherosclerosis.
Results
Since 2000, 1249 patients have been entered into the cohort. There have been 97 vascular events in 72 patients. These include: myocardial infarction (13), angina (15), congestive heart failure (24), peripheral vascular disease (8), transient ischemic attack (13), stroke (23), pacemaker insertion (1). Fifty of the events were attributed to active lupus, 31events in 22 patients were attributed to atherosclerosis, and 16 to other causes. Time from diagnosis to first atherosclerotic event was 2.0 ± 1.5 years. Compared to patients followed for 2 years without atherosclerosis events (615), at enrolment patients with AVE were more frequently Caucasian, male, older at diagnosis of SLE, obese, smokers, hypertensive and had a family history of coronary artery disease. On multivariate analysis only male gender and older age at diagnosis were associated factors.
Conclusion
In an inception cohort with SLE followed for up to 8 years there were 97 vascular events but only 31 were attributable to atherosclerosis. Patients with atherosclerotic events were more likely to be male and to be older at diagnosis of SLE.
doi:10.1002/acr.20122
PMCID: PMC2989413  PMID: 20535799
19.  Prospective Analysis Of Neuropsychiatric Events In An International Disease Inception Cohort of SLE Patients 
Annals of the rheumatic diseases  2009;69(3):529-535.
Objectives
To determine the frequency, accrual, attribution and outcome of neuropsychiatric (NP) events and impact on quality of life over 3 years in a large inception cohort of SLE patients.
Methods
The study was conducted by the Systemic Lupus International Collaborating Clinics. Patients were enrolled within 15 months of SLE diagnosis. NP events were identified using the ACR case definitions and decision rules were derived to determine the proportion of NP disease attributable to SLE. The outcome of NP events was recorded and patient perceived impact determined by the SF-36.
Results
There were 1206 patients (89.6% female) with a mean (±SD) age of 34.5±13.2 years. The mean disease duration at enrollment was 5.4±4.2 months. Over a mean follow-up of 1.9±1.2 years 486/1206 (40.3%) patients had ≥1 NP events which were attributed to SLE in 13.0%–23.6% of patients using two a priori decision rules. The frequency of individual NP events varied from 47.1% (headache) to 0% (myasthenia gravis). The outcome was significantly better for those NP events attributed to SLE especially if they occurred within 1.5 years of the diagnosis of SLE. Patients with NP events, regardless of attribution, had significantly lower summary scores for both mental and physical health over the study.
Conclusions
NP events in SLE patients are variable in frequency, most commonly present early in the disease course and adversely impact patients’ quality of life over time. Events attributed to non-SLE causes are more common than those due to SLE, although the latter have a more favourable outcome.
doi:10.1136/ard.2008.106351
PMCID: PMC2929162  PMID: 19359262
Lupus; Neuropsychiatric; Prospective; Inception cohort
20.  International Collaboration between US and Thailand on a Clinical Trial of Treatment for HIV-associated Cryptococcal Meningitis 
Contemporary clinical trials  2009;31(1):34-43.
Background
International clinical trials can provide scientific and logistic benefits in spite of the many challenges. Determining whether a country, especially a developing country, is an appropriate location for the research should include in-country consultation and partnering to assess its social value for the population; that treatments are relevant for the population under study; and that the research infrastructure and ethical oversight are adequate. Collaboration increases the likelihood of study success and helps ensure that benefits accrue to recruited populations and their community.
Purpose
This paper describes our experiences on a bi-national study and may provide guidance for those planning to engage in future collaborations.
Methods
A Thai and United States team collaborated to develop and implement a Phase II clinical trial for HIV-associated cryptococcal meningitis to assess safety and tolerability of combination therapy versus standard treatment. Clinical and cultural differences, regulatory hurdles and operational issues were addressed before and during the study to ensure a successful collaboration between the 2 groups.
Results
The international multicenter study allowed for more rapid enrollment, reduced costs to complete the study, sharing of the benefits of research, greater generalizability of results and capacity building in Thailand; quality metrics in Thailand were equivalent to or better than those in the U.S.
Conclusions
Conducting successful clinical trials internationally requires early and ongoing collaboration to ensure the study meets sites’ requirements and expectations, conforms to varying national regulations, adheres to data quality standards and is responsive to the health needs of studied populations.
doi:10.1016/j.cct.2009.11.002
PMCID: PMC2861565  PMID: 19897055
controlled clinical trials; cryptococcal meningitis; international health problems; collaboration
21.  Oxidative stress, mitochondrial dysfunction and calcium overload in human lamina cribrosa cells from glaucoma donors 
Molecular Vision  2011;17:1182-1191.
Purpose
Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH.
Methods
Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca2+]i) levels were evaluated by flow cytometry using the JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca2+ transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively.
Results
Intracellular ROS production was increased in GLC compared to NLC (27.19±7.05 µM MDA versus 14.59±0.82 µM MDA, p<0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p=0.02) compared to NLC control. MMP was lower in GLC (57.5±6.8%) compared to NLC (41.8±5.3%). [Ca2+]i levels were found to be higher (p<0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca2+/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca2+/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H202) reduced expression of Na+/Ca2+ exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT–PCR.
