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.

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.

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.

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.

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.

Transgene-mediated skeletal phenotypic variation in zebrafish.

When considering relationships between genotype and phenotype we frequently ignore the fact that the genome of a typical animal, notably including that of a fish and a human, harbours a huge amount of foreign DNA. Such DNA, in the form of transposable elements, can affect genome function in a major way, and transgene biology needs to be included in our understanding of the genome. Here we examine an unexpected phenotypic effect of the chromosomally integrated transgene fli1a-F-hsp70l:Gal4VP16 that serves as a model for transgene function generally. We examine larval fras1 mutant zebrafish (Danio rerio). Gal4VP16 is a potent transcriptional activator that is already well known for toxicity and mediating unusual transcriptional effects. In the presence of the transgene, phenotypes in the neural crest-derived craniofacial skeleton, notably fusions and shape changes associated with loss of function fras1 mutations, are made more severe, as we quantify by scoring phenotypic penetrance, the fraction of mutants expressing the trait. A very interesting feature is that the enhancements are highly specific for fras1 mutant phenotypes, occurring in the apparent absence of more widespread changes. Except for the features due to the fras1 mutation, the transgene-bearing larvae appear generally healthy and to be developing normally. The transgene behaves as a genetic partial dominant: a single copy is sufficient for the enhancements, yet, for some traits, two copies may exert a stronger effect. We made new strains bearing independent insertions of the fli1a-F-hsp70l:Gal4VP16 transgene in new locations in the genome, and observed increased severities of the same phenotypes as observed for the original insertion. This finding suggests that sequences within the transgene, for example Gal4VP16, are responsible for the enhancements, rather than the effect on neighbouring host sequences (such as an insertional mutation). The specificity and biological action underlying the traits are subjects of considerable interest for further investigation, as we discuss. Our findings show that work with transgenes needs to be undertaken with caution and attention to detail.

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.

Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.

Heightened phenotypic variation among mutant animals is a well-known, but poorly understood phenomenon. One hypothetical mechanism accounting for mutant phenotypic variation is progenitor cells variably choosing between two alternative fates during development. Zebrafish mef2cab1086 mutants develop tremendously variable ectopic bone in their hyoid craniofacial skeleton. Here, we report evidence that a key component of this phenotype is variable fate switching from ligament to bone. We discover that a 'track' of tissue prone to become bone cells is a previously undescribed ligament. Fate-switch variability is heritable, and comparing mutant strains selectively bred to high and low penetrance revealed differential mef2ca mutant transcript expression between high and low penetrance strains. Consistent with this, experimental manipulation of mef2ca mutant transcripts modifies the penetrance of the fate switch. Furthermore, we discovered a transposable element that resides immediately upstream of the mef2ca locus and is differentially DNA methylated in the two strains, correlating with differential mef2ca expression. We propose that variable transposon epigenetic silencing underlies the variable mef2ca mutant bone phenotype, and could be a widespread mechanism of phenotypic variability in animals.

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.

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.

Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton.

Phenotypic robustness requires a process of developmental buffering that is largely not understood, but which can be disrupted by mutations. Here we show that in mef2ca(b1086) loss of function mutant embryos and early larvae, development of craniofacial hyoid bones, the opercle (Op) and branchiostegal ray (BR), becomes remarkably unstable; the large magnitude of the instability serves as a positive attribute to learn about features of this developmental buffering. The OpBR mutant phenotype variably includes bone expansion and fusion, Op duplication, and BR homeosis. Formation of a novel bone strut, or a bone bridge connecting the Op and BR together occurs frequently. We find no evidence that the phenotypic stability in the wild type is provided by redundancy between mef2ca and its co-ortholog mef2cb, or that it is related to the selector (homeotic) gene function of mef2ca. Changes in dorsal-ventral patterning of the hyoid arch also might not contribute to phenotypic instability in mutants. However, subsequent development of the bone lineage itself, including osteoblast differentiation and morphogenetic outgrowth, shows marked variation. Hence, steps along the developmental trajectory appear differentially sensitive to the loss of buffering, providing focus for the future study.

HCMV-infected cells maintain efficient nucleotide excision repair of the viral genome while abrogating repair of the host genome.

Many viruses subvert the host cell's ability to mount and complete various DNA damage responses (DDRs) after infection. HCMV infection of permissive fibroblasts activates host DDRs at the time of viral deposition and during replication, but the DDRs remain uncompleted without arrest or apoptosis. We believe this was in part due to partitioning of the damage response and double strand break repair components. After extraction of soluble proteins, the localization of these components fell into three groups: specifically associated with the viral replication centers (RCs), diffused throughout the nucleoplasm and excluded from the RCs. Others have shown that cells are incapable of processing exogenously introduced damage after infection. We hypothesized that the inability of the cells to process damage might be due to the differential association of repair components within the RCs and, in turn, potentially preferential repair of the viral genome and compromised repair of the host genome. To test this hypothesis we used multiple strategies to examine repair of UV-induced DNA damage in mock and virus-infected fibroblasts. Comet assays indicated that repair was initiated, but was not completed in infected cells. Quantitative analysis of immunofluorescent localization of cyclobutane pyrimidine dimers (CPDs) revealed that after 24 h of repair, CPDs were significantly reduced in viral DNA, but not significantly changed in the infected host DNA. To further quantitate CPD repair, we developed a novel dual-color Southern protocol allowing visualization of host and viral DNA simultaneously. Combining this Southern methodology with a CPD-specific T4 endonuclease V alkaline agarose assay to quantitate repair of adducts, we found efficient repair of CPDs from the viral DNA but not host cellular DNA. Our data confirm that NER functions in HCMV-infected cells and almost exclusively repairs the viral genome to the detriment of the host's genome.

