Predictors of outcome following an epidural steroid injection for disc-related sciatica: a Delphi consensus study.

PURPOSE: Trial data shows modest reductions in leg pain, disability and surgery avoidance following epidural steroid injections (ESI) for severe sciatica. Despite their common use, there is no clear evidence about which patients are more likely to benefit from ESI. The aim of this study was to generate consensus on potential predictors of outcome following ESI for disc-related sciatica. METHODS: A list of potential predictors of outcome was generated during a consensus meeting of seven experts. The items were subsequently presented in a two round on-line Delphi study to generate consensus among experts on which items are potential predictors of outcome. Consensus was defined as 70% agreement among participants. RESULTS: Sixty-one items were generated during the consensus meeting. Of ninety experts invited to participate in the on-line Delphi study, 44 (48%) and 33 (73%) took part in rounds one and two respectively. Twenty-eight additional items suggested by participants in round one were included in round two. Overall, 14 items reached consensus reflecting domains of health, medication use, pain intensity, psychosocial factors, imaging findings and type of injection. CONCLUSION: Based on expert consensus, items that can be routinely collected in clinical practice were identified as potential predictors of outcomes following ESI.

Improvement in functional outcome 1 year after nonsurgical multidisciplinary treatment for chronic pain after total knee arthroplasty: A prospective cohort study.

PURPOSE: The purpose of this study was to evaluate a multidisciplinary intervention developed for patients with debilitating chronic pain after total knee arthroplasty (TKA) unresponsive to existing treatment options. METHODS: A treatment-based prospective cohort study was caried out in 30 TKA patients with debilitating chronic pain at least 1 year after TKA. The treatment was a multidisciplinary intervention. Main inclusion criteria: no indication for surgery. Primary outcome was function measured by KOOS-PS, OKS, OKS-APQ and WORQ. Secondary outcome measures were pain, fear of movement, self-efficacy, quality of life (QoL), health care and pain medication use, work rehabilitation and patient satisfaction. The assessments took place pre- and directly posttreatment, at 1, 3 and 12 months follow-up. The clinical relevance was assessed by predefined minimal important clinical change (MCIC). RESULTS: At baseline patients were on average 64.7 (±7.9) years old, 67% were female, and they had knee pain for 42 (10-360) months. The results at 12-month follow-up: first, a significant improvement was shown in function, pain, fear of movement, self-efficacy and QoL. Second, in 38.5%-69.2% of patients clinical relevant improvement was shown for functional outcome, 31% for pain, and 50% for self-efficacy. Third, 42% of patients reported 'no healthcare use in the past three months'. CONCLUSION: One year after a multidisciplinary treatment a clinically relevant improvement was shown in terms of function, pain, self-efficacy and QoL. It seems to be a promising treatment option in this difficult-to-treat patient group with debilitating chronic pain after TKA. Future research should examine the effect of the treatment in a larger study population, considering a control group, and focusing on the working population and evaluating cost-efficacy. LEVEL OF EVIDENCE: Level II.

Predictors of outcome in sciatica patients following an epidural steroid injection: the POiSE prospective observational cohort study protocol.

INTRODUCTION: Sciatica can be very painful and, in most cases, is due to pressure on a spinal nerve root from a disc herniation with associated inflammation. For some patients, the pain persists, and one management option is a spinal epidural steroid injection (ESI). The aim of an ESI is to relieve leg pain, improve function and reduce the need for surgery. ESIs work well in some patients but not in others, but we cannot identify these patient subgroups currently. This study aims to identify factors, including patient characteristics, clinical examination and imaging findings, that help in predicting who does well and who does not after an ESI. The overall objective is to develop a prognostic model to support individualised patient and clinical decision-making regarding ESI. METHODS: POiSE is a prospective cohort study of 439 patients with sciatica referred by their clinician for an ESI. Participants will receive weekly text messages until 12 weeks following their ESIand then again at 24 weeks following their ESI to collect data on leg pain severity. Questionnaires will be sent to participants at baseline, 6, 12 and 24 weeks after their ESI to collect data on pain, disability, recovery and additional interventions. The prognosis for the cohort will be described. The primary outcome measure for the prognostic model is leg pain at 6 weeks. Prognostic models will also be developed for secondary outcomes of disability and recovery at 6 weeks and additional interventions at 24 weeks following ESI. Statistical analyses will include multivariable linear and logistic regression with mixed effects model. ETHICS AND DISSEMINATION: The POiSE study has received ethical approval (South Central Berkshire B Research Ethics Committee 21/SC/0257). Dissemination will be guided by our patient and public engagement group and will include scientific publications, conference presentations and social media.

