Atypical teratoid rhabdoid tumours (ATRTs)-a 21-year institutional experience.

PURPOSE: Atypical teratoid/rhabdoid tumours (ATRTs) are malignant embryonal tumours of childhood that affect the central nervous system (CNS). We aim to determine which factors, including patient age, extent of resection (EOR), presence of distal metastasis and use of adjuvant therapies, affect overall survival in children with atypical teratoid/rhabdoid tumours (ATRTs) treated at this single centre. METHODS: Retrospective cohort review of patients with histological diagnosis of ATRT treated over 21 years (1999-2020) was conducted. Data on demographics, tumour location, presence of metastasis, use of adjuvant therapy, extent of resection (EOR), complications, neurological outcome post-surgery, and overall survival were collected. Kaplan-Meier survival analysis was performed. RESULTS: A total of 45 children (mean age 2 years) underwent 64 operations. 25 patients were <1 year of age. Gross-total resection (GTR) pre-adjuvant therapy was achieved in 15, near-total resection (NTR) in 15, subtotal resection (STR) in 9, and biopsy in 6 children. Most children had good neurological outcomes post-operatively (28/45 with GOS 5). Fourteen patients survived longer than 4 years. Survival analysis showed a significant difference in median survival in favour of GTR and localised disease. There was no significant difference in median survival between patients <1 year vs >1 year of age (p=0.84). CONCLUSION: We find that presence of metastasis was an important factor in poor survival in patients with ATRT. GTR, where possible, may confer significant survival benefit in ATRT. Children aged <1 year appear to have performed as well as those >1 year and therefore should still be considered for radical surgery.

Visual outcomes and predictors in optic pathway glioma: a single centre study.

BACKGROUND/AIMS: Optic pathway gliomas (OPGs) may cause progressive visual loss despite chemotherapy. Newer, less toxic treatments might be given earlier, depending on visual prognosis. We aimed to investigate the prognostic value of visual evoked potentials (VEP) and optical coherence tomography (OCT). METHODS: A retrospective study of OPG patients (treated 2003-2017) was conducted. Primary outcome was PEDIG category visual acuity in better and worse eyes (good < = 0.2, moderate 0.3-0.6 and poor > = 0.7 logMAR). Binary logistic regression analysis was used to identify predictors of these outcomes. RESULTS: 60 patients (32 Neurofibromatosis type 1 [NF1] and 28 sporadic) had median presentation age 49 months (range 17-183) (NF1) and 27 months (range 4-92) (sporadic). Median follow up was 82 months (range 12-189 months). At follow up 24/32 (75%) of NF1 children and 14/28 (50%) of sporadic children had good better eye visual acuity and 11/32 (34%) of NF1 children and 15/28 (54%) of sporadics had poor worse eye acuity. Mean peripapillary retinal nerve fibre layer (RNFL) thickness predicted good better eye final acuity (OR 0.799, 95%CI 0.646-0.987, p = 0.038). Presenting with visual symptoms (OR 0.22 95% CI 0.001-0.508, p = 0.017) and poorer VEP scores (OR 2.35 95% CI 1.1-5.03, p = 0.027) predicted poor worse eye final acuity. 16 children had homonymous hemianopias at follow up, predicted by poor presenting binocular VEP score (OR 1.449 95%CI 1.052-1.995, p = 0.02). CONCLUSIONS: We found that both RNFL thickness on OCT and VEP were useful in predicting future visual acuity and vision and potentially in planning treatment. We had a high prevalence of homonymous hemianopia.

Following the trend in maxillofacial surgery literature.

Evidence-based medicine relies on the integration of high-quality research with clinical expertise and patient values. The hierarchy of evidence allows the clinician to assign value to research based on the methodological quality of the study design and its applicability to the clinical question. Improvements in the quality of research in oral and maxillofacial surgery aim to strengthen evidence-based medicine and patient care. Analysis of the trends in maxillofacial surgery publications can identify the strengths and weaknesses of the current body of research, and direct researchers to areas that require improvement. The aim of this study was to review the proportion of the types of articles published in the British Journal of Oral and Maxillofacial Surgery (BJOMS) and the International Journal of Oral and Maxillofacial Surgery (IJOMS) between January 2010 and December 2019. These data were compared with a previously published review that summarised the proportion published in 2000 - 2009. The topics chosen for meta-analysis and the number of qualitative studies were also summarised. In total, 4931 articles were reviewed over the 10-year period. Compared with the previous 10 years, there was an increase in randomised controlled trials and meta-analyses, and a reduction in case series and case reports. Implantology and dentoalveolar surgery were the most common topics chosen for meta-analysis. Overall, the trend in the maxillofacial literature is towards a higher quality body of research.