Conclusions
Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca2+]i.
PMCID: PMC3102025  PMID: 21617752
22.  Use of a 360-Degree Evaluation in the Outpatient Setting: The Usefulness of Nurse, Faculty, Patient/Family, and Resident Self-Evaluation 
Background
Faculty have traditionally evaluated resident physician professionalism and interpersonal skills without input from patients, family members, nurses, or the residents themselves. The objective of our study was to use “360-degree evaluations,” as suggested by the Accreditation Council for Graduate Medical Education (ACGME), to determine if nonfaculty ratings of resident professionalism and interpersonal skills differ from faculty ratings.
Methods
Pediatrics residents were enrolled in a hospital-based resident continuity clinic during a 5-week period. Patient/families (P/Fs), faculty (MD [doctor of medicine]), nurses (RNs [registered nurses]), and residents themselves (self) completed evaluator-specific evaluations after each clinic session by using a validated 10-item questionnaire with a 5-point Likert scale. The average Likert score was tallied for each questionnaire. Mean Likert scale scores for each type of rater were compared by using analysis of variance, text with pair-wise comparisons when appropriate. Agreement between rater types was measured by using the Pearson correlation.
Results
A total of 823 evaluations were completed for 66 residents (total eligible residents, 69; 95% participation). All evaluators scored residents highly (mean Likert score range, 4.4 to 4.9). However, MDs and RNs scored residents higher than did P/Fs (mean scores: MD, 4.77, SD [standard deviation], 0.32; RN, 4.85, SD, 0.30; P/F, 4.53, SD, 0.96; P < .0001). MD and RN scores also were higher than residents' self-evaluation scores, but there was no difference between self-scores and P/F scores (average resident self-score, 4.44, SD, 0.43; P < .0001 compared to MD and RN; P  =  .19 compared to P/F). Correlation coefficients between all combinations of raters ranged from −0.21 to 0.21 and none were statistically significant.
Conclusion
Our study found high ratings for resident professionalism and interpersonal skills. However, different members of the health care team rated residents differently, and ratings are not correlated. Our results provide evidence for the potential value of 360-degree evaluations.
doi:10.4300/JGME-D-10-00013.1
PMCID: PMC2951785  PMID: 21976094
23.  Problems with Radiotherapy After Immediate Breast Reconstruction 
doi:10.1308/003588408X321783b
PMCID: PMC2727834  PMID: 18990288
24.  Altered CD45 expression in C77G carriers influences immune function and outcome of hepatitis C infection 
Journal of Medical Genetics  2006;43(8):678-684.
Background
A polymorphism in exon 4 (C77G) of CD45 that alters CD45 splicing has been associated with autoimmune and infectious diseases in humans.
Objective
To investigate the effect of C77G in hepatitis C virus (HCV) infected individuals and study the phenotype and function of peripheral blood mononuclear cells (PBMC) from healthy and hepatitis C infected C77G carriers.
Results
C77G individuals showed an increased proportion of primed CD45RA and effector memory CD8 T cells and more rapid activation of the lymphocyte specific protein tyrosine kinase (Lck) following CD3 stimulation. Transgenic mice with CD45 expression mimicking that in human C77G variants had more activated/memory T cells, more rapid proliferative responses, and activation of Lck.
Conclusions
Changes in CD45 isoform expression can alter immune function in human C77G variants and CD45 transgenic mice. The C77G allele may influence the outcome of HCV infection.
doi:10.1136/jmg.2005.040485
PMCID: PMC2564592  PMID: 16505159
CD45; C77G variant; hepatitis C; immune response
25.  First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin 
Gut  2005;54(7):980-986.
Background: Transferrin receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in humans cause type 3 haemochromatosis and a targeted mutation in mice leads to iron overload with a similar phenotype. We have previously described the generation of a complete TfR2-knockout (KO) mouse.
Aims: The aims of this study were to determine the phenotype and analyse expression of iron related molecules in the liver, duodenum, and spleen of homozygous TfR2-KO, heterozygous, and wild-type mice.
Methods: Serum and tissue iron levels were determined in 10 week old male mice. Expression of iron related mRNA transcripts were analysed in the liver, duodenum, and spleen using real time polymerase chain reaction. Expression of iron related proteins in the liver were analysed by immunoblotting and immunohistochemistry.
Results: Homozygous TfR2-KO mice had no TfR2 protein expression and developed significant iron overload typical of TfR2 associated haemochromatosis. In the liver of TfR2-KO mice there was no upregulation of hepcidin mRNA or prohepcidin protein in response to iron loading.
Conclusions: Our results suggest that TfR2 is required for iron regulated expression of hepcidin and is involved in a pathway related to Hfe and hemojuvelin.
doi:10.1136/gut.2004.062018
PMCID: PMC1774629  PMID: 15951546
transferrin receptor 2; hepcidin; haemochromatosis; iron metabolism; knockout mouse

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