HCMV Infection Reduces Nidogen-1 Expression, Contributing to Impaired Neural Rosette Development in Brain Organoids.

Human cytomegalovirus (HCMV) is a leading cause of birth defects in humans. These birth defects include microcephaly, sensorineural hearing loss, vision loss, and cognitive impairment. The process by which the developing fetus incurs these neurological defects is poorly understood. To elucidate some of these mechanisms, we have utilized HCMV-infected induced pluripotent stem cells (iPSCs) to generate in vitro brain organoids, modeling the first trimester of fetal brain development. Early during culturing, brain organoids generate neural rosettes. These structures are believed to model neural tube formation. Rosette formation was analyzed in HCMV-infected and mock-infected brain organoids at 17, 24, and 31 days postinfection. Histological analysis revealed fewer neural rosettes in HCMV-infected compared to mock-infected organoids. HCMV-infected organoid rosettes incurred multiple structural deficits, including increased lumen area, decreased ventricular zone depth, and decreased cell count. Immunofluorescent (IF) analysis found that nidogen-1 (NID1) protein expression in the basement membrane surrounding neural rosettes was greatly reduced by virus infection. IF analysis also identified a similar downregulation of laminin in basement membranes of HCMV-infected organoid rosettes. Knockdown of NID1 alone in brain organoids impaired their development, leading to the production of rosettes with increased lumen area, decreased structural integrity, and reduced laminin localization in the basement membrane, paralleling observations in HCMV-infected organoids. Our data strongly suggest that HCMV-induced downregulation of NID1 impairs neural rosette formation and integrity, likely contributing to many of HCMV's most severe birth defects. IMPORTANCE HCMV infection in pregnant women continues to be the leading cause of virus-induced neurologic birth defects. The mechanism through which congenital HCMV (cCMV) infection induces pathological changes to the developing fetal central nervous system (CNS) remains unclear. Our lab previously reproduced identified clinical defects in HCMV-infected infants using a three dimensional (3D) brain organoid model. In this new study, we have striven to discover very early HCMV-induced changes in developing brain organoids. We investigated the development of neural tube-like structures, neural rosettes. HCMV-infected rosettes displayed multiple structural abnormalities and cell loss. HCMV-infected rosettes displayed reduced expression of the key basement membrane protein, NID1. We previously found NID1 to be specifically targeted in HCMV-infected fibroblasts and endothelial cells. Brain organoids generated from NID1 knockdown iPSCs recapitulated the structural defects observed in HCMV-infected rosettes. Findings in this study revealed HCMV infection induced early and dramatic structural changes in 3D brain organoids. We believe our results suggest a major role for infection-induced NID1 downregulation in HCMV-induced CNS birth defects.

Human Cytomegalovirus Utilizes Multiple Viral Proteins to Regulate the Basement Membrane Protein Nidogen 1.