Allogeneic stem cell transplantation for refractory acute myeloid leukemia in pediatric patients: the UK experience.

We report outcomes for 44 children who underwent stem cell transplantation (SCT) for refractory AML in the UK between 2000 and 2012. Median age at SCT was 11.5 years. Twenty-three patients had primary refractory and 21 relapsed refractory AML. Refractory disease was confirmed by cytogenetics/molecular genetics in 24 cases. Median follow-up of the whole cohort is 6.8 years (2.1-14.9 years). Thirty patients (68%) achieved a CR following SCT. Transplant-related mortality at 1 year was 18%. Acute GVHD incidence was 52% (grade ⩾III 19%), chronic 7%. Relapse was the major cause of treatment failure and occurred in 32% of patients at a median of 61 days post SCT. Five-year overall survival and leukemia-free survival (LFS) were 43% (95% CI 31-61%). All patients with favorable cytogenetics (n=6) are alive in CR. Outcomes in patients with primary refractory disease were equivalent to those with relapsed refractory AML. Blast percentage ⩽30% in the BM pre-SCT, myeloablative conditioning and acute GVHD proved to be favorable prognostic features. We could stratify patients according to age ⩾10 years and >30% blasts in BM pre-SCT. Patients with none/one of these risk factors were highly salvageable (5 years LFS 53%) whereas those with both factors had a very poor prognosis (5 years LFS 10%). This may facilitate decision making on whether it is appropriate to consider transplant in such patients.

Melanoma in congenital melanocytic naevi.

Congenital melanocytic naevi (CMN) are a known risk factor for melanoma, with the greatest risk currently thought to be in childhood. There has been controversy over the years about the incidence of melanoma, and therefore over the clinical management of CMN, due partly to the difficulties of histological diagnosis and partly to publishing bias towards cases of malignancy. Large cohort studies have demonstrated that melanoma risk in childhood is related to the severity of the congenital phenotype. New understanding of the genetics of CMN offers the possibility of improvement in diagnosis of melanoma, identification of those at highest risk, and new treatment options. We review the world literature and our centre's experience over the last 25 years, including the molecular characteristics of melanoma in these patients and new melanoma incidence and outcome data from our prospective cohort. Management strategies are proposed for presentation of suspected melanoma of the skin and the central nervous system in patients with CMN, including use of oral mitogen-activated protein kinase kinase inhibitors in NRAS-mutated tumours.

Attitudes, access and anguish: a qualitative interview study of staff and patients' experiences of diabetic retinopathy screening.

OBJECTIVE: To examine the experiences of patients, health professionals and screeners; their interactions with and understandings of diabetic retinopathy screening (DRS); and how these influence uptake. DESIGN: Purposive, qualitative design using multiperspectival, semistructured interviews and thematic analysis. SETTING: Three UK Screening Programme regions with different service-delivery modes, minority ethnic and deprivation levels across rural, urban and inner-city areas, in general practitioner practices and patients' homes. PARTICIPANTS: 62 including 38 patients (22 regular-screening attenders, 16 non-regular attenders) and 24 professionals (15 primary care professionals and 9 screeners). RESULTS: Antecedents to attendance included knowledge about diabetic retinopathy and screening; antecedents to non-attendance included psychological, pragmatic and social factors. Confusion between photographs taken at routine eye tests and DRS photographs was identified. The differing regional invitation methods and screening locations were discussed, with convenience and transport safety being over-riding considerations for patients. Some patients mentioned significant pain and visual disturbance from mydriasis drops as a deterrent to attendance. CONCLUSIONS: In this, the first study to consider multiperspectival experiential accounts, we identified that proactive coordination of care involving patients, primary care and screening programmes, prior to, during and after screening is required. Multiple factors, prior to, during and after screening, are involved in the attendance and non-attendance for DRS. Further research is needed to establish whether patient self-management educational interventions and the pharmacological reformulation of shorter acting mydriasis drops, may improve uptake of DRS. This might, in turn, reduce preventable vision loss and its associated costs to individuals and their families, and to health and social care providers, reducing current inequalities.

