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1.  Meropenem versus piperacillin-tazobactam for definitive treatment of bloodstream infections due to ceftriaxone non-susceptible Escherichia coli and Klebsiella spp (the MERINO trial): study protocol for a randomised controlled trial 
Trials  2015;16:24.
Gram-negative bacteria such as Escherichia coli or Klebsiella spp. frequently cause bloodstream infections. There has been a worldwide increase in resistance in these species to antibiotics such as third generation cephalosporins, largely driven by the acquisition of extended-spectrum beta-lactamase or plasmid-mediated AmpC enzymes. Carbapenems have been considered the most effective therapy for serious infections caused by such resistant bacteria; however, increased use creates selection pressure for carbapenem resistance, an emerging threat arising predominantly from the dissemination of genes encoding carbapenemases. Recent retrospective data suggest that beta-lactam/beta-lactamase inhibitor combinations, such as piperacillin-tazobactam, may be non-inferior to carbapenems for the treatment of bloodstream infection caused by extended-spectrum beta-lactamase-producers, if susceptible in vitro. This study aims to test this hypothesis in an effort to define carbapenem-sparing alternatives for these infections.
The study will use a multicentre randomised controlled open-label non-inferiority trial design comparing two treatments, meropenem (standard arm) and piperacillin-tazobactam (carbapenem-sparing arm) in adult patients with bacteraemia caused by E. coli or Klebsiella spp. demonstrating non-susceptibility to third generation cephalosporins. Recruitment is planned to occur in sites across three countries (Australia, New Zealand and Singapore). A total sample size of 454 patients will be required to achieve 80% power to determine non-inferiority with a margin of 5%. Once randomised, definitive treatment will be for a minimum of 4 days, but up to 14 days with total duration determined by treating clinicians. Data describing demographic information, antibiotic use, co-morbid conditions, illness severity, source of infection and other risk factors will be collected. Vital signs, white cell count, use of vasopressors and days to bacteraemia clearance will be recorded up to day 7. The primary outcome measure will be mortality at 30 days, with secondary outcomes including days to clinical and microbiological resolution, microbiological failure or relapse, isolation of a multi-resistant organism or Clostridium difficile infection.
Trial registration
The MERINO trial is registered under the Australian New Zealand Clinical Trials Register (ANZCTR), reference number: ACTRN12613000532707 (registered 13 May 2013) and the US National Institute of Health register, reference number: NCT02176122 (registered 24 June 2014).
Electronic supplementary material
The online version of this article (doi:10.1186/s13063-014-0541-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4311465  PMID: 25623485
Extended-spectrum beta-lactamase; ESBL; Plasmid-AmpC; Therapy; Resistance; Beta-lactam/beta-lactamase inhibitor; Carbapenem; Clinical trial
2.  Gastroenteritis due to typhoidal Salmonella: a decade of observation at an urban and a rural diarrheal disease hospital in Bangladesh 
BMC Infectious Diseases  2014;14(1):435.
The study aimed to compare the socio-demographic, host and clinical characteristics, seasonality and antimicrobial susceptibility of Typhoidal Salmonella (Salmonella enterica serovar Typhi and Paratyphi) (TS) with diarrhea between urban and rural Bangladesh.
Relevant information of 77/25,767 (0.30%) and 290/17,622 (1.65%) patients positive with TS (in stool) were extracted from the data archive of Diarrheal Disease Surveillance System of icddr,b (urban Dhaka and rural Matlab Hospitals respectively) during 2000–2012. Comparison group (diarrhea patients negative for TS) was randomly selected from the database (1:3 ratio). Two poisson regression models were investigated for modelling seasonal effects on the number of cases.
Salmonella Typhi was more frequently isolated in Dhaka than Matlab (57% vs. 5%, p < 0.001); while Salmonella Paratyphi was more frequent in Matlab than Dhaka (96% vs. 43%; p < 0.001). Fever [adj. OR-5.86 (95% CI: 2.16, 15.94)], antimicrobial use at home [5.08 (2.60, 9.90)], and fecal red blood cells [2.53 (1.38, 4.64)] were significantly associated with detection of TS in stool of patient from Dhaka. For Matlab, the correlates were, vomiting [1.88 (1.35, 2.64)], fecal macrophage [1.89 (1.29, 2.74)] in addition to fever and duration of diarrhea and antimicrobial use. At Dhaka, all Salmonella Typhi isolates were susceptible to ceftriaxone; while in Dhaka and Matlab however, for ciprofloxacin it was 45% and 91%, respectively. Susceptibility to chloramphenicol, ampicillin, trimethoprim-sulphamethoxazole and nalidixic acid ranged from 12%-58%. Salmonella Paratyphi were susceptible to ceftriaxone (99%). A significant seasonal trend and year difference (before and after 2007) for Matlab was observed (p < 0.001 for all effects). Dhaka does not show significant year or seasonal effects (p = 0.07 for years and p = 0.81 and p = 0.18 for the cos and sin components, respectively). While not significant, two seasonal peaks were observed in Dhaka (January-February and September-November); while a single peak (August-November) was observed in Matlab.
