TRAF2 facilitates Vaccinia virus replication by promoting rapid virus entry.

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is a pivotal intracellular mediator of signaling pathways downstream of TNFR1 and -2 with known pro- and antiviral effects. We investigated its role in the replication of the prototype poxvirus vaccinia virus (VACV). Loss of TRAF2 expression, either through small interfering RNA treatment of HeLa cells or through genetic knockout in murine embryonic fibroblasts (MEFs), led to significant reductions in VACV growth following low-multiplicity infection. In single-cycle infections, there was delayed production of both early and late VACV proteins as well as accelerated virus-induced alterations to cell morphology, indicating that TRAF2 influences early stages of virus replication. Consistent with an early role, uncoating assays showed normal virus attachment but delayed virus entry in the absence of TRAF2. Although alterations to c-Jun N-terminal kinase (JNK) signaling were apparent in VACV-infected TRAF2(−/−) MEFs, treatment of wild-type cells with a JNK inhibitor did not affect virus entry. Instead, treatment with an inhibitor of endosomal acidification greatly reduced virus entry into TRAF2(−/−) MEFs, suggesting that VACV is reliant on the endosomal route of entry in the absence of TRAF2. Thus, TRAF2 is a proviral factor for VACV that plays a role in promoting efficient viral entry, most likely via the plasma membrane. IMPORTANCE Tumor necrosis factor receptor-associated factors (TRAFs) are key facilitators of intracellular signaling with roles in innate and adaptive immunity and stress responses. We have discovered that TRAF2 is a proviral factor in vaccinia virus replication in both HeLa cells and mouse embryonic fibroblasts and that its influence is exercised through promotion of efficient virus entry

Permissive and Restricted Virus Infection of Murine Embryonic Stem Cells

Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence

A Loss of Function Analysis of Host Factors Influencing Vaccinia virus Replication by RNA Interference

Vaccinia virus (VACV) is a large, cytoplasmic, double-stranded DNA virus that requires complex interactions with host proteins in order to replicate. To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. The experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellular proteins involved in cell-to-cell spread of VACV. The screen revealed 153 pro- and 149 anti-viral HFs that strongly influenced VACV replication. These HFs were investigated further by comparisons with transcriptional profiling data sets and HFs identified in RNAi screens of other viruses. In addition, functional and pathway analysis of the entire screen was carried out to highlight cellular mechanisms involved in VACV replication. This revealed, as anticipated, that many pro-viral HFs are involved in translation of mRNA and, unexpectedly, suggested that a range of proteins involved in cellular transcriptional processes and several DNA repair pathways possess anti-viral activity. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. This advancement in our understanding of the VACV life cycle provides a reliable knowledge base for the improvement of poxvirus-based vaccine vectors and development of anti-viral theraputics

Radiocarbon Analyses Quantify Peat Carbon Losses With Increasing Temperature in a Whole Ecosystem Warming Experiment

Climate warming is expected to accelerate peatland degradation and release rates of carbon dioxide (CO2) and methane (CH4). Spruce and Peatlands Responses Under Changing Environments is an ecosystem-scale climate manipulation experiment, designed to examine peatland ecosystem response to climate forcings. We examined whether heating up to +9 °C to 3 m-deep in a peat bog over a 7-year period led to higher C turnover and CO2 and CH4 emissions, by measuring 14C of solid peat, dissolved organic carbon (DOC), CH4, and dissolved CO2 (DIC). DOC, a major substrate for heterotrophic respiration, increased significantly with warming. There was no 7-year trend in the DI14 C of the ambient plots which remained similar to their DO14 C. At +6.75 °C and +9 °C, the 14C of DIC, a product of microbial respiration, initially resembled ambient plots but became more depleted over 7 years of warming. We attributed the shifts in DI14 C to the increasing importance of solid phase peat as a substrate for microbial respiration and quantified this shift via the radiocarbon mass balance. The mass-balance model revealed increases in peat-supported respiration of the catotelm depths in heated plots over time and relative to ambient enclosures, from a baseline of 20%–25% in ambient enclosures, to 35%–40% in the heated plots. We find that warming stimulates microorganisms to respire ancient peat C, deposited under prior climate (cooler) conditions. This apparent destabilization of the large peat C reservoir has implications for peatland-climate feedbacks especially if the balance of the peatland is tipped from net C sink to C source. Plain Language Summary Since the end of the last glacial period, about 20 thousand years ago, peatlands have taken up carbon and now store an amount nearly equivalent to the quantity in the atmosphere. Microorganisms consume and respire that peat C releasing it back to the atmosphere as CO2 and CH4. Until now, many studies have shown that microorganisms prefer to consume the most recently fixed carbon and that the deeply buried ancient peat carbon reservoir is relatively stable. However, climate warming is expected to upset that balance. The Spruce and Peatlands Responses Under Changing Environments is large-scale experimental warming of a Minnesota peatland designed to study these effects. We conducted radiocarbon analysis of the peat and the microbially produced CO2 and dissolved organic carbon in ambient and heated areas of the peatland and show that at warmer temperatures more of the ancient peat carbon is being mobilized and respired to CO2. This is troubling as it signifies a positive feedback loop wherein warming stimulates peat to produce more CO2 which further exacerbates climate change