Functional analysis of nuclear localization signals in VP1-2 homologues from all herpesvirus subfamilies.

UNLABELLED: The herpes simplex virus (HSV) tegument protein VP1-2 contains an N-terminal nuclear localization signal (NLS) that is critical for capsid routing to the nuclear pore. Here we analyzed positionally conserved determinants in VP1-2 homologues from each of the alpha, beta, and gamma classes of human herpesviruses. The overall architectures of the VP1-2s were similar, with a conserved N-terminal ubiquitin-specific protease domain separated from an internal region by a linker that was quite poorly conserved in length and sequence. Within this linker region all herpesviruses contained a conserved, highly basic motif which nevertheless exhibited distinct class-specific features. The motif in HSV functioned as a monopartite NLS, while in varicella-zoster virus (VZV) activity required an adjacent basic section defining the motif as a bipartite NLS. Neither the beta- nor gammaherpesvirus VP1-2 motifs were identified by prediction algorithms, but they nevertheless functioned as efficient NLS motifs both in heterologous transfer assays and in HSV VP1-2. Furthermore, though with different efficiencies and with the exception of human herpesvirus 8 (HHV-8), these chimeric variants rescued the replication defect of an HSV mutant lacking its NLS motif. We demonstrate that the lysine at position 428 of HSV is critical for replication, with a single alanine substitution being sufficient to abrogate NLS function and virus growth. We conclude that the basic motifs of each of the VP1-2 proteins are likely to confer a similar function in capsid entry in the homologous setting and that while there is flexibility in the exact type of motif employed, specific individual residues are critical for function. IMPORTANCE: To successfully infect cells, all herpesviruses, along with many other viruses, e.g., HIV, hepatitis B virus, and influenza virus, must navigate through the cytoplasmic environment and dock with nuclear pores for transport of their genomes into the nucleus. However, we still have a limited understanding of the detailed mechanisms involved. Insight into these events is needed and could offer opportunities for therapeutic intervention. This work investigated the role of a specific determinant in the structural protein VP1-2 in herpesvirus entry. We examined this determinant in representative VP1-2s from all herpesvirus subfamilies, demonstrated NLS function, dissected key residues, and showed functional relevance in rescuing replication of the mutant blocked in capsid navigation to the pore. The results are important and strongly support our conclusions of the generality that these motifs are crucial for entry of all herpesviruses. They also facilitate future analysis on selective host interactions and possible routes to disrupt function.

The influence of target stoichiometry on early cell adhesion of co-sputtered calcium-phosphate surfaces.

The nature of the initial interaction between calcium phosphate (Ca-P) thin films and osteoblasts can be influenced by a number of different properties including the phase, crystallinity, stoichiometry and composition of the surface. There is still a strong interest in developing and studying Ca-P surfaces that have the ability to accurately control the osteoblast response. Radio frequency (RF) magnetron sputtering is a technique that allows for accurate control of the properties of deposited Ca-P coatings and has been studied extensively because of this fact. In this work, Ca-P coatings were co-deposited using RF magnetron sputtering in order to study the effect of changing the target stoichiometry on the initial in vitro behavior of MG63 osteoblast-like cells. The samples produced were analysed both as-deposited and after thermal annealing to 500 °C. After annealing XPS analyses of the samples co-deposited using tricalcium phosphate (TCP) materials gave a Ca/P ratio of 1.71 ± 0.01, as compared to those co-deposited from hydroxyapatite (HA) materials, with a Ca/P of 1.82 ± 0.06. In addition to this, the curve fitted XPS data indicated the presence of low levels of carbonate in the coatings. Despite this the XRD results for all of the annealed coatings were shown to be characteristic of pure HA with a preferred 002 orientation. The atomic force microscopy results also highlighted that both types of coatings had surface features of a similar size (200-220 nm). Both surfaces exhibited a degree of surface degradation, even after 1 h of cell culture. However, the TCP derived surfaces showed an enhanced osteoblastic cell response in terms of cell adhesion and cell proliferation in the earlier stages of cell culture than the surfaces deposited from HA. An improvement in the initial cell attachment and a potential for increased cell proliferation rates is viewed as a highly advantageous result in relation to controlling the osteoblast response on these surfaces.