Proportion of serovar distribution of TS and their clinical characteristics, antimicrobial susceptibility and seasonal pattern were different among diarrhea patients in urban Dhaka and rural Matlab of Bangladesh.
PMCID: PMC4132926  PMID: 25098316
Bangladesh; Diarrhea; Rural; Typhoidal Salmonella; Urban
4.  Cost-effectiveness analysis of vaccinating children in Malawi with RTS,S vaccines in comparison with long-lasting insecticide-treated nets 
Malaria Journal  2014;13:66.
New RTS,S malaria vaccines may soon be licensed, yet its cost-effectiveness is unknown. Before the widespread introduction of RTS,S vaccines, cost-effectiveness studies are needed to help inform governments in resource-poor settings about how best to prioritize between the new vaccine and existing malaria interventions.
A Markov model simulated malaria progression in a hypothetical Malawian birth cohort. Parameters were based on published data. Three strategies were compared: no intervention, vaccination at one year, and long-lasting, insecticide-treated nets (LLINs) at birth. Both health service and societal perspectives were explored. Health outcomes were measured in disability-adjusted life years (DALYs) averted and costed in 2012 US$. Incremental cost-effectiveness ratios (ICERs) were calculated and extensive sensitivity analyses were conducted. Three times GDP per capita ($1,095) per DALY averted was used for a cost-effectiveness threshold, whilst one times GDP ($365) was considered ‘very cost-effective’.
From a societal perspective the vaccine strategy was dominant. It averted 0.11 more DALYs than LLINs and 0.372 more DALYs than the no intervention strategy per person, while costing $10.04 less than LLINs and $59.74 less than no intervention. From a health service perspective the vaccine’s ICER was $145.03 per DALY averted, and thus can be considered very cost-effective. The results were robust to changes in all variables except the vaccine and LLINs’ duration of efficacy. Vaccines remained cost-effective even at the lowest assumed efficacy levels of 49.6% (mild malaria) and 14.2% (severe malaria), and the highest price of $15. However, from a societal perspective, if the vaccine duration efficacy was set below 2.69 years or the LLIN duration of efficacy was greater than 4.24 years then LLINs became the more cost-effective strategy.
The results showed that vaccinating Malawian children with RTS,S vaccines was very cost-effective from both a societal and a health service perspective. This result was robust to changes in most variables, including vaccine price and vaccine efficacy, but was sensitive to the duration of efficacy of the vaccine and LLINs. Given the best evidence currently available, vaccines can be considered as a very cost-effective component of Malawi’s future malaria control programmes. However, long-term follow-up studies on both interventions are needed.
PMCID: PMC4016032  PMID: 24564883
RTS,S vaccine; Malaria vaccine; Cost-effectiveness analysis; Long-lasting insecticide-treated net; Bed net; Malawi; Malaria
5.  Concurrent and Simultaneous Polydrug Use: Latent Class Analysis of an Australian Nationally Representative Sample of Young Adults 
Background: Alcohol use and illicit drug use peak during young adulthood (around 18–29 years of age), but comparatively little is known about polydrug use in nationally representative samples of young adults. Drawing on a nationally representative cross-sectional survey (Australian National Drug Strategy Household Survey), this study examines polydrug use patterns and associated psychosocial risk factors among young adults (n = 3,333; age 19–29).
Method: The use of a broad range of licit and illicit drugs were examined, including alcohol, tobacco, cannabis, cocaine, hallucinogens, ecstasy, ketamine, GHB, inhalants, steroids, barbiturates, meth/amphetamines, heroin, methadone/buprenorphine, other opiates, painkillers, and tranquilizers/sleeping pills. Latent class analysis was employed to identify patterns of polydrug use.
Results: Polydrug use in this sample was best described using a 5-class solution. The majority of young adults predominantly used alcohol only (52.3%), alcohol and tobacco (34.18%). The other classes were cannabis, ecstasy, and licit drug use (9.4%), cannabis, amphetamine derivative, and licit drug use (2.8%), and sedative and alcohol use (1.3%). Young adult males with low education and/or high income were most at risk of polydrug use.
Conclusion: Almost half of young adults reported polydrug use, highlighting the importance of post-high school screening for key risk factors and polydrug use profiles, and the delivery of early intervention strategies targeting illicit drugs.