Nidogen 1 (NID1) is an important basement membrane protein secreted by many cell types. We previously found that human cytomegalovirus (HCMV) infection rapidly induced chromosome 1 breaks and that the basement membrane protein NID1, encoded near the 1q42 break site, was downregulated. We have now determined that the specific breaks in and of themselves did not regulate NID1, rather interactions between several viral proteins and the cellular machinery and DNA regulated NID1. We screened a battery of viral proteins present by 24 hours postinfection (hpi) when regulation was induced, including components of the incoming virion and immediate early (IE) proteins. Adenovirus (Ad) delivery of the tegument proteins pp71 and UL35 and the IE protein IE1 influenced steady-state (ss) NID1 levels. IE1's mechanism of regulation was unclear, while UL35 influenced proteasomal regulation of ss NID1. Real-time quantitative PCR (RT-qPCR) experiments determined that pp71 downregulated NID1 transcription. Surprisingly, WF28-71, a fibroblast clone that expresses minute quantities of pp71, suppressed NID1 transcription as efficiently as HCMV infection, resulting in the near absence of ss NID1. Sequence analysis of the region surrounding the 1q42 break sites and NID1 promoter revealed CCCTC-binding factor (CTCF) binding sites. Chromatin immunoprecipitation experiments determined that pp71 and CTCF were both bound at these two sites during HCMV infection. Expression of pp71 alone replicated this binding. Binding was observed as early as 1 hpi, and colocalization of pp71 and CTCF occurred as quickly as 15 min postinfection (pi) in infected cell nuclei. In fibroblasts where CTCF was knocked down, Adpp71 infection did not decrease NID1 transcription nor ss NID1 protein levels. Our results emphasize another aspect of pp71 activity during infection and identify this viral protein as a key contributor to HCMV's efforts to eliminate NID1. Further, we show, for the first time, direct interaction between pp71 and the cellular genome. IMPORTANCE We have found that human cytomegalovirus (HCMV) utilizes multiple viral proteins in multiple pathways to regulate a ubiquitous cellular basement membrane protein, nidogen-1 (NID1). The extent of the resources and the redundant methods that the virus has evolved to affect this control strongly suggest that its removal provides a life cycle advantage to HCMV. Our discoveries that one of the proteins that HCMV uses to control NID1, pp71, binds directly to the cellular DNA and can exert control when present in vanishingly small quantities may have broad implications in a wide range of infection scenarios. Dysregulation of NID1 in an immunocompetent host is not known to manifest complications during infection; however, in the naive immune system of a developing fetus, disruption of this developmentally critical protein could initiate catastrophic HCMV-induced birth defects.

Non-surgical treatment for adult spinal deformity: results of an intensive combined physical and psychological programme for patients with adult spinal deformity and chronic low back pain-a treatment-based cohort study.

OBJECTIVES: No evidence-based treatment exists for adult spinal deformity (ASD) patients with chronic low back pain (CLBP). AIM OF THIS STUDY: evaluate a combined physical and psychological programme (CPPP) for ASD patients with CLBP and to compare this with a non-ASD-cohort with CLBP. METHODS: Data were extracted from the database of CLBP-patients for whom surgery is not an option and completed CPPP. Two cohorts were selected: an ASD-cohort (n = 80) based on a Cobb angle of > 10° and a consecutive age- and gender-matched non-ASD-cohort (n = 240). PRIMARY OUTCOME: functional status (Oswestry Disability Index; ODI). SECONDARY OUTCOMES: pain intensity, self-efficacy and quality of life. ASSESSMENTS: pre and post treatment, one-month and one-year follow-up (FU). CLINICAL RELEVANCE: minimal important clinical change (MCIC; ODI 10 points), patient acceptable symptom state (PASS; ODI ≤ 22). RESULTS: Demographics ASD-cohort: 79% female, mean age 50.9 (± 14.1) years, mean CLBP duration 15.5 (± 12.5) years, mean Cobb angle 21.4 (± 9.4)°. Non-ASD-cohort: not significantly different. Both cohorts improved in functional status (F[1,318] = 142.982, p < .001; r = 0.31). The ASD-cohort improved from mean ODI 39.5(± 12.0) at baseline to mean ODI 31.8(± 16.5) at one-year FU. CLINICAL RELEVANCE: 51% of the ASD patients reached MCIC and 33% reached a PASS. An interaction effect is shown between time and both cohorts (F[1,318] = 8.2, p = .004; r = 0.03); however, not clinically relevant. All secondary outcomes: improvement at one-year FU. CONCLUSION: This is the first study showing beneficial outcomes of a non-surgical treatment in selected ASD patients with longstanding CLBP. Improvement is shown in functional status, and appeared equivalent to the non-ASD cohort. LEVEL OF EVIDENCE 1: Diagnostic: individual cross-sectional studies with the consistently applied reference standard and blinding.