A Nuclear localization signal in herpesvirus protein VP1-2 is essential for infection via capsid routing to the nuclear pore.

To initiate infection, herpesviruses must navigate to and transport their genomes across the nuclear pore. VP1-2 is a large structural protein of the virion that is conserved in all herpesviruses and plays multiple essential roles in virus replication, including roles in early entry. VP1-2 contains an N-terminal basic motif which functions as an efficient nuclear localization signal (NLS). In this study, we constructed a mutant HSV strain, K.VP1-2ΔNLS, which contains a 7-residue deletion of the core NLS at position 475. This mutant fails to spread in normal cells but can be propagated in complementing cell lines. Electron microscopy (EM) analysis of infection in noncomplementing cells demonstrated capsid assembly, cytoplasmic envelopment, and the formation of extracellular enveloped virions. Furthermore, extracellular virions isolated from noncomplementing cells had similar profiles and abundances of structural proteins. Virions containing VP1-2ΔNLS were able to enter and be transported within cells. However, further progress of infection was prevented, with at least a 500- to 1,000-fold reduction in the efficiency of initiating gene expression compared to that in the revertant. Ultrastructural and immunofluorescence analyses revealed that the K.VP1-2ΔNLS mutant was blocked at the microtubule organizing center or immediately upstream of nuclear pore docking and prior to gene expression. These results indicate that the VP1-2 NLS is not required for the known assembly functions of the protein but is a key requirement for the early routing to the nuclear pore that is necessary for successful infection. Given its conservation, we propose that this motif may also be critical for entry of other classes of herpesviruses.

Autocatalytic activity of the ubiquitin-specific protease domain of herpes simplex virus 1 VP1-2.

The herpes simplex virus (HSV) tegument protein VP1-2 is essential for virus entry and assembly. VP1-2 also contains a highly conserved ubiquitin-specific protease (USP) domain within its N-terminal region. Despite conservation of the USP and the demonstration that it can act on artificial substrates such as polyubiquitin chains, identification of the relevance of the USP in vivo to levels or function of any substrate remains limited. Here we show that HSV VP1-2 USP can act on itself and is important for stability. VP1-2 N-terminal variants encompassing the core USP domain itself were not affected by mutation of the catalytic cysteine residue (C65). However, extending the N-terminal region resulted in protein species requiring USP activity for accumulation. In this context, C65A mutation resulted in a drastic reduction in protein levels which could be stabilized by proteosomal inhibition or by the presence of normal C65. The functional USP domain could increase abundance of unstable variants, indicating action at least in part, in trans. Interestingly, full-length variants containing the inactive USP, although unstable when expressed in isolation, were stabilized by virus infection. The catalytically inactive VP1-2 retained complementation activity of a VP1-2-negative virus. Furthermore, a recombinant virus expressing a C65A mutant VP1-2 exhibited little difference in single-step growth curves and the kinetics and abundance of VP1-2 or a number of test proteins. Despite the absence of a phenotype for these replication parameters, the USP activity of VP1-2 may be required for function, including its own stability, under certain circumstances.

Comparative in vitro cytotoxicity study of carbon nanotubes and titania nanostructures on human lung epithelial cells.

The aim of this study is to assess in vitro cytotoxic effects of titania nanostructures and carbon nanotubes (CNTs) by exposing A549 lung epithelial cell line to these materials. Titania nanotubes (TiNTs) were grown by hydrothermal treatment of TiO(2) nanoparticles, followed by annealing them at 400°C. The titania nanostructures obtained on annealing (mixture of nanotubes and nanorods) were hollow and open ended, containing 3-5 layers of titania sheets, with an internal diameter ∼3-5 nm and external diameter ∼8-10 nm, and a specific surface area of 265 m(2)/g. As-supplied single walled (SWCNTs) and microwave plasma enhanced chemical vapour deposition (MPCVD) grown multi walled carbon nanotubes (MWCNTs) were used in this study. The lengths and diameters of the SWCNTs were 5-10nm and 0.5-3 nm respectively. The lengths and diameters of the MWCNTs were 25-30 µm and 10-30 nm respectively. The cell viability was evaluated using the MTT (3-(4,-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium) assay. No significant cytotoxic effects of titania nanostructures were observed over a period of a week of testing time, while the presence of CNTs in some cases demonstrated significant cytotoxic effects. Finally, possible reason of cytotoxicity is discussed in the light of microstructures of materials.

A single mutation responsible for temperature-sensitive entry and assembly defects in the VP1-2 protein of herpes simplex virus.

Evidence for an essential role of the herpes simplex virus type 1 (HSV-1) tegument protein VP1-2 originated from the analysis of the temperature-sensitive (ts) mutant tsB7. At the nonpermissive temperature (NPT), tsB7 capsids accumulate at the nuclear pore, with defective genome release and substantially reduced virus gene expression. We compared the UL36 gene of tsB7 with that of the parental strain HFEM or strain 17 and identified four amino acid substitutions, 1061D → G, 1453Y → H, 2273Y → H, and 2558T → I. We transferred the UL36 gene from tsB7, HFEM, or strain 17 into a KOS background. While KOS recombinants containing the HFEM or strain 17 UL36 gene exhibited no ts defect, recombinants containing the tsB7 UL36 VP1-2 exhibited a 5-log deficiency at the NPT. Incubation at the NPT resulted in little or no virus gene expression, though limited expression could be detected in a highly delayed fashion. Using shift-down regimes, gene expression recovered and recapitulated the time course normally observed, indicating that the initial block was in a reversible pathway. Using temperature shift-up regimes, a second defect later in the replication cycle was also observed in the KOS.ts viruses. We constructed a further series of recombinants which contained subsets of the four substitutions. A virus containing the wild-type (wt) residue at position 1453 and with the other three residues being from tsB7 VP1-2 exhibited wt plaquing efficiency. Conversely, a virus containing the three wt residues but the single Y → H change at position 1453 from tsB7 exhibited a 4- to 5-log drop in plaquing efficiency and was defective at both early and late stages of infection.

Deposition of substituted apatites with anticolonizing properties onto titanium surfaces using a novel blasting process.

A series of doped apatites have been deposited onto titanium (V) substrates using a novel ambient temperature blasting process. The potential of these deposited doped apatites as non-colonizing osteoconductive coatings has been evaluated in vitro. XPS, EDX, and gravimetric analysis demonstrated that a high degree of coating incorporation was observed for each material. The modified surfaces were found to produce osteoblast proliferation comparable to, or better than, a hydroxyapatite finish. Promising levels of initial microbial inhibition were observed from the Sr- and Ag-doped surfaces, with the strontium showing prolonged ability to reduce bacteria numbers over a 30-day period. Ion elution profiles have been characterized and linked to the microbial response and based on the results obtained, mechanisms of kill have been suggested. In this study, the direct contact of coated substrate surfaces with microbes was observed to be a significant contributing factor to the antimicrobial performance and the anticolonizing activity. The silver substituted apatite was observed to out-perform both the SrA and ZnA in terms of biofilm inhibition.

Characterization of the herpes simplex virus (HSV)-1 tegument protein VP1-2 during infection with the HSV temperature-sensitive mutant tsB7.

VP1-2, encoded by the UL36 gene of herpes simplex virus (HSV), is a large structural protein, conserved across the family Herpesviridae, that is assembled into the tegument and is essential for virus replication. Current evidence indicates that VP1-2 is a central component in the tegumentation and envelopment processes and that it also possesses important roles in capsid transport and entry. However, any detailed mechanistic understanding of VP1-2 function(s) remains limited. This study characterized the replication of HSV-1 tsB7, a temperature-sensitive mutant restricted at the non-permissive temperature due to a defect in VP1-2 function. A tsB7 virus expressing green fluorescent protein-fused VP16 protein was used to track the accumulation and location of a major tegument protein. After infection at the permissive temperature and shift to the non-permissive temperature, the production of infectious virus ceased. VP1-2 accumulated in altered cytosolic clusters, together with VP16 and other virion proteins. Furthermore, correlating with the results of immunofluorescence, electron microscopy demonstrated abnormal cytosolic capsid clustering and a block in envelopment. As VP1-2 encompasses a ubiquitin-specific protease domain, the occurrence of ubiquitin-conjugated proteins during tsB7 infection was also examined at the non-permissive temperature. A striking overaccumulation was observed of ubiquitin-specific conjugates in cytoplasmic clusters, overlapping and adjacent to the VP1-2 clusters. These results are discussed in relation to the possible functions of VP1-2 in the assembly pathway and the nature of the defect in tsB7.