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.

Predictive factors for successful clinical outcome 1 year after an intensive combined physical and psychological programme for chronic low back pain.

PURPOSE: The aim of this longitudinal study is to determine the factors which predict a successful 1-year outcome from an intensive combined physical and psychological (CPP) programme in chronic low back pain (CLBP) patients. METHODS: A prospective cohort of 524 selected consecutive CLBP patients was followed. Potential predictive factors included demographic characteristics, disability, pain and cognitive behavioural factors as measured at pre-treatment assessment. The primary outcome measure was the oswestry disability index (ODI). A successful 1-year follow-up outcome was defined as a functional status equivalent to 'normal' and healthy populations (ODI ≤22). The 2-week residential programme fulfills the recommendations in international guidelines. For statistical analysis we divided the database into two equal samples. A random sample was used to develop a prediction model with multivariate logistic regression. The remaining cases were used to validate this model. RESULTS: The final predictive model suggested being 'in employment' at pre-treatment [OR 3.61 (95 % CI 1.80-7.26)] and an initial 'disability score' [OR 0.94 (95 % CI 0.92-0.97)] as significant predictive factors for a successful 1-year outcome (R (2) = 22 %; 67 % correctly classified). There was no predictive value from measures of psychological distress. CONCLUSION: CLBP patients who are in work and mild to moderately disabled at the start of a CPP programme are most likely to benefit from it and to have a successful treatment outcome. In these patients, the disability score falls to values seen in healthy populations. This small set of factors is easily identified, allowing selection for programme entry and triage to alternative treatment regimes.

Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation.

Mef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart.

FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution.

BACKGROUND: The vertebrate craniofacial skeleton may exhibit anatomical complexity and diversity, but its genesis and evolution can be understood through careful dissection of developmental programs at cellular resolution. Resources are lacking that include introductory overviews of skeletal anatomy coupled with descriptions of craniofacial development at cellular resolution. In addition to providing analytical guidelines for other studies, such an atlas would suggest cellular mechanisms underlying development. DESCRIPTION: We present the Fish Face Atlas, an online, 3D-interactive atlas of craniofacial development in the zebrafish Danio rerio. Alizarin red-stained skulls scanned by fluorescent optical projection tomography and segmented into individual elements provide a resource for understanding the 3D structure of the zebrafish craniofacial skeleton. These data provide the user an anatomical entry point to confocal images of Alizarin red-stained zebrafish with transgenically-labelled pharyngeal arch ectomesenchyme, chondrocytes, and osteoblasts, which illustrate the appearance, morphogenesis, and growth of the mandibular and hyoid cartilages and bones, as viewed in live, anesthetized zebrafish during embryonic and larval development. Confocal image stacks at high magnification during the same stages provide cellular detail and suggest developmental and evolutionary hypotheses. CONCLUSION: The FishFace Atlas is a novel learning tool for understanding craniofacial skeletal development, and can serve as a reference for a variety of studies, including comparative and mutational analyses.

Language and social/emotional problems identified at a universal developmental assessment at 30 months.

BACKGROUND: Preschool language and neurodevelopmental problems often persist and impede learning. The aims of the current study are to assess the uptake of a new universal 30 month health visitor contact and to quantify the prevalence of language delay and social/emotional difficulties. METHODS: All families of 30 month old children in four Glasgow localities were offered a visit from their health visitor. Structured data were collected relating to language, social and emotional development using three instruments; The Strengths and Difficulties Questionnaire (SDQ), the abbreviated Sure Start Language Measure and a two-item language screen. RESULTS: From an eligible population of 543 children, there was a 90% return rate of contact forms from the health visitors, and assessments were completed on 78% of eligible children. Visit completion rates did not differ significantly by socio-economic status. 3-8% of children were reported to have language delay depending on the method of assessment. 8.8% of children scored in the "abnormal" range of SDQ total difficulties scores and 31.1% had an abnormality in at least one subscale. There was substantial overlap between language delay and abnormal scores on the SDQ. CONCLUSIONS: Universal assessment of neurodevelopmental function at 30 months identified a significant proportion of children, including those previously considered at low risk, with both language and social/emotional difficulties. Further work is required to assess the precise nature of these difficulties and to assess the potential impact on services.