Targeting self-efficacy more important than dysfunctional behavioral cognitions in patients with longstanding chronic low back pain; a longitudinal study.

BACKGROUND: Multidisciplinary pain management programs based on cognitive behavioral training (CBT) principles have been shown moderately effective in improving daily functioning in patients with chronic low back pain (CLBP). To optimize health-related outcomes as daily functioning, a clear understanding of the working mechanisms of these programs is warranted. Being confident to achieve a desired outcome, i.e. self-efficacy, is suggested to be a more potent determinant for beneficial treatment outcomes than restructuring the patient's dysfunctional behavioral cognitions (pain catastrophizing and fear of movement [FoM]), but the evidence is scarce. The objective of this study was twofold: 1) to determine whether a two-week pain management program resulted in post-treatment improvements in self-efficacy and decreased dysfunctional behavioral cognitions in patients with CLBP, and 2) to examine the unique contribution of self-efficacy to improvement in post-treatment disability. METHODS: A secondary analysis of an historical cohort study was performed, including 524 patients (59% females). PRIMARY OUTCOME: functional status (Oswestry Disability Index v2.1a). SECONDARY OUTCOMES: catastrophizing (Pain Catastrophizing Scale), FoM (Tampa Scale for Kinesiophobia), and self-efficacy (Pain Self-Efficacy Questionnaire). ASSESSMENTS: pre-, post-treatment, 1, and 12-months follow-up. Paired Student's t-tests were applied and clinical relevancy of improvements was described using minimal clinical important changes. Adjusted multivariate linear regression analyses were performed to explore the unique contribution of self-efficacy. RESULTS: The mean age of patients was 46 (SD = 9.5) years and they had longstanding CLBP (mean 12.5 [SD = 10.8] years). Mean functional status, self-efficacy, and dysfunctional behavioral cognitions improved significantly at post-treatment, with improvements maintained at 12 months follow-up. Post-treatment relevant improvements in self-efficacy and dysfunctional behavioral cognitions ranged from 62.4% (FoM) to 68.7% (self-efficacy). Post-treatment self-efficacy improved the model explaining post-treatment functional disability (basic model R2 = 0.49, F(6,517) = 83.67, p < 0.001; final model R2 = 0.57, F(8,515) = 85.20, p < 0.001). This was further substantiated by the relative contribution (standardized betas) of self-efficacy: 5.67 times more than catastrophizing and 9.75 times more than FoM. CONCLUSIONS: Targeting self-efficacy contributes to fast improvement in functional status for selected and motivated patients with persistent CLBP. In pain management programs and (online) self-management programs for CLBP, targeting patients' self-efficacy should have a prominent place.

Human Cytomegalovirus Interactions with the Basement Membrane Protein Nidogen 1.

In 2000, we reported that human cytomegalovirus (HCMV) induced specific damage on chromosome 1. The capacity of the virus to induce DNA breaks indicated potent interaction between viral proteins and these loci. We have fine mapped the 1q42 breaksite. Transcriptional analysis of genes encoded in close proximity revealed virus-induced downregulation of a single gene, nidogen 1 (NID1). Beginning between 12 and 24 hours postinfection (hpi) and continuing throughout infection, steady-state (ss) NID1 protein levels were decreased in whole-cell lysates and secreted supernatants of human foreskin fibroblasts. Addition of the proteasomal inhibitor MG132 to culture medium stabilized NID1 in virus-infected cells, implicating infection-activated proteasomal degradation of NID1. Targeting of NID1 via two separate pathways highlighted the virus' emphasis on NID1 elimination. NID1 is an important basement membrane protein secreted by many cell types, including the endothelial cells (ECs) lining the vasculature. We found that ss NID1 was also reduced in infected ECs and hypothesized that virus-induced removal of NID1 might offer HCMV a means of increased distribution throughout the host. Supporting this idea, transmigration assays of THP-1 cells seeded onto NID1-knockout (KO) EC monolayers demonstrated increased transmigration. NID1 is expressed widely in the developing fetal central and peripheral nervous systems (CNS and PNS) and is important for neuronal migration and neural network excitability and plasticity and regulates Schwann cell proliferation, migration, and myelin production. We found that NID1 expression was dramatically decreased in clinical samples of infected temporal bones. While potentially beneficial for virus dissemination, HCMV-induced elimination of NID1 may underlie negative ramifications to the infected fetus.IMPORTANCE We have found that HCMV infection promotes the elimination of the developmentally important basement membrane protein nidogen 1 (NID1) from its host. The virus both decreased transcription and induced degradation of expressed protein. Endothelial cell (EC) secretion of basement membrane proteins is critical for vascular wall integrity, and infection equivalently affected NID1 protein levels in these cells. We found that the absence of NID1 in an EC monolayer allowed increased transmigration of monocytes equivalent to that observed after infection of ECs. The importance of NID1 in development has been well documented. We found that NID1 protein was dramatically reduced in infected inner ear clinical samples. We believe that HCMV's attack on host NID1 favors viral dissemination at the cost of negative developmental ramifications in the infected fetus.

Correction to: Trial of healthy relationship initiatives for the very early years (THRIVE), evaluating Enhanced Triple P for Baby and Mellow Bumps for those with additional social and care needs during pregnancy and their infants who are at higher risk of maltreatment: study protocol for a randomised controlled trial.

Following publication of the original article [1], it has been brought to our attention that an error was slipped into the article's title.

Trial of healthy relationship initiatives for the very early years (THRIVE), evaluating Enhanced Triple P for Baby and Mellow Bumps additional social and care needs during pregnancy and their infants who are at higher risk of maltreatment: study protocol for a randomised controlled trial.

BACKGROUND: Growing evidence suggests that experiences in the early years play a major role in children's development in terms of health, wellbeing and educational attainment. The Trial of healthy relationship initiatives for the very early years (THRIVE) aims to evaluate two antenatal group interventions, Enhanced Triple P for Baby and Mellow Bumps, designed for those with additional health or social care needs in pregnancy. As both interventions aim to improve maternal mental health and parenting skills, we hypothesise that in the longer term, participation may lead to an improvement in children's life trajectories. METHODS: THRIVE is a three-arm, longitudinal, randomised controlled trial aiming to recruit 500 pregnant women with additional health or social care needs. Participants will be referred by health and social care professionals, predominately midwives. Consenting participants will be block randomised to one of the three arms: Enhanced Triple P for Baby plus care as usual, Mellow Bumps plus care as usual or care as usual. Groups will commence when participants are between 20 and 34 weeks pregnant. DISCUSSION: The population we aim to recruit are traditionally referred to as "hard to reach", therefore we will monitor referrals received from maternity and social care pathways and will be open to innovation to boost referral rates. We will set geographically acceptable group locations for participants, to limit challenges we foresee for group participation and retention. We anticipate the results of the trial will help inform policy and practice in supporting women with additional health and social care needs during antenatal and early postnatal periods. This is currently a high priority for the Scottish and UK Governments. TRIAL REGISTRATION: International Standard Randomised Controlled Trials Number (ISRCTN) Registry, ISRCTN:21656568 . Registered on 28 February 2014 (registered retrospectively (by 3 months)).

Human Cytomegalovirus Compromises Development of Cerebral Organoids.

Congenital human cytomegalovirus (HCMV) infection causes a broad spectrum of central and peripheral nervous system disorders, ranging from microcephaly to hearing loss. These ramifications mandate the study of virus-host interactions in neural cells. Neural progenitor cells are permissive for lytic infection. We infected two induced pluripotent stem cell (iPSC) lines and found these more primitive cells to be susceptible to infection but not permissive. Differentiation of infected iPSCs induced de novo expression of viral antigens. iPSCs can be cultured in three dimensions to generate cerebral organoids, closely mimicking in vivo development. Mock- or HCMV-infected iPSCs were subjected to a cerebral organoid generation protocol. HCMV IE1 protein was detected in virus-infected organoids at 52 days postinfection. Absent a significant effect on organoid size, infection induced regions of necrosis and the presence of large vacuoles and cysts. Perhaps more in parallel with the subtler manifestations of HCMV-induced birth defects, infection dramatically altered neurological development of organoids, decreasing the number of developing and fully formed cortical structure sites, with associated changes in the architectural organization and depth of lamination within these structures, and manifesting aberrant expression of the neural marker ß-tubulin III. Our observations parallel published descriptions of infected clinical samples, which often contain only sparse antigen-positive foci yet display areas of focal necrosis and cellular loss, delayed maturation, and abnormal cortical lamination. The parallels between pathologies present in clinical specimens and the highly tractable three-dimensional (3D) organoid system demonstrate the utility of this system in modeling host-virus interactions and HCMV-induced birth defects.IMPORTANCE Human cytomegalovirus (HCMV) is a leading cause of central nervous system birth defects, ranging from microcephaly to hearing impairment. Recent literature has provided descriptions of delayed and abnormal maturation of developing cortical tissue in infected clinical specimens. We have found that infected induced pluripotent stem cells can be differentiated into three-dimensional, viral protein-expressing cerebral organoids. Virus-infected organoids displayed dramatic alterations in development compared to those of mock-infected controls. Development in these organoids closely paralleled observations in HCMV-infected clinical samples. Infection induced regions of necrosis, the presence of larger vacuoles and cysts, changes in the architectural organization of cortical structures, aberrant expression of the neural marker ß-tubulin III, and an overall reduction in numbers of cortical structure sites. We found clear parallels between the pathologies of clinical specimens and virus-infected organoids, demonstrating the utility of this highly tractable system for future investigations of HCMV-induced birth defects.

Moving regional health services planning and management to a population-based approach: implementation of the Regional Operating Model (ROM) in Victoria, Australia.

Various jurisdictions are moving towards population-based approaches to plan and manage healthcare services. The evidence on the implementation of these models remains limited. The aim of this study is to evaluate the effect of a regional operating model (ROM) on internal functioning and stakeholder engagement of a regional office. Semi-structured interviews and focus groups with staff members and stakeholders of the North West Metropolitan Regional office in Victoria, Australia, were conducted. Overall, the ROM was perceived as relevant to staff and stakeholders. However, creating shared objectives and priorities across a range of organisations remained a challenge. Area-based planning and management is seen as simplifying management of contracts; however, reservations were expressed about moving from specialist to more generalist approaches. A clearer articulation of the knowledge, skills and competencies required by staff would further support the implementation of the model. The ROM provides a platform for public services and stakeholders to discuss, negotiate and deliver on shared outcomes at the regional level. It provides an integrated managerial platform to improve service delivery and avoid narrow programmatic approaches.

Scoping of models to support population-based regional health planning and management: comparison with the regional operating model in Victoria, Australia.

Objective The aim of the present study was to try to understand the breadth and comprehensiveness of a regional operating model (ROM) developed within the Victorian Department of Health's North West Metropolitan Region office in Melbourne, Australia. Methods A published literature search was conducted, with additional website scanning, snowballing technique and expert consultation, to identify existing operating models. An analytical grid was developed covering 16 components to evaluate the models and assess the exhaustiveness of the ROM. Results From the 34 documents scoped, 10 models were identified to act as a direct comparator to the ROM. These concerned models from Australia (n=5) and other comparable countries (Canada, UK). The ROM was among the most exhaustive models, covering 13 of 16 components. It was one of the few models that included intersectoral actions and levers of influence. However, some models identified more precisely the planning tools, prioritisation criteria and steps, and the allocation mechanisms. Conclusions The review finds that the ROM appears to provide a wide coverage of aspects of planning and integrates into a single model some of the distinctive elements of the other models scoped. What is known about the topic? Various jurisdictions are moving towards a population-based approach to manage public services with regard to the provision of individual medical and social care. Various models have been proposed to guide the planning of services from a population health perspective. What does this paper add? This paper assesses the coverage of attributes of operating models supporting a population health planning approach to the management of services at the regional or local level. It provides a scoping of current models proposed to organise activities to ensure an integrated approach to the provision of services and compares the scoped models to a model recently implemented in Victoria, Australia. What are the implications for practitioners? This paper highlights the relative paucity of operating models describing in concrete terms how to manage medical and social services from a population perspective and encourages organisations that are accountable for securing population health to clearly articulate their own operating model. It outlines strengths and potential gaps in current models.

The absence of p53 during Human Cytomegalovirus infection leads to decreased UL53 expression, disrupting UL50 localization to the inner nuclear membrane, and thereby inhibiting capsid nuclear egress.

Our electron microscopy study (Kuan et al., 2016) found HCMV nuclear capsid egress was significantly reduced in p53 knockout cells (p53KOs), correlating with inhibited formation of infoldings of the inner nuclear membrane (IINMs). Molecular examination of these phenomena has found p53KOs expressed UL97 and phosphorylated lamins, however the lamina failed to remodel. The nuclear egress complex (NEC) protein UL50 was expressed in almost all cells. UL50 re-localized to the inner nuclear membrane (INM) in ~90% of wt cells, but only ~35% of p53KOs. UL53 expression was significantly reduced in p53KOs, and cells lacking UL50 nuclear staining, expressed no UL53. Re-introduction of p53 into p53KOs largely recovered UL53 positivity and UL50 nuclear re-localization. Nuclear rim located UL50/53 puncta, which co-localized with the major capsid protein, were largely absent in p53KOs. We believe these puncta were IINMs. In the absence of p53, UL53 expression was inhibited, disrupting formation of the NEC/IINMs, and reducing functional virion secretion.

Human Cytomegalovirus nuclear egress and secondary envelopment are negatively affected in the absence of cellular p53.

Human Cytomegalovirus (HCMV) infection is compromised in cells lacking p53, a transcription factor that mediates cellular stress responses. In this study we have investigated compromised functional virion production in cells with p53 knocked out (p53KOs). Infectious center assays found most p53KOs released functional virions. Analysis of electron micrographs revealed modestly decreased capsid production in infected p53KOs compared to wt. Substantially fewer p53KOs displayed HCMV-induced infoldings of the inner nuclear membrane (IINMs). In p53KOs, fewer capsids were found in IINMs and in the cytoplasm. The deficit in virus-induced membrane remodeling within the nucleus of p53KOs was mirrored in the cytoplasm, with a disproportionately smaller number of capsids re-enveloped. Reintroduction of p53 substantially recovered these deficits. Overall, the absence of p53 contributed to inhibition of the formation and function of IINMs and re-envelopment of the reduced number of capsids able to reach the cytoplasm.

Infection of a Single Cell Line with Distinct Strains of Human Cytomegalovirus Can Result in Large Variations in Virion Production and Facilitate Efficient Screening of Virus Protein Function.