KISS AGNs in the Soft X-ray Band: Correlation with the ROSAT All-Sky Survey

We present a study of the X-ray properties of a volume-limited sample of optically selected emission-line galaxies. The sample is derived from a correlation between the KPNO International Spectroscopic Survey (KISS), an H-alpha-selected objective-prism survey of AGNs and starbursting galaxies, and the ROSAT All-Sky Survey (RASS). After elimination of all spurious matches, we identify 18 ROSAT-detected X-ray sources within the KISS sample in the 0.1-2.4 keV band. Due to soft X-ray selection effects, the majority of the ROSAT sources are Seyfert 1 galaxies. The majority (54%) of the ROSAT-KISS Seyferts are classified as narrow-line Seyfert 1 objects, a relatively high percentage compared to previous objective-prism-selected Seyfert galaxy samples. We estimate the X-ray luminosities of the ROSAT-detected KISS objects and derive volume emissivities of 6.63 x 10^38 ergs/s/Mpc^3 and 1.45 x 10^38 ergs/s/Mpc^3 for the 30 deg and 43 deg survey strips, respectively. For those KISS AGNs not detected by RASS, we use the median L_X/L_H-alpha ratio derived from a previous study to estimate L_X. The total 0.5-2 keV volume emissivity we predict for the overall KISS AGN sample is sufficient to account for 22.1 +/- 8.9% of the soft X-ray background (XRB), averaged over both survey strips. The KISS AGN sample is made up predominantly of intermediate-luminosity Seyfert 2's and LINERs, which tend to be weak soft X-ray sources. They may, however, represent a much more significant contribution to the hard XRB.Comment: 15 pages, including 5 figures and 2 tables. Accepted for publication in December 2002 A

A novel isolator-based system promotes viability of human embryos during laboratory processing

In vitro fertilisation (IVF) and related technologies are arguably the most challenging of all cell culture applications. The starting material is a single cell from which one aims to produce an embryo capable of establishing a pregnancy eventually leading to a live birth. Laboratory processing during IVF treatment requires open manipulations of gametes and embryos, which typically involves exposure to ambient conditions. To reduce the risk of cellular stress, we have developed a totally enclosed system of interlinked isolator-based workstations designed to maintain oocytes and embryos in a physiological environment throughout the IVF process. Comparison of clinical and laboratory data before and after the introduction of the new system revealed that significantly more embryos developed to the blastocyst stage in the enclosed isolator-based system compared with conventional open-fronted laminar flow hoods. Moreover, blastocysts produced in the isolator-based system contained significantly more cells and their development was accelerated. Consistent with this, the introduction of the enclosed system was accompanied by a significant increase in the clinical pregnancy rate and in the proportion of embryos implanting following transfer to the uterus. The data indicate that protection from ambient conditions promotes improved development of human embryos. Importantly, we found that it was entirely feasible to conduct all IVF-related procedures in the isolator-based workstations

Intentional and unintentional medication non-adherence in psoriasis: The role of patients’ medication beliefs and habit strength