Ghrelin and the differential regulation of des-acyl (DSG) and oct-anoyl ghrelin (OTG) in human adipose tissue (AT).

OBJECTIVES: Ghrelin, an important central acting orexigenic hormone, is predominantly secreted in the gastrointestinal tract. However little is known about the action of ghrelin in human adipose tissue (AT). AIM: To study the expression of ghrelin in AT, the effects of octanoyl-(OTG) and des-acyl (DSG) ghrelin on lipolysis and lipogenesis, leptin release and potential peripheral signalling through the Y1 receptor. METHODS: Ex vivo human AT was obtained from women undergoing elective surgery (46 (mean +/- SD) 6.8 years, body mass index (BMI): 25.6 +/- 5.0 kg/m(2), n = 20). Abdominal-subcutaneous (AbdSc) adipocytes were isolated and treated with recombinant human (rh) OTG and DSG to assess lipid metabolism leptin release and the influence of Y1-receptor blocker. RESULTS: Ghrelin was expressed in AbdScAT and negatively correlated with BMI (lean: 3.6 +/- 0.74 optical-density-units (OD), obese: 1.64 +/- 0.45 OD, *P < 0.05). Only DSG significantly suppressed glycerol release (Control (C): 286 +/- 58 microl/l; DSG 1 nm: 224 +/- 38 microl/l downward arrow*; DSG 100 nm: 172 +/- 13 microl/l downward arrow*,* downward arrow P < 0.05, n = 7) and reduced hormone sensitive lipase expression (C: 1.0 +/- 0.3 OD; DSG 1 nm: 0.8 +/- 0.3 OD downward arrow*; DSG 100 nm: 0.6 +/- 0.1 OD downward arrow*, n = 4). However, both isoforms increased lipoprotein lipase expression (C: 1.0 +/- 0.3OD; DSG 100 nm: 0.2 +/- 0.4 OD upward arrow*; OTG 100 nm: 2.5 +/- 0.3 OD upward arrow*, n = 4), whilst blockade of Y1 eliminated this effect in both. Leptin was down-regulated by DSG only (DSG 1 nm: 5.3 +/- 0.7 ng/ml; DSG 100 nm: 4.1 +/- 0.7 ng/ml*) and was significant after BMI adjustment (P = 0.029). CONCLUSION: Ghrelin was expressed in human AbdSc AT. In vitro, both OGT and DSG appear to mediate fat deposition with the lipogenic effects in part mediated by the Y1 receptor, whilst the influence of DSG affected lipolysis, lipogenesis and leptin secretion. Taken together, these studies support a local action for ghrelin isoforms on lipid and adipokine metabolism that further supports a cross talk between organs.

Identification of a highly conserved, functional nuclear localization signal within the N-terminal region of herpes simplex virus type 1 VP1-2 tegument protein.

VP1-2 is a large structural protein assembled into the tegument compartment of the virion, conserved across the herpesviridae, and essential for virus replication. In herpes simplex virus (HSV) and pseudorabies virus, VP1-2 is tightly associated with the capsid. Studies of its assembly and function remain incomplete, although recent data indicate that in HSV, VP1-2 is recruited onto capsids in the nucleus, with this being required for subsequent recruitment of additional structural proteins. Here we have developed an antibody to characterize VP1-2 localization, observing the protein in both cytoplasmic and nuclear compartments, frequently in clusters in both locations. Within the nucleus, a subpopulation of VP1-2 colocalized with VP26 and VP5, though VP1-2-positive foci devoid of these components were observed. We note a highly conserved basic motif adjacent to the previously identified N-terminal ubiquitin hydrolase domain (DUB). The DUB domain in isolation exhibited no specific localization, but when extended to include the adjacent motif, it efficiently accumulated in the nucleus. Transfer of the isolated motif to a test protein, beta-galactosidase, conferred specific nuclear localization. Substitution of a single amino acid within the motif abolished the nuclear localization function. Deletion of the motif from intact VP1-2 abrogated its nuclear localization. Moreover, in a functional assay examining the ability of VP1-2 to complement growth of a VP1-2-ve mutant, deletion of the nuclear localization signal abolished complementation. The nuclear localization signal may be involved in transport of VP1-2 early in infection or to late assembly sites within the nucleus or, considering the potential existence of VP1-2 cleavage products, in selective localization of subdomains to different compartments.