PMCID: PMC3860005  PMID: 24350230
young adults; polydrug use; latent class analysis; cluster; risk and protective factors; simultaneous
6.  Histopathologic Evaluation of Patent Ductus Arteriosus Stents After Hybrid Stage I Palliation 
Pediatric cardiology  2011;32(4):10.1007/s00246-010-9870-y.
The aim of this study was to determine the histopathology of patent ductus arteriosus (PDA) in-stent stenosis after hybrid stage I palliation. The hybrid approach to palliation of hypoplastic left heart syndrome can be complicated by the development of in-stent stenosis of the PDA. This may obstruct retrograde aortic arch flow, decrease systemic circulation, and lead to interstage interventional procedures. Stented PDA samples removed from eight patients undergoing comprehensive stage II repair were examined by way of radiography and histochemistry (hematoxylin and eosin, Movat pentachrome, α-smooth muscle actin, and proliferating cell nuclear antigen). A retrospective chart review of the patients was also performed. PDA stents were in place in the PDA for a mean period of 169 ± 28 days in patients who had a mean age of 176 ± 30 days at the time of stent removal. Stent deployment caused chronic inflammation, caused fibrin deposition, and induced vascular smooth muscle–cell (VSMC) proliferation in the area immediately surrounding the stent struts. The neointimal region was composed largely of smooth muscle cells that appeared to be fully differentiated by the lack of PCNA staining. Neointimal thickening occurs in the PDA after stent placement for hybrid palliation of HLHS and is the result of inflammation, extracellular matrix deposition, and smooth muscle–cell proliferation in the peristrut region. This finding suggests that proliferating VSMCs in the peristrut region may provide the impetus for inward neointimal formation and therefore the manifestation of in-stent stenosis.
PMCID: PMC3822434  PMID: 21298382
Ductus arteriosus; Stent; Neointima; In-stent stenosis
7.  Mental health issues decrease diabetes-specific quality of life independent of glycaemic control and complications: findings from Australia’s living with diabetes cohort study 
While factors associated with health-related quality of life for people with chronic diseases including diabetes are well researched, far fewer studies have investigated measures of disease-specific quality of life. The purpose of this study is to assess the impact of complications and comorbidities on diabetes-specific quality of life in a large population-based cohort of type 2 diabetic patients.
The Living with Diabetes Study recruited participants from the National Diabetes Services Scheme in Australia. Data were collected via a mailed self-report questionnaire. Diabetes-specific quality of life was measured using the Audit of Diabetes-Dependent Quality of Life (ADDQoL) questionnaire. The analyses are for 3609 patients with type 2 diabetes. Regression models with adjustment for control variables investigated the association of complications and comorbidities with diabetes-specific quality of life. Next, the most parsimonious model for diabetes-specific quality of life after controlling for important covariates was examined.
The expected associations with better diabetes-specific quality of life were evident, such as increased income, not on insulin, better glycaemic control and older age. However, being single and having been diagnosed with cancer were also associated with better ADDQoL. Additionally, poorer diabetes-specific quality of life was strongly sensitive to the presence of diabetes complications and mental health conditions such as depression, anxiety and schizophrenia. These relationships persisted after adjustment for gender, age, duration of diabetes, treatment regimen, sampling region and other treatment and socio-demographic variables.
A greater appreciation of the complexities of diabetes-specific quality of life can help tailor disease management and self-care messages given to patients. Attention to mental health issues may be as important as focusing on glycaemic control and complications. Therefore clinicians’ ability to identify and mange mental health issues and/or refer patients is critical to improving patients’ diabetes-specific quality of life.
PMCID: PMC3853250  PMID: 24131673
Diabetes-specific quality of life; Audit of Diabetes-Dependent Quality of Life (ADDQoL); Type 2 diabetes; Adults
8.  At-risk but viable myocardium in a large animal model of non ST-segment elevation acute coronary syndrome: cardiovascular magnetic resonance with ex vivo validation 
Patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) have varying degrees of salvageable myocardium at risk of irreversible injury. We hypothesized that a novel model of NSTE-ACS produces acute myocardial injury, measured by increased T2 cardiovascular magnetic resonance (CMR), without significant necrosis by late gadolinium enhancement (LGE).
In a canine model, partial coronary stenosis was created and electrodes placed on the epicardium. Myocardial T2, an indicator of at-risk myocardium, was measured pre- and post-tachycardic pacing.
Serum troponin-I (TnI) was not detectable in unoperated sham animals but averaged 1.97 ± 0.72 ng/mL in model animals. Coronary stenosis and pacing produced significantly higher T2 in the affected vs. the remote myocardium (53.2 ± 4.9 vs. 43.6 ± 2.8 ms, p < 0.01) with no evident injury by LGE. Microscopy revealed no significant irreversible cellular injury. Relative respiration rate (RRR) of affected vs. remote myocardial tissue was significantly lower in model vs. sham animals (0.72 ± 0.07 vs. 1.04 ± 0.07, p < 0.001). Lower RRR corresponded to higher final TnI levels (R2 = 0.83, p = 0.004) and changes in CaMKIID and mitochondrial gene expression.
A large animal NSTE-ACS model with mild TnI elevation and without ST elevation, similar to the human syndrome, demonstrates signs of acute myocardial injury by T2-CMR without significant irreversible damage. Reduced tissue respiration and associated adaptations of critical metabolic pathways correspond to increased myocardial injury by serum biomarkers in this model. T2-CMR as a biomarker of at-risk but salvageable myocardium warrants further consideration in preclinical and clinical studies of NSTE-ACS.
PMCID: PMC3852225  PMID: 24107555
Myocardial ischemia; Oxygen consumption; Cardiovascular magnetic resonance; Canine model; Mitochondria
9.  Ribonuclease 7, an antimicrobial peptide up-regulated during infection, contributes to microbial defense of the human urinary tract 
Kidney international  2013;83(4):615-625.
The mechanisms that maintain sterility in the urinary tract are incompletely understood; however, recent studies stress the importance of antimicrobial peptides in protecting the urinary tract from infection. Ribonuclease 7 (RNase 7), a potent antimicrobial peptide contributing to urinary tract sterility, is expressed by intercalated cells in the renal collecting tubules and is present in the urine at levels sufficient to kill bacteria at baseline. Here, we characterize the expression and function of RNase 7 in the human urinary tract during infection. Both quantitative real-time PCR and ELISA assays demonstrated increases in RNASE7 expression in the kidney along with kidney and urinary RNase 7 peptide concentrations with infection. While immunostaining localized RNase 7 production to the intercalated cells of the collecting tubule during sterility, its expression during pyelonephritis was found to increase throughout the nephron but not in glomeruli or the interstitium. Recombinant RNase 7 exhibited antimicrobial activity against uropathogens at low micromolar concentrations by disrupting the microbial membrane as determined by atomic force microscopy. Thus, RNase 7 expression is increased in the urinary tract with infection, and has antibacterial activity against uropathogens at micromolar concentrations.
PMCID: PMC3612368  PMID: 23302724
Ribonuclease 7; Antimicrobial Peptide; Pyelonephritis; Urinary Tract Infection; Intercalated Cells; Innate Immunity; Immunology
10.  Unusual case of a vanishing bronchus of the left allograft in a lung transplant recipient 
Annals of Thoracic Medicine  2013;8(4):229-230.
We present an interesting case of a complete vanishing of the left main bronchus in a lung transplant recipient who had a successful outcome due to acute respiratory support with venovenous extracorporeal membrane oxygenation in order to perform airway dilation.
PMCID: PMC3821284  PMID: 24250738
Acute; extracorporeal membrane oxygenation; left main bronchus; lung transplantation; vanishing; venovenous
11.  Screening and Improving the Recombinant Nitrilases and Application in Biotransformation of Iminodiacetonitrile to Iminodiacetic Acid 
PLoS ONE  2013;8(6):e67197.
In this study, several nitrilase genes from phylogenetically distinct organisms were expressed and purified in E. coli in order to study their ability to mediate the biotransformation of nitriles. We identified three nitrilases: Acidovorax facilis nitrilase (AcN); Alcaligenes fecalis nitrilase (AkN); and Rhodococcus rhodochrous nitrilase (RkN), which catalyzed iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA). AcN demonstrated 8.8-fold higher activity for IDAN degradation as compared to AkN and RkN. Based on homology modeling and previously described ‘hot spot’ mutations, several AcN mutants were screened for improved activity. One mutant M3 (F168V/L201N/S192F) was identified, which demonstrates a 41% enhancement in the conversion as well as a 2.4-fold higher catalytic efficiency towards IDAN as compared to wild-type AcN.
PMCID: PMC3695085  PMID: 23826231
12.  Type 2 diabetes: a cohort study of treatment, ethnic and social group influences on glycated haemoglobin 
BMJ Open  2012;2(5):e001477.
To assess whether in people with poorly controlled type 2 diabetes (HbA1c>7.5%) improvement in HbA1c varies by ethnic and social group.
Prospective 2-year cohort of type 2 diabetes treated in general practice.
Setting and participants
All patients with type 2 diabetes in 100 of the 101 general practices in two London boroughs. The sample consisted of an ethnically diverse group with uncontrolled type 2 diabetes aged 37–71 years in 2007 and with HbA1c recording in 2008–2009.
Outcome measure
Change from baseline HbA1c in 2007 and achievement of HbA1c control in 2008 and 2009 were estimated for each ethnic, social and treatment group using multilevel modelling.
The sample consisted of 6104 people; 18% were white, 63% south Asian, 16% black African/Caribbean and 3% other ethnic groups. HbA1c was lower after 1 and 2 years in all ethnic groups but south Asian people received significantly less benefit from each diabetes treatment. After adjustment, south Asian people were found to have 0.14% less reduction in HbA1c compared to white people (95% CI 0.04% to 0.24%) and white people were 1.6 (95% CI 1.2 to 2.0) times more likely to achieve HbA1c controlled to 7.5% or less relative to south Asian people. HbA1c reduction and control in black African/Caribbean and white people did not differ significantly. There was no evidence that social deprivation influenced HbA1c reduction or control in this cohort.
In all treatment groups, south Asian people with poorly controlled diabetes are less likely to achieve controlled HbA1c, with less reduction in mean HbA1c than white or black African/Caribbean people.
PMCID: PMC3488709  PMID: 23087015
diabetes & endocrinology; primary care; therapeutics; public health
13.  Current challenges in software solutions for mass spectrometry-based quantitative proteomics 
Amino Acids  2012;43(3):1087-1108.
Mass spectrometry-based proteomics has evolved as a high-throughput research field over the past decade. Significant advances in instrumentation, and the ability to produce huge volumes of data, have emphasized the need for adequate data analysis tools, which are nowadays often considered the main bottleneck for proteomics development. This review highlights important issues that directly impact the effectiveness of proteomic quantitation and educates software developers and end-users on available computational solutions to correct for the occurrence of these factors. Potential sources of errors specific for stable isotope-based methods or label-free approaches are explicitly outlined. The overall aim focuses on a generic proteomic workflow.
PMCID: PMC3418498  PMID: 22821268
LC–MS; Quantitative proteomics; Quantification software; Stable isotope labeling; Label-free
14.  Improved identification of O-linked glycopeptides from ETD data with optimized scoring for different charge states and cleavage specificities 
Amino acids  2010;41(2):321-328.
This article describes the effect of re-interrogation of electron-transfer dissociation (ETD) data with newly developed analytical tools. MS/MS-based characterization of O-linked glycopeptides is discussed using data acquired from a complex mixture of O-linked glycopep-tides, featuring mucin core 1-type carbohydrates with and without sialic acid, as well as after partial deglycosylation to leave only the core GalNAc units (Darula and Medzihradszky in Mol Cell Proteomics 8:2515, 2009). Information content of collision-induced dissociation spectra generated in collision cell (in QqTOF instruments) and in ion traps is compared. Interpretation of the corresponding ETD data using Protein Prospector is also presented. Search results using scoring based on the frequency of different fragment ions occurring in ETD spectra of tryptic peptides are compared with results obtained after ion weightings were adjusted to accommodate differential ion frequencies in spectra of differing charge states or cleavage specificities. We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria. This study illustrates that “old” proteomic data may yield significant new information when re-interrogated with new, improved tools.
PMCID: PMC3102200  PMID: 20652609
Mass spectrometry; Electron-transfer dissociation (ETD); Collision-induced dissociation (CID); O-linked glycopeptides; O-glycosylation; Database search
15.  Ethnic and social disparity in glycaemic control in type 2 diabetes; cohort study in general practice 2004–9 
To determine whether ethnic group differences in glycated haemoglobin (HbA1c) changed over a 5-year period in people on medication for type 2 diabetes.
Open cohort in 2004–9.
Electronic records of 100 of the 101 general practices in two inner London boroughs.
People aged 35 to 74 years on medication for type 2 diabetes.
Main outcome measures
Mean HbA1c and proportion with HbA1c controlled to ≤7.5%.
In this cohort of 24,111 people, 22% were White, 58% South Asian and 17% Black African/Caribbean. From 2004 to 2009 mean HbA1c improved from 8.2% to 7.8% for White, from 8.5% to 8.0% for Black African/Caribbean and from 8.5% to 8.0% for South Asian people. The proportion with HbA1c controlled to 7.5% or less, increased from 44% to 56% in White, 38% to 53% in Black African/Caribbean and 34% to 48% in South Asian people. Ethnic group and social deprivation were independently associated with HbA1c. South Asian and Black African/Caribbean people were treated more intensively than White people.
HbA1c control improved for all ethnic groups between 2004–9. However, South Asian and Black African/Caribbean people had persistently worse control despite more intensive treatment and significantly more improvement than White people. Higher social deprivation was independently associated with worse control.
PMCID: PMC3407404  PMID: 22396467
16.  Novel X-linked glomerulopathy associated with a COL4A5 missense mutation in a noncollagenous interruption 
Kidney international  2010;79(1):120-127.
We report a novel COL4A5 mutation causing rapid progression to end stage renal disease in males despite the absence of clinical and biopsy findings associated with Alport syndrome. Affected males had proteinuria, variable hematuria, early progression to end stage renal disease; and renal biopsy findings which included global and segmental glomerulosclerosis, mesangial hypercellularity and basement membrane immune complex deposition.
Exon sequencing of the COL4A5 locus identified a thymine to guanine transversion at nucleotide 665, resulting in a phenylalanine to cysteine missense mutation at codon 222. This mutation was confirmed in 4 affected males and 4 female obligate carriers, but was absent in 6 asymptomatic male family members and 198 unrelated individuals. α5(IV) collagen staining in renal biopsies from affected males was normal.
The phenylalanine at position 222 is 100% conserved among vertebrates. This is the first description of a mutation in a non-collagenous interruption associated with severe renal disease, providing evidence for the importance of this structural motif. The range of phenotypes associated with COL4A5 mutations is more diverse than previously realized. COL4A5 mutation analysis should be considered when glomerulonephritis presents in an X-linked inheritance pattern, even with a distinct presentation from Alport syndrome.
PMCID: PMC3248803  PMID: 20881942
17.  Structural and Functional Studies of A. oryzae Cutinase: Enhanced Thermostability and Hydrolytic Activity of Synthetic Ester and Polyester Degradation 
Journal of the American Chemical Society  2009;131(43):15711-15716.
Cutinases are responsible for hydrolysis of the protective cutin lipid polyester matrix in plants and thus have been exploited for hydrolysis of small molecule esters and polyesters. Here we explore the reactivity, stability, and structure of Aspergillus oryzae cutinase and compare it to the well-studied enzyme from Fusarium solani. Two critical differences are highlighted in the crystallographic analysis of the A. oryzae structure: (i) an additional disulfide bond and (ii) a topologically favored catalytic triad with a continuous and deep groove. These structural features of A. oryzae cutinase are proposed to result in improved hydrolytic activity and altered substrate specificity profile, enhanced thermostability and remarkable reactivity towards the degradation of the synthetic polyester, polycaprolactone. The results presented here provide insight into engineering new cutinase-inspired biocatalysts with tailor-made properties.
PMCID: PMC2796240  PMID: 19810726
A. oryzae; cutinase; hydrolysis; substrate specificity; thermostability; disulfide bond; polyester degradation
18.  Improved identification of O-linked glycopeptides from ETD data with optimized scoring for different charge states and cleavage specificities 
Amino Acids  2010;41(2):321-328.
This article describes the effect of re-interrogation of electron-transfer dissociation (ETD) data with newly developed analytical tools. MS/MS-based characterization of O-linked glycopeptides is discussed using data acquired from a complex mixture of O-linked glycopeptides, featuring mucin core 1-type carbohydrates with and without sialic acid, as well as after partial deglycosylation to leave only the core GalNAc units (Darula and Medzihradszky in Mol Cell Proteomics 8:2515, 2009). Information content of collision-induced dissociation spectra generated in collision cell (in QqTOF instruments) and in ion traps is compared. Interpretation of the corresponding ETD data using Protein Prospector is also presented. Search results using scoring based on the frequency of different fragment ions occurring in ETD spectra of tryptic peptides are compared with results obtained after ion weightings were adjusted to accommodate differential ion frequencies in spectra of differing charge states or cleavage specificities. We show that the improved scoring is more than doubled the glycopeptide assignments under very strict acceptance criteria. This study illustrates that “old” proteomic data may yield significant new information when re-interrogated with new, improved tools.
Electronic supplementary material
The online version of this article (doi:10.1007/s00726-010-0692-2) contains supplementary material, which is available to authorized users.
PMCID: PMC3102200  PMID: 20652609
Mass spectrometry; Electron-transfer dissociation (ETD); Collision-induced dissociation (CID); O-linked glycopeptides; O-glycosylation; Database search
19.  A cluster randomized controlled cross-over bed net acceptability and preference trial in Solomon Islands: community participation in shaping policy for malaria elimination 
Malaria Journal  2009;8:298.
A key component of the malaria elimination strategy in Solomon Islands (SI) is widespread coverage of long-lasting insecticidal nets (LLINs). The success of this strategy is dependent on LLIN acceptability and compliance. There has been unresolved debate among policy makers and donors as to which type of LLIN would be most appropriate for large-scale distribution in SI, and anecdotal reports of a lack of acceptability of certain brands of LLINs. A cluster randomized controlled crossover bed net acceptability and preference trial was therefore carried out from July to September, 2008 to inform policy and to facilitate community engagement and participation in the selection of the most appropriate LLIN for use in SI.
A three-stage sampling method was used to randomly select the study population from Malaita Province, SI. Three brands of LLINs were assessed in this study: Olyset®, PermaNet® and DuraNet®. Bed net acceptability and preference were evaluated through surveys at three defined time points after short and longer-term trial of each LLIN.
The acceptability of PermaNet® after short-term use (96.5%) was significantly greater than Olyset® (67.3%, p < 0.001) and DuraNet® (69.8%, p < 0.001). The acceptability of DuraNet® and Olyset® after short-term use was not significantly different at the 5% level. LLINs that were perceived not to prevent mosquito bites were significantly less acceptable than LLINs that were perceived to prevent mosquito bites (OR 0.15; 95%CI 0.03 to 0.6). LLINs that allow a pleasant night's sleep (OR 6.3; 95%CI:3.3-12.3) and have a soft texture (OR 5.7; 95%CI:1.9-20.5) were considered more acceptable than those that did not. Olyset®'s acceptability decreased over time and this was due to net wrinkling/shrinkage after washing resulting in reduced efficiency in preventing mosquito bites. The increase in DuraNet® acceptability was a result of a reduction in minor adverse events following longer-term use.
This research was conducted to inform LLIN procurement as part of the national malaria control and elimination programme in SI. The success of malaria elimination in the Pacific and elsewhere relies on provision of acceptable interventions, consideration of local-level realities and engagement of communities in strategy development.
Trial Registrations
Clinical trials ACTRN12608000322336
PMCID: PMC2803192  PMID: 20015402
20.  In Vivo MRI Atherosclerotic Plaque Characterization Using Magnetic Susceptibility Distinguishes Symptom-Producing Plaques 
JACC. Cardiovascular imaging  2008;1(1):49-57.
We investigated iron's role in atherosclerosis and plaque instability with a novel approach to in vivo atherosclerotic plaque characterization using noninvasive, noncontrast magnetic resonance-based T2* measurement. We validated this approach using ex vivo plaque analyses to establish that T2* reflects intraplaque iron composition.
Iron catalyzes free radical production, a key step for lipid peroxidation and atherosclerosis development. The parameter T2* measures tissue magnetic susceptibility, historically has been used to quantify hepatic and myocardial iron. To date, T2* measurement has not been previously developed for in vivo plaque characterization in patients with atherosclerosis.
Thirty-nine patients referred for carotid endarterectomy were prospectively enrolled to undergo preoperative carotid MRI and postoperative analysis of the explanted plaque. Clinical history of any symptoms attributable to each carotid lesion was recorded.
MRI could not be completed in 4 subjects due to claustrophobia, and three patients scanned prior to the use of a neck stabilizer had motion artifact precluding quantification. In the remaining subjects, symptomatic compared to asymptomatic patients had significantly lower plaque T2* values (20.0±1.8 vs. 34.4±2.7 ms, respectively, p<0.001). Analytical methods demonstrated similar total iron (138.6±36.5 vs. 165.8±48.3 mg/kg, p=NS) but less low-molecular weight Fe(III) (7.3±3.8 vs. 17.7±4.0 nmol/mg, p<0.05) in the explanted plaques of symptomatic versus asymptomatic patients, respectively, consistent with a shift in iron from Fe(III) to higher amounts of T2*-shortening forms of iron. Mass spectroscopy also showed significantly lower calcium (37.5±10.8 vs. 123.6±19.3 g/kg, p<0.01) and higher copper (3.2±0.5 vs. 1.7±0.1 mg/kg, p<0.01) in plaques from symptomatic patients.
In vivo measurement of intraplaque T2* using MRI is feasible and reproducible, and distinguishes symptom-producing from non-symptom producing plaques in patients with carotid artery atherosclerosis. Symptom-producing plaques demonstrated characteristic changes in iron forms by ex vivo analysis, supporting the dynamic presence of iron in the microenvironment of atherosclerotic plaque.
PMCID: PMC2729432  PMID: 19356405
iron; atherosclerosis; magnetic resonance imaging; electron paramagnetic resonance; mass spectroscopy; stroke
21.  Diffuse Lung Disease in Young Children 
Rationale: Considerable confusion exists regarding nomenclature, classification, and management of pediatric diffuse lung diseases due to the relative rarity and differences in the spectrum of disease between adults and young children.
Objectives: A multidisciplinary working group was formed to: (1) apply consensus terminology and diagnostic criteria for disorders presenting with diffuse lung disease in infancy; and (2) describe the distribution of disease entities, clinical features, and outcome in young children who currently undergo lung biopsy in North America.
Methods: Eleven centers provided pathologic material, clinical data, and imaging from all children less than 2 years of age who underwent lung biopsy for diffuse lung disease from 1999 to 2004.
Measurements and Main Results: Multidisciplinary review categorized 88% of 187 cases. Disorders more prevalent in infancy, including primary developmental and lung growth abnormalities, neuroendocrine cell hyperplasia of infancy, and surfactant-dysfunction disorders, constituted the majority of cases (60%). Lung growth disorders were often unsuspected clinically and under-recognized histologically. Cases with known surfactant mutations had characteristic pathologic features. Age at biopsy and clinical presentation varied among categories. Pulmonary hypertension, presence of a primary developmental abnormality, or ABCA3 mutation was associated with high mortality, while no deaths occurred in cases of pulmonary interstitial glycogenosis, or neuroendocrine cell hyperplasia of infancy.
Conclusions: This retrospective cohort study identifies a diverse spectrum of lung disorders, largely unique to young children. Application of a classification scheme grouped clinically distinct patients with variable age of biopsy and mortality. Standardized terminology and classification will enhance accurate description and diagnosis of these disorders.
PMCID: PMC2176101  PMID: 17885266
infant; pulmonary; interstitial lung disease; surfactant; neuroendocrine hyperplasia
22.  Impact of Low and High Tidal Volumes on the Rat Alveolar Epithelial Type II Cell Proteome 
Rationale: Mechanical ventilation with high tidal volumes leads to increased permeability, generation of inflammatory mediators, and damage to alveolar epithelial cells (ATII).
Objectives: To identify changes in the ATII proteome after two different ventilation strategies in rats.
Methods: Rats (n = 6) were ventilated for 5 hours with high- and low tidal volumes (Vts) (high Vt: 20 ml/kg; low Vt: 6 ml/kg). Pooled nonventilated rats served as control animals. ATII cells were isolated and lysed, and proteins were tryptically cleaved into peptides. Cellular protein content was evaluated by peptide labeling of the ventilated groups with 18O. Samples were fractionated by cation exchange chromatography and identified using electrospray tandem mass spectrometry. Proteins identified by 15 or more peptides were statistically compared using t tests corrected for the false discovery rate.
Measurements and Main Results: High Vt resulted in a significant increase in airspace neutrophils without an increase in extravascular lung water. Compared with low-Vt samples, high-Vt samples showed a 32% decrease in the inositol 1,4,5-trisphosphate 3 receptor (p < 0.01), a 34% decrease in Na+, K+-ATPase (p < 0.01), and a significantly decreased content in ATP synthase chains. Even low-Vt samples displayed significant changes, including a 66% decrease in heat shock protein 90-β (p < 0.01) and a 67% increase in mitochondrial pyruvate carboxylase (p < 0.01). Significant differences were found in membrane, acute phase, structural, and mitochondrial proteins.
Conclusions: After short-term exposure to high-Vt ventilation, significant reductions in membrane receptors, ion channel proteins, enzymes of the mitochondrial energy system, and structural proteins in ATII cells were present. The data supports the two-hit concept that an unfavorable ventilatory strategy may make the lung more vulnerable to an additional insult.
PMCID: PMC1899270  PMID: 17363773
acute lung injury; alveolar epithelium; corticosterone
23.  The international men's health movement  
BMJ : British Medical Journal  2001;323(7320):1014-1015.
PMCID: PMC1121528  PMID: 11691743
24.  Molecular Characterization of the Toxic Cyanobacterium Cylindrospermopsis raciborskii and Design of a Species-Specific PCR 
Cylindrospermopsis raciborskii is a toxic-bloom-forming cyanobacterium that is commonly found in tropical to subtropical climatic regions worldwide, but it is also recognized as a common component of cyanobacterial communities in temperate climates. Genetic profiles of C. raciborskii were examined in 19 cultured isolates originating from geographically diverse regions of Australia and represented by two distinct morphotypes. A 609-bp region of rpoC1, a DNA-dependent RNA polymerase gene, was amplified by PCR from these isolates with cyanobacterium-specific primers. Sequence analysis revealed that all isolates belonged to the same species, including morphotypes with straight or coiled trichomes. Additional rpoC1 gene sequences obtained for a range of cyanobacteria highlighted clustering of C. raciborskii with other heterocyst-producing cyanobacteria (orders Nostocales and Stigonematales). In contrast, randomly amplified polymorphic DNA and short tandemly repeated repetitive sequence profiles revealed a greater level of genetic heterogeneity among C. raciborskii isolates than did rpoC1 gene analysis, and unique band profiles were also found among each of the cyanobacterial genera examined. A PCR test targeting a region of the rpoC1 gene unique to C. raciborskii was developed for the specific identification of C. raciborskii from both purified genomic DNA and environmental samples. The PCR was evaluated with a number of cyanobacterial isolates, but a PCR-positive result was only achieved with C. raciborskii. This method provides an accurate alternative to traditional morphological identification of C. raciborskii.
PMCID: PMC91826  PMID: 10618244
25.  Febrile Convulsions 
The Ulster Medical Journal  1967;36(2):155-161.
PMCID: PMC2385109  PMID: 20476449

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