Medication non-adherence is a missed opportunity for therapeutic benefit. We assessed “real-world” levels of self-reported non-adherence to conventional and biologic systemic therapies used for psoriasis and evaluated psychological and biomedical factors associated with non-adherence using multivariable analyses. Latent profile analysis was used to investigate whether patients can be categorized into groups with similar medication beliefs. Latent profile analysis categorizes individuals with similar profiles on a set of continuous variables into discrete groups represented by a categorical latent variable. Eight hundred and eleven patients enrolled in the British Association of Dermatologists Biologic Interventions Register were included. Six hundred and seventeen patients were using a self-administered systemic therapy; 22.4% were classified as “non-adherent” (12% intentionally and 10.9% unintentionally). Patients using an oral conventional systemic agent were more likely to be non-adherent compared to those using etanercept or adalimumab (29.2% vs. 16.4%; P ≤ 0.001). Latent profile analysis supported a three-group model; all groups held strong beliefs about their need for systemic therapy but differed in levels of medication concerns. Group 1 (26.4% of the sample) reported the strongest concerns, followed by Group 2 (61%), with Group 3 (12.6%) reporting the weakest concerns. Group 1 membership was associated with intentional non-adherence (odds ratio = 2.27, 95% confidence interval = 1.16−4.47) and weaker medication-taking routine or habit strength was associated with unintentional non-adherence (odds ratio = 0.92, 95% confidence interval = 0.89−0.96). Medication beliefs and habit strength are modifiable targets for strategies to improve adherence in psoriasis

Cognitive behavioural therapy for clozapine-resistant schizophrenia: the FOCUS RCT

Background: Clozapine (clozaril, Mylan Products Ltd) is a first-choice treatment for people with schizophrenia who have a poor response to standard antipsychotic medication. However, a significant number of patients who trial clozapine have an inadequate response and experience persistent symptoms, called clozapine-resistant schizophrenia (CRS). There is little evidence regarding the clinical effectiveness of pharmacological or psychological interventions for this population. Objectives: To evaluate the clinical effectiveness and cost-effectiveness of cognitive–behavioural therapy (CBT) for people with CRS and to identify factors predicting outcome. Design: The Focusing on Clozapine Unresponsive Symptoms (FOCUS) trial was a parallel-group, randomised, outcome-blinded evaluation trial. Randomisation was undertaken using permuted blocks of random size via a web-based platform. Data were analysed on an intention-to-treat (ITT) basis, using random-effects regression adjusted for site, age, sex and baseline symptoms. Cost-effectiveness analyses were carried out to determine whether or not CBT was associated with a greater number of quality-adjusted life-years (QALYs) and higher costs than treatment as usual (TAU). Setting: Secondary care mental health services in five cities in the UK. Participants: People with CRS aged up to 16 years, with an International Classification of Diseases, Tenth Revision (ICD-10) schizophrenia spectrum diagnoses and who are experiencing psychotic symptoms. Interventions: Individual CBT included up to 30 hours of therapy delivered over 9 months. The comparator was TAU, which included care co-ordination from secondary care mental health services. Main outcome measures: The primary outcome was the Positive and Negative Syndrome Scale (PANSS) total score at 21 months and the primary secondary outcome was PANSS total score at the end of treatment (9 months post randomisation). The health benefit measure for the economic evaluation was the QALY, estimated from the EuroQol-5 Dimensions, five-level version (EQ-5D-5L), health status measure. Service use was measured to estimate costs. Results: Participants were allocated to CBT (n = 242) or TAU (n = 245). There was no significant difference between groups on the prespecified primary outcome [PANSS total score at 21 months was 0.89 points lower in the CBT arm than in the TAU arm, 95% confidence interval (CI) –3.32 to 1.55 points; p = 0.475], although PANSS total score at the end of treatment (9 months) was significantly lower in the CBT arm (–2.40 points, 95% CI –4.79 to –0.02 points; p = 0.049). CBT was associated with a net cost of £5378 (95% CI –£13,010 to £23,766) and a net QALY gain of 0.052 (95% CI 0.003 to 0.103 QALYs) compared with TAU. The cost-effectiveness acceptability analysis indicated a low likelihood that CBT was cost-effective, in the primary and sensitivity analyses (probability < 50%). In the CBT arm, 107 participants reported at least one adverse event (AE), whereas 104 participants in the TAU arm reported at least one AE (odds ratio 1.09, 95% CI 0.81 to 1.46; p = 0.58). Conclusions: Cognitive–behavioural therapy for CRS was not superior to TAU on the primary outcome of total PANSS symptoms at 21 months, but was superior on total PANSS symptoms at 9 months (end of treatment). CBT was not found to be cost-effective in comparison with TAU. There was no suggestion that the addition of CBT to TAU caused adverse effects. Future work could investigate whether or not specific therapeutic techniques of CBT have value for some CRS individuals, how to identify those who may benefit and how to ensure that effects on symptoms can be sustained. Trial registration: Current Controlled Trials ISRCTN99672552

A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome