Overcoming the false-minima problem in direct methods: Structure determination of the packaging enzyme P4 from bacteriophage φ13

The problems encountered during the phasing and structure determination of the packaging enzyme P4 from bacteriophage φ13 using the anomalous signal from selenium in a single-wavelength anomalous dispersion experiment (SAD) are described. The oligomeric state of P4 in the virus is a hexamer (with sixfold rotational symmetry) and it crystallizes in space group C2, with four hexamers in the crystallographic asymmetric unit. Current state-of-the-art ab initio phasing software yielded solutions consisting of 96 atoms arranged as sixfold symmetric clusters of Se atoms. However, although these solutions showed high correlation coefficients indicative that the substructure had been solved, the resulting phases produced uninterpretable electron-density maps. Only after further analysis were correct solutions found (also of 96 atoms), leading to the eventual identification of the positions of 120 Se atoms. Here, it is demonstrated how the difficulties in finding a correct phase solution arise from an intricate false-minima problem. © 2005 International Union of Crystallography - all rights reserved

Speech audiometry test with picture-related sentence lists in Modern Greek for partially hearing children

Objective: The aim of this study was to develop Greek Sentence Based Speech Audiometry Test in quiet test for Hearing Impaired (HI) children (G-SEBSAT). Methods: Seventy-six children were recruited following approval by the local ethics committee and after obtaining informed consent from their parents. The collection of vocabulary was based on showing pictures selected from popular reading materials in Greek to hearing impaired HI children. A grammatical content analysis was carried out to determine the average syntactic and morphological structures of the sentences used by the HI children. Ten picture related sentence lists were developed based on the vocabulary and the grammatical analysis, and recorded by a male native speaker of standard Modern Greek. These were presented to both normal hearing (NH) and HI children, and the average speech response threshold (SRT) as well as the slope of the SRT curve at the SRT level of 50% correct responses (S50) were recorded in both groups. Sentence lists were validated with respect to the variability of their difficulty within each group, as well the test-retest variability of the respective SRT scores. Results: The average speech response threshold (SRT) across all lists for HI children was 65.27 dB and the slope of the SRT curve at the SRT level of 50% correct responses was 3.11%/dB. The corresponding results across all lists for NH children were 17.66 dB and 9.7%/dB, respectively. The SRT of HI children were strongly positively correlated, in a statistically significant manner with the PTA in both the test and the retest sessions (test: r=0.750, P < 0.0005; retest: r=0.753, P < 0.0005). The Spearman correlation of the rankings of SRT values and the slope values was 0.998 and 0.997 respectively for the HI and 0.939 and 0.88, for the NH group, indicating very low variability across the test and retest sessions. In addition, the analysis of variance (ANOVA) of the average SRT in NH children and the SRT residuals in the HI group indicated that the different sentences were of the same difficulty within each group. ((F(9,81)=0.401, p=0.930 and (F(9,93)=2.241, p=0.025 respectively). Conclusions: A validated sentence based speech audiometry test was created in Greek for the first time. SRT and S50 values for both NH and HI children are comparable to similar tests developed in other languages

The influence of paediatric HIV infection on circulating B cell subsets and CXCR5+ T helper cells

Antiretroviral therapy (ART) only partially restores HIV-induced alterations in lymphocyte populations. We assessed B and T cell phenotypes in a cohort of children from a single centre in the United Kingdom with perinatally acquired HIV compared to healthy controls. The majority of HIV infected children (44 of 56) were on fully suppressive combination ART. Children with perinatally acquired HIV had significantly lower memory B and CD4(+) CD45RO(+) CXCR5(+) [follicular T helper cell (Tfh)-like] T cell percentages. Detectable viraemia was associated with higher CD21(-) (activated and exhausted/tissue-like memory) B cells. A greater proportion of life spent on suppressive ART was associated with higher memory B cell percentages. These results suggest that early and sustained suppressive ART may preserve B and T cell phenotypes in perinatally acquired HIV and limit deficits in humoral immunity. A lower proportion of circulating Tfh-like cells in HIV infected children appears to be independent of HIV treatment history and ongoing HIV viraemia and warrants further investigation

Serological response to 13-valent pneumococcal conjugate vaccine in children and adolescents with perinatally acquired HIV infection

BACKGROUND: Children with perinatally acquired HIV (paHIV) remain at an increased risk of pneumococcal infection despite highly active antiretroviral therapy (HAART). Beyond infancy, responses to pneumococcal conjugate vaccine (PCV) remain under-investigated. There are currently no published data on serological response to 13-valent PCV (PCV13) in the HIV-infected populations. METHODS: We measured pneumococcal serotype-specific IgG in 48 paHIV-infected child patients (CP), 27 young adult healthy controls (AHC) and 30 child healthy controls (CHC). Opsonophagocytic assay (OPA) titres for three PCV13-exclusive serotypes were measured in a subset of children. Serotype-specific IgG was repeated 1 and 6 months following PCV13 vaccination of CP and AHC groups. OPA titres for four serotypes were measured at the 1-month time-point. RESULTS: The majority of CP, CHC and AHC had serotype-specific IgG above 0.35 μg/ml at baseline, although OPA activity was undetectable for two of the three serotypes studied. Baseline IgG concentrations were significantly lower in CP than AHC for a proportion of serotypes and were strongly predictive of responses to vaccine. After adjusting for baseline, postvaccination IgG concentrations were comparable, although responses to some serotypes were impaired for CP. OPA correlated well with IgG after vaccination. Detectable HIV viral load was associated with significantly lower IgG concentration and OPA titre. CONCLUSION: Children with paHIV mount a robust serological response to PCV13 for most but not all vaccine serotypes. Viral load suppression with HAART and higher baseline IgG concentration are associated with higher postvaccination antibody levels. This has implications for HAART treatment and vaccination practices

The development of a theory-based intervention to promote appropriate disclosure of a diagnosis of dementia

Background: The development and description of interventions to change professional practice are often limited by the lack of an explicit theoretical and empirical basis. We set out to develop an intervention to promote appropriate disclosure of a diagnosis of dementia based on theoretical and empirical work. Methods: We identified three key disclosure behaviours: finding out what the patient already knows or suspects about their diagnosis; using the actual words 'dementia' or 'Alzheimer's disease' when talking to the patient; and exploring what the diagnosis means to the patient. We conducted a questionnaire survey of older peoples' mental health teams (MHTs) based upon theoretical constructs from the Theory of Planned Behaviour (TPB) and Social Cognitive Theory (SCT) and used the findings to identify factors that predicted mental health professionals' intentions to perform each behaviour. We selected behaviour change techniques likely to alter these factors. Results: The change techniques selected were: persuasive communication to target subjective norm; behavioural modelling and graded tasks to target self-efficacy; persuasive communication to target attitude towards the use of explicit terminology when talking to the patient; and behavioural modelling by MHTs to target perceived behavioural control for finding out what the patient already knows or suspects and exploring what the diagnosis means to the patient. We operationalised these behaviour change techniques using an interactive 'pen and paper' intervention designed to increase intentions to perform the three target behaviours. Conclusion : It is feasible to develop an intervention to change professional behaviour based upon theoretical models, empirical data and evidence based behaviour change techniques. The next step is to evaluate the effect of such an intervention on behavioural intention. We argue that this approach to development and reporting of interventions will contribute to the science of implementation by providing replicable interventions that illuminate the principles and processes underlying change.This project is funded by UK Medical Research Council, Grant reference number G0300999. Jeremy Grimshaw holds a Canada Research Chair in Health Knowledge Transfer and Uptake. Jill Francis is funded by the Chief Scientist Office of the Scottish Government Health Directorate. The views expressed in this study are those of the authors

Membrane-Assisted Viral DNA Ejection

Genome packaging and delivery are fundamental steps in the replication cycle of all viruses. Icosahedral viruses with linear double-stranded DNA (dsDNA) usually package their genome into a preformed, rigid procapsid using the power generated by a virus-encoded packaging ATPase. The pressure and stored energy due to this confinement of DNA at a high density is assumed to drive the initial stages of genome ejection. Membrane-containing icosahedral viruses, such as bacteriophage PRD1, present an additional architectural complexity by enclosing their genome within an internal membrane vesicle. Upon adsorption to a host cell, the PRD1 membrane remodels into a proteo-lipidic tube that provides a conduit for passage of the ejected linear dsDNA through the cell envelope. Based on volume analyses of PRD1 membrane vesicles captured by cryo-electron tomography and modeling of the elastic properties of the vesicle, we propose that the internal membrane makes a crucial and active contribution during infection by maintaining the driving force for DNA ejection and countering the internal turgor pressure of the host. These novel functions extend the role of the PRD1 viral membrane beyond tube formation or the mere physical confinement of the genome. The presence and assistance of an internal membrane might constitute a biological advantage that extends also to other viruses that package their linear dsDNA to high density within an internal vesicle

N-{3-[2-(4-Fluoro­phen­oxy)eth­yl]-2,4-dioxo-1,3-diaza­spiro­[4.5]decan-7-yl}-4-methyl­benzamide

In the title compound, C24H26FN3O4, the two aromatic rings form a dihedral angle of 88.81 (15)°. The cyclo­hexane ring adopts a chair conformation and the five-membered ring is essentially planar, with a maximum deviation from planarity of 0.041 (2) Å. The crystal structure displays inter­molecular C—H⋯O and N—H⋯O hydrogen bonds

N-[3-(4-Fluoro­benz­yl)-2,4-dioxo-1,3-diaza­spiro­[4.5]dec-8-yl]-2-methyl­benzene­sulfonamide

In the title compound, C22H24FN3O4S, the cyclo­hexane ring adopts a chair conformation and the five-membered ring is essentially planar, with a maximum deviation of 0.040 (2) Å. The dihedral angles between the five-membered ring and the tolyl and fluoro­benzene rings are 56.74 (12) and 89.88 (12)°, respectively. The two terminal benzene rings make a dihedral angle of 63.53 (12)°. The crystal structure displays inter­molecular C—H⋯O and N—H⋯O hydrogen bonds. An intra­molecular C—H⋯O hydrogen bond also occurs

N-{3-[2-(4-Fluoro­phen­oxy)eth­yl]-2,4-dioxo-1,3-diaza­spiro­[4.5]decan-7-yl}-4-meth­oxy­benzene­sulfonamide

In the title compound, C23H26FN3O6S, the two terminal aromatic rings form a dihedral angle of 49.26 (12)°. The cyclo­hexane ring adopts a chair conformation and the five-membered ring is essentially planar, with a maximum deviation from planarity of 0.0456 (19) Å. The dihedral angles between the five-membered ring and the meth­oxy­benzene and fluoro­benzene rings are 33.56 (11) and 81.94 (12)°, respectively. The crystal structure displays N—H⋯O hydrogen bonds as well as weak inter­molecular C—H⋯O inter­actions