Characterisation of calcium phosphate/titanium dioxide hybrid coatings.

The role of titanium dioxide (TiO2) as a means to engender enhanced stability into calcium phosphate (Ca-P) coatings has been well recognised. Several different methods have been used to create such Ca-P/TiO2 hybrid layers on a range of substrates. This paper reports the properties of a Ca-P/TiO2 system created by the sputter deposition of hydroxyapatite onto a titanium surface and the subsequent thermal diffusion of TiO2 through the porous Ca-P layer. The role of temperature in determining the surface contribution from TiO2 has been determined. Coatings annealed up to 600 degrees C did not exhibit any hybrid nature in the uppermost surface, however the coatings annealed to 700 degrees C did show the presence of both HA and rutile TiO2. The surfaces annealed to 800 degrees C were predominantly rutile TiO2. It was also observed that the Ca/P ratio decreased with increasing annealing temperature and that the coating annealed to 700 degrees C had a value of 1.82 +/- 0.07, which was closest to stoichiometric HA. Furthermore, the coatings that were annealed to 700 degrees C displayed a Ca-P/TiO2 hybrid nature, specifically in their uppermost surface and supported the growth and proliferation of osteoblast-like cells more readily when compared to the HA coatings or the rutile TiO2 surfaces.

A series of systematic reviews to inform a decision analysis for sampling and treating infected diabetic foot ulcers.

OBJECTIVES: To review systematically the evidence on the performance of diagnostic tests used to identify infection in diabetic foot ulcers (DFUs) and of interventions to treat infected DFUs. To use estimates derived from the systematic reviews to create a decision analytic model in order to identify the most effective method of diagnosing and treating infection and to identify areas of research that would lead to large reductions in clinical uncertainty. DATA SOURCES: Electronic databases covering period from inception of the database to November 2002. REVIEW METHODS: Selected studies were assessed against validated criteria and described in a narrative review. The structure of a decision analytic model was derived for two groups of patients in whom diagnostic tests were likely to be used. RESULTS: Three studies that investigated the performance of diagnostic tests for infection on populations including people with DFUs found that there was no evidence that single items on a clinical examination checklist were reliable in identifying infection in DFUs, that wound swabs perform poorly against wound biopsies, and that semi-quantitative analysis of wound swabs may be a useful alternative to quantitative analysis. However, few people with DFUs were included, so it was not possible to tell whether diagnostic performance differs for DFUs relative to wounds of other aetiologies. Twenty-three studies investigated the effectiveness (n = 23) or cost-effectiveness (n = 2) of antimicrobial agents for DFUs. Eight studied intravenous antibiotics, five oral antibiotics, four different topical agents such as dressings, four subcutaneous granulocyte colony stimulating factor (G-CSF), one evaluated oral and topical Ayurvedic preparations and one compared topical sugar versus antibiotics versus standard care. The majority of trials were underpowered and were too dissimilar to be pooled. There was no strong evidence for recommending any particular antimicrobial agent for the prevention of amputation, resolution of infection or ulcer healing. Topical pexiganan cream may be as effective as oral antibiotic treatment with ofloxacin for the resolution of local infection. Ampicillin and sulbactam were less costly than imipenem and cilastatin, a growth factor (G-CSF) was less costly than standard care and cadexomer iodine dressings may be less costly than daily dressings. A decision analytic model was derived for two groups of people, those for whom diagnostic testing would inform treatment--people with ulcers which do not appear infected but whose ulcer is not progressing despite optimal concurrent treatment--and those in whom a first course of antibiotics (prescribed empirically) have failed. There was insufficient information from the systematic reviews or interviews with experts to populate the model with transition probabilities for the sensitivity and specificity of diagnosis of infection in DFUs. Similarly, there was insufficient information on the probabilities of healing, amputation or death in the intervention studies for the two populations of interest. Therefore, we were unable to run the model to inform the most effective diagnostic and treatment strategy. CONCLUSIONS: The available evidence is too weak to be able to draw reliable implications for practice. This means that, in terms of diagnosis, infection in DFUs cannot be reliably identified using clinical assessment. This has implications for determining which patients need formal diagnostic testing for infection, on whether empirical treatment with antibiotics (before the results of diagnostic tests are available) leads to better outcomes, and on identifying the optimal methods of diagnostic testing. With respect to treatment, it is not known whether treatment with systemic or local antibiotics leads to better outcomes or whether any particular agent is more effective. Limited evidence suggests that both G-CSF and cadexomer iodine dressings may be less expensive than 'standard' care, that ampicillin/sulbactam may be less costly than imipenem/cilastatin, and that an unlicensed cream (pexiganan) may be as effective as oral ofloxacin. Further research is needed to ascertain the characteristics of infection in people with DFUs that influence healing and amputation outcomes, to determine whether detecting infection prior to treatment offers any benefit over empirical therapy, and to establish the most effective and cost-effective methods for detecting infection, as well as the relative effectiveness and cost-effectiveness of antimicrobial interventions for DFU infection.

