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1.  Identification of Aph1, a Phosphate-Regulated, Secreted, and Vacuolar Acid Phosphatase in Cryptococcus neoformans 
mBio  2014;5(5):e01649-14.
ABSTRACT
Cryptococcus neoformans strains isolated from patients with AIDS secrete acid phosphatase, but the identity and role of the enzyme(s) responsible have not been elucidated. By combining a one-dimensional electrophoresis step with mass spectrometry, a canonically secreted acid phosphatase, CNAG_02944 (Aph1), was identified in the secretome of the highly virulent serotype A strain H99. We created an APH1 deletion mutant (Δaph1) and showed that Δaph1-infected Galleria mellonella and mice survived longer than those infected with the wild type (WT), demonstrating that Aph1 contributes to cryptococcal virulence. Phosphate starvation induced APH1 expression and secretion of catalytically active acid phosphatase in the WT, but not in the Δaph1 mutant, indicating that Aph1 is the major extracellular acid phosphatase in C. neoformans and that it is phosphate repressible. DsRed-tagged Aph1 was transported to the fungal cell periphery and vacuoles via endosome-like structures and was enriched in bud necks. A similar pattern of Aph1 localization was observed in cryptococci cocultured with THP-1 monocytes, suggesting that Aph1 is produced during host infection. In contrast to Aph1, but consistent with our previous biochemical data, green fluorescent protein (GFP)-tagged phospholipase B1 (Plb1) was predominantly localized at the cell periphery, with no evidence of endosome-mediated export. Despite use of different intracellular transport routes by Plb1 and Aph1, secretion of both proteins was compromised in a Δsec14-1 mutant. Secretions from the WT, but not from Δaph1, hydrolyzed a range of physiological substrates, including phosphotyrosine, glucose-1-phosphate, β-glycerol phosphate, AMP, and mannose-6-phosphate, suggesting that the role of Aph1 is to recycle phosphate from macromolecules in cryptococcal vacuoles and to scavenge phosphate from the extracellular environment.
IMPORTANCE
Infections with the AIDS-related fungal pathogen Cryptococcus neoformans cause more than 600,000 deaths per year worldwide. Strains of Cryptococcus neoformans isolated from patients with AIDS secrete acid phosphatase; however, the identity and role of the enzyme(s) are unknown. We have analyzed the secretome of the highly virulent serotype A strain H99 and identified Aph1, a canonically secreted acid phosphatase. By creating an APH1 deletion mutant and an Aph1-DsRed-expressing strain, we demonstrate that Aph1 is the major extracellular and vacuolar acid phosphatase in C. neoformans and that it is phosphate repressible. Furthermore, we show that Aph1 is produced in cryptococci during coculture with THP-1 monocytes and contributes to fungal virulence in Galleria mellonella and mouse models of cryptococcosis. Our findings suggest that Aph1 is secreted to the environment to scavenge phosphate from a wide range of physiological substrates and is targeted to vacuoles to recycle phosphate from the expendable macromolecules.
doi:10.1128/mBio.01649-14
PMCID: PMC4172073  PMID: 25227465
2.  Frequency and reasons for outcome reporting bias in clinical trials: interviews with trialists 
Objectives To provide information on the frequency and reasons for outcome reporting bias in clinical trials.
Design Trial protocols were compared with subsequent publication(s) to identify any discrepancies in the outcomes reported, and telephone interviews were conducted with the respective trialists to investigate more extensively the reporting of the research and the issue of unreported outcomes.
Participants Chief investigators, or lead or coauthors of trials, were identified from two sources: trials published since 2002 covered in Cochrane systematic reviews where at least one trial analysed was suspected of being at risk of outcome reporting bias (issue 4, 2006; issue 1, 2007, and issue 2, 2007 of the Cochrane library); and a random sample of trial reports indexed on PubMed between August 2007 and July 2008.
Setting Australia, Canada, Germany, the Netherlands, New Zealand, the United Kingdom, and the United States.
Main outcome measures Frequency of incomplete outcome reporting—signified by outcomes that were specified in a trial’s protocol but not fully reported in subsequent publications—and trialists’ reasons for incomplete reporting of outcomes.