UNLABELLED: Previously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM(-) lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM(-) lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM(-) line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM(-) cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production. In silico comparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints. IMPORTANCE: Human cytomegalovirus (HCMV) infection of multiple cell lines lacking ataxia telangiectasia mutated (ATM) protein produced wild-type levels of infectious virus. Interactions between virus strains and the microenvironment of individual ATM(-) lines determined the efficiency of virion production. Infection of one ATM(-) cell line, GM05823, produced large titer differentials dependent on the strain used, Towne or AD169. This discrepancy resolved a disagreement in the literature of a requirement for ATM expression and HCMV reproduction. The titer differentials in GM08523 cells were due, in part, to a decreased capacity of AD169 virions to enter the cell and traffic genomes to the nucleus. In silico comparison of the Towne, AD169, and related variant strains' genomes was coupled with serial iterations of a virus entry experiment, narrowing 28 candidate proteins responsible for the phenotype down to 4. Reintroduction of UL148 significantly rescued genome trafficking. Differential behavior of virus strains can be exploited to elucidate gene function.

Development of a triage tool for neurodevelopmental risk in children aged 30 months.

Neurodevelopmental and neuropsychiatric disorders in young children predict educational, health and social problems. Early identification may significantly reduce this burden but relevant tools largely lack validation. We aimed to develop and evaluate the predictive validity of a simple screening tool for neurodevelopmental problems in a community sample of 30 month old children. A sample of children was selected from a community cohort screened at 30 months by health visitors using the Sure Start Language Measure (SSLM) and the Strengths and Difficulties Questionnaire (SDQ) in 2011. Predictive validity was assessed by comparing screening results with detailed psychometric data from the same sample 1-2 years later. Screening performance using different thresholds was explored using Receiver Operating Characteristic (ROC) with ROC area under the curve (AUC) and bootstrapping techniques. The SSLM predicted both language disorder identified by the New Reynell Developmental Language Scales (NRDLS) at follow-up (AUC 0.905) and global developmental delay assessed by the Griffiths Mental Development Scales (AUC 0.983). The SDQ administered at 30 months predicted psychiatric disorders identified by the Development and Wellbeing Assessment (DAWBA) at follow-up (AUC 0.821). Using optimal cut-offs for the SDQ and SSLM at 30 months, both tools together had sensitivity 87%; specificity 64%; positive predictive value 31%; and negative predictive value 97% in the prediction of any kind of neurodevelopmental problem 1-2 years later. The combined measure reported here is not yet sufficient as a stand-alone population screening tool for neurodevelopmental disorders. The SSLM and SDQ did however show promise in identifying preschool children at risk of ongoing language, psychiatric disorders and global developmental delay 1-2 years later but with fairly high false positive rates. Given that current developmental risk prediction in resource-poor settings is little better than random assignment, the SDQ and SSLM may aid clinical judgement when used as interim triage tools for practitioners with no specialist knowledge, in the context of longitudinal follow-up arrangements.

Comparing early years and childhood experiences and outcomes in Scotland, England and three city-regions: a plausible explanation for Scottish 'excess' mortality?

BACKGROUND: Negative early years and childhood experiences (EYCE), including socio-economic circumstances, parental health and parenting style, are associated with poor health outcomes both in childhood and adulthood. It has also been proposed that EYCE were historically worse in Scottish areas, especially Glasgow and the Clyde Valley, compared to elsewhere in the UK and that this variation can provide a partial explanation for the excess of ill health and mortality observed among those Scottish populations. METHODS: Multiple logistic regression analysis was applied to two large, representative, British birth cohorts (the NCDS58 and the BCS70), to test the independent association of area of residence at ages 7 and 5 with risk of behavioural problems, respiratory problems and reading/vocabulary problems at the same age. Cohort members resident in Scotland were compared with those who were resident in England, while those resident in Glasgow and the Clyde Valley were compared with those resident in Merseyside and Greater Manchester. RESULTS: After adjustment for a range of relevant variables, the risk of adverse childhood outcomes was found to be either no different, or lower, in the Scottish areas. At a national level, the study reinforces the combined association of socio-economic circumstances, parental health (especially maternal mental health) and parenting with child health outcomes. CONCLUSION: Based on these samples, the study does not support the hypothesis that EYCE were worse in Scotland and Glasgow and the Clyde Valley. It seems, therefore (based on these data), less likely that the roots of the excess mortality observed in the Scottish areas can be explained by these factors.