Characterization of VP22 in herpes simplex virus-infected cells.

We examine biochemical characteristics of the herpes simplex virus (HSV) tegument protein VP22 by gel filtration, glycerol sedimentation, and chemical cross-linking experiments and use time course radiolabeling and immunoprecipitation assays to analyze its synthesis and interaction with other infected-cell proteins. VP22 was expressed as a delayed early protein with optimal synthesis requiring DNA replication. In immunoprecipitation assays, VP22 was found in association with several additional proteins including VP16 and a kinase activity likely to be that of UL13. Furthermore, in sizing chromatography experiments, VP22 was present in several higher-order complexes in infected cells. From gel filtration analysis the major form of VP22 migrated with a molecular mass of approximately 160 kDa, consistent with its presence as a tetramer, or a dimer complexed with other proteins, with a fraction of the protein migrating at larger molecular mass. In vitro-synthesized VP22 sedimented in a size range consistent with a mixture of tetramers and dimers. Short N- or C-terminal deletions resulted in migration almost exclusively as dimers, indicating that VP22, in the absence of additional virus-encoded proteins, could form higher-order assemblies, most likely tetramers, but that both N-and C-terminal determinants were required for stabilizing such assemblies. Consistent with this we found that isolated proteins encompassing either the N-terminal or C-terminal region of VP22 sedimented as dimers, and that the purified C-terminal domain could be cross-linked into dimeric structures. These results are discussed with regard to possible virus and host interactions involved in VP22 recruitment into virus particles.

Plasma ghrelin following cure of Helicobacter pylori.

BACKGROUND: In the Western world, the incidence of oesophageal adenocarcinoma has increased over the last 30 years coinciding with a decrease in the prevalence of Helicobacter pylori. Trends of increasing oesophageal adenocarcinoma can be linked causally to increasing gastro-oesophageal reflux disease (GORD) which can be linked to an increasingly obese population. However, there is no plausible biological mechanism of association between H. pylori, obesity, and GORD. Ghrelin, a peptide produced in the stomach, which regulates appetite, food intake, and body composition, was studied in H. pylori positive asymptomatic subjects. METHODS: Plasma ghrelin, leptin, and gastrin were measured for six hours after an overnight fast, before and after cure of H. pylori in 10 subjects. Twenty four hour intragastric acidity was also assessed. RESULTS: After cure, median (95% confidence intervals) integrated plasma ghrelin increased from 1160.5 (765.5-1451) pg/ml x h to 1910.4 (1675.6-2395.6) pg/ml x h (p=0.002, Wilcoxon's rank sum test), a 75% increase. This was associated with a 14% increase in 24 hour intragastric acidity (p=0.006) and non-significant changes in leptin and gastrin. There was a significant positive correlation between plasma ghrelin and intragastric acidity (r(s) 0.44, p=0.05, Spearman's rank correlation). CONCLUSIONS: After H. pylori cure, plasma ghrelin increased profoundly in asymptomatic subjects. This could lead to increased appetite and weight gain, and contribute to the increasing obesity seen in Western populations where H. pylori prevalence is low. This plausible biological mechanism links H pylori, through increasing obesity and GORD, to the increase in oesophageal adenocarcinoma observed in the West.