Results 268 trials were identified for inclusion (183 from the cohort of Cochrane systematic reviews and 85 from PubMed). Initially, 161 respective investigators responded to our requests for interview, 130 (81%) of whom agreed to be interviewed. However, failure to achieve subsequent contact, obtain a copy of the study protocol, or both meant that final interviews were conducted with 59 (37%) of the 161 trialists. Sixteen trial investigators failed to report analysed outcomes at the time of the primary publication, 17 trialists collected outcome data that were subsequently not analysed, and five trialists did not measure a prespecified outcome over the course of the trial. In almost all trials in which prespecified outcomes had been analysed but not reported (15/16, 94%), this under-reporting resulted in bias. In nearly a quarter of trials in which prespecified outcomes had been measured but not analysed (4/17, 24%), the “direction” of the main findings influenced the investigators’ decision not to analyse the remaining data collected. In 14 (67%) of the 21 randomly selected PubMed trials, there was at least one unreported efficacy or harm outcome. More than a quarter (6/21, 29%) of these trials were found to have displayed outcome reporting bias.
Conclusion The prevalence of incomplete outcome reporting is high. Trialists seemed generally unaware of the implications for the evidence base of not reporting all outcomes and protocol changes. A general lack of consensus regarding the choice of outcomes in particular clinical settings was evident and affects trial design, conduct, analysis, and reporting.
doi:10.1136/bmj.c7153
PMCID: PMC3016816  PMID: 21212122
3.  Risk of recurrence after a first seizure and implications for driving: further analysis of the Multicentre study of early Epilepsy and Single Seizures 
Objective To determine for how long after a first unprovoked seizure a driver must be seizure-free before the risk of recurrence in the next 12 months falls below 20%, enabling them to regain their driving licence.
Design Randomised controlled trial: Multicentre study of early Epilepsy and Single Seizures (MESS).
Setting UK hospital outpatient clinics from 1 January 1993 to 31 December 2000.
Participants People entered MESS if they had had one or more unprovoked seizures and both the participant and the clinician were uncertain about the need to start antiepileptic drug treatment. The subset of people used for this analysis comprised participants aged at least 16 years with a single unprovoked seizure.
Main outcome measure Risk of seizure recurrence in the 12 months after a seizure-free period of 6, 12, 18, or 24 months from the date of the first (index) seizure. Regression modelling was used to investigate how antiepileptic treatment and several clinical factors influence the risk of seizure recurrence.
Results At six months after the index seizure the risk of recurrence in the next 12 months for those who start antiepileptic drugs was significantly below 20% (unadjusted risk 14%, 95% confidence interval 10% to 18%). For patients who did not start treatment the risk estimate was less than 20% but the upper limit of the confidence interval was greater than 20% (18%, 13% to 23%). Multivariable analyses identified subgroups with a significantly greater than 20% risk of seizure recurrence in the 12 months after a six month seizure-free period, such as those with a remote symptomatic seizure with abnormal electroencephalogram results.
Conclusion After a single unprovoked seizure this reanalysis of MESS provides estimates of seizure recurrence risks that will inform policy and guidance about regaining an ordinary driving licence. Further guidance is needed as to how such data should be utilised; in particular, whether a population approach should be taken with a focus on the unadjusted results or whether attempts should be made to individualise risk. Guidance is also required as to whether the focus should be on risk estimates only or on the confidence interval as well. If the focus is on the estimate only our unadjusted estimates suggest that treated and untreated patients are eligible to drive after being seizure-free for six months. If the focus is also on confidence intervals, direction is needed as to whether a conservative or liberal approach should be taken.
Trial registration Current Controlled Trials ISRCTN98767960.
doi:10.1136/bmj.c6477
PMCID: PMC2998675  PMID: 21147743
4.  Melatonin for sleep problems in children with neurodevelopmental disorders: randomised double masked placebo controlled trial 
Objective To assess the effectiveness and safety of melatonin in treating severe sleep problems in children with neurodevelopmental disorders.
Design 12 week double masked randomised placebo controlled phase III trial.
Setting 19 hospitals across England and Wales.
Participants 146 children aged 3 years to 15 years 8 months were randomised. They had a range of neurological and developmental disorders and a severe sleep problem that had not responded to a standardised sleep behaviour advice booklet provided to parents four to six weeks before randomisation. A sleep problem was defined as the child not falling asleep within one hour of lights out or having less than six hours’ continuous sleep.
Interventions Immediate release melatonin or matching placebo capsules administered 45 minutes before the child’s bedtime for a period of 12 weeks. All children started with a 0.5 mg capsule, which was increased through 2 mg, 6 mg, and 12 mg depending on their response to treatment.
Main outcome measures Total sleep time at night after 12 weeks adjusted for baseline recorded in sleep diaries completed by the parent. Secondary outcomes included sleep onset latency, assessments of child behaviour, family functioning, and adverse events. Sleep was measured with diaries and actigraphy.
Results Melatonin increased total sleep time by 22.4 minutes (95% confidence interval 0.5 to 44.3 minutes) measured by sleep diaries (n=110) and 13.3 (−15.5 to 42.2) measured by actigraphy (n=59). Melatonin reduced sleep onset latency measured by sleep diaries (−37.5 minutes, −55.3 to −19.7 minutes) and actigraphy (−45.3 minutes, −68.8 to −21.9 minutes) and was most effective for children with the longest sleep latency (P=0.009). Melatonin was associated with earlier waking times than placebo (29.9 minutes, 13.6 to 46.3 minutes). Child behaviour and family functioning outcomes showed some improvement and favoured use of melatonin. Adverse events were mild and similar between the two groups.
Conclusions Children gained little additional sleep on melatonin; though they fell asleep significantly faster, waking times became earlier. Child behaviour and family functioning outcomes did not significantly improve. Melatonin was tolerable over this three month period. Comparisons with slow release melatonin preparations or melatonin analogues are required.
Trial registration ISRCT No 05534585.
doi:10.1136/bmj.e6664
PMCID: PMC3489506  PMID: 23129488
5.  Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. 
Journal of Bacteriology  1994;176(3):656-664.
Melanin production is a major virulence factor for Cryptococcus neoformans, an organism causing life-threatening infections in an estimated 10% of AIDS patients. In order to characterize the events involved in melanin synthesis, an enzyme having diphenol oxidase activity was purified and its gene was cloned. The enzyme was purified as a glycosylated 75-kDa protein which migrated at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after deglycosylation by endoglycosidase F. Substrate specificity resembled that of a laccase in that it oxidized multiple diphenolic and diamino compounds. Dopamine was shown by mass spectroscopy to be oxidized to decarboxy dopachrome, an intermediate of melanin synthesis. The enzyme contained 4.1 +/- 0.1 mol of copper per mol. It resembled a laccase in its absorbance spectrum, containing a peak of 610 nm and the shoulder at 320 nm, corresponding to the absorbance of a type I and type III copper, respectively. The cloned gene of C. neoformans laccase (CNLAC1) contained a single open reading frame encoding a polypeptide 624 amino acids in length. The encoded polypeptide contained a presumptive leader sequence, on the basis of its relative hydrophobicity and by comparison of the sequence to that of the N-terminal sequence of the purified enzyme. CNLAC1 also contained 14 introns ranging from 52 to 340 bases long. Transcriptional activity of CNLAC1 was found to be derepressed in the absence of glucose and to correspond to an increase in enzymatic activity.
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PMCID: PMC205102  PMID: 8300520
6.  Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization. 
Journal of Bacteriology  1992;174(21):6992-6996.
In order to isolate the structural gene involved in sucrose utilization, we screened a sucrose-induced Candida albicans cDNA library for clones expressing alpha-glucosidase activity. The C. albicans maltase structural gene (CAMAL2) was isolated. No other clones expressing alpha-glucosidase activity. were detected. A genomic CAMAL2 clone was obtained by screening a size-selected genomic library with the cDNA clone. DNA sequence analysis reveals that CAMAL2 encodes a 570-amino-acid protein which shares 50% identity with the maltase structural gene (MAL62) of Saccharomyces carlsbergensis. The substrate specificity of the recombinant protein purified from Escherichia coli identifies the enzyme as a maltase. Northern (RNA) analysis reveals that transcription of CAMAL2 is induced by maltose and sucrose and repressed by glucose. These results suggest that assimilation of sucrose in C. albicans relies on an inducible maltase enzyme. The family of genes controlling sucrose utilization in C. albicans shares similarities with the MAL gene family of Saccharomyces cerevisiae and provides a model system for studying gene regulation in this pathogenic yeast.
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PMCID: PMC207379  PMID: 1400249

Results 1-6 (6)