Why don't children in Uganda have tics? A mixed-methods study of beliefs, knowledge, and attitudes of health professionals.

BACKGROUND: Tourette syndrome (TS) is reported in all cultures, although is speculated to be rare among those of Sub-Saharan African descent. A lack of research exploring TS in the context of Sub-Saharan Africa has meant that it is not yet established whether this apparent rarity is due to a true low prevalence or if identification of the condition merely is unrecognized. The present study aimed to explore health professionals' knowledge and attitudes of the identification, diagnosis and management of TS in Uganda. METHOD: A mixed-methods design was used to collect data from 152 Ugandan healthcare professionals by survey. Of these, 6 professionals took part in semi-structured interviews. Data gathered were analyzed with descriptive statistics and qualitatively using thematic analysis. RESULTS: Professionals' views and experiences led to conclusions addressing (a) perceived challenges in diagnosing and treating TS in a Ugandan healthcare setting, (b) the role of cultural factors in help-seeking behaviors, and (c) ways by which efforts can be made to build capacity in awareness and clinical care. CONCLUSIONS: The findings suggest that TS is present in Uganda, but a number of factors hinder detection rates causing it to be under-reported in the population. This study highlights the need for more focused and adequate training for all healthcare professionals in Uganda and education campaigns to increase awareness among the general public. KEY PRACTITIONER MESSAGE: Little is currently known about the presentation of or understanding by professionals around Tourette syndrome in Sub-Saharan Africa. This study found health professionals in Uganda indicated a number of factors which interact and negatively impact recognition rates of tic disorders. This included limited training, exposure, and misconceptions, contributing to a lack of clinical awareness and attention. They also reported a lack of help-seeking behaviors due to parental perceptions and Ugandan traditional, cultural and religious beliefs. Professionals perceive that there are likely large numbers of undiagnosed and untreated children with Tourette syndrome in Uganda. This supports existing literature regarding the diagnostic challenges of neurodevelopmental disorders in Africa and provides an alternative explanation for the assumption that tic disorders may be rare and possible absent in Sub-Saharan Africa. There is a need to develop adequate training regarding tic disorders for all healthcare professionals working in Uganda as well as education campaigns for the general public to increase awareness.

Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.

A crucial role for the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specification.

Mammalian development is regulated by the interplay of tissue-specific and ubiquitously expressed transcription factors, such as Sp1. Sp1 knockout mice die in utero with multiple phenotypic aberrations, but the underlying molecular mechanism of this differentiation failure has been elusive. Here, we have used conditional knockout mice as well as the differentiation of mouse ES cells as a model with which to address this issue. To this end, we examined differentiation potential, global gene expression patterns and Sp1 target regions in Sp1 wild-type and Sp1-deficient cells representing different stages of hematopoiesis. Sp1(-/-) cells progress through most embryonic stages of blood cell development but cannot complete terminal differentiation. This failure to fully differentiate is not seen when Sp1 is knocked out at later developmental stages. For most Sp1 target and non-target genes, gene expression is unaffected by Sp1 inactivation. However, Cdx genes and multiple Hox genes are stage-specific targets of Sp1 and are downregulated at an early stage. As a consequence, expression of genes involved in hematopoietic specification is progressively deregulated. Our work demonstrates that the early absence of active Sp1 sets a cascade in motion that culminates in a failure of terminal hematopoietic differentiation and emphasizes the role of ubiquitously expressed transcription factors for tissue-specific gene regulation. In addition, our global side-by-side analysis of the response of the transcriptional network to perturbation sheds a new light on the regulatory hierarchy of hematopoietic specification.

The Role of the Ubiquitously Expressed Transcription Factor Sp1 in Tissue-specific Transcriptional Regulation and in Disease.

Sp1 belongs to the 26 member strong Sp/KLF family of transcription factors. It is a paradigm for a ubiquitously expressed transcription factor and is involved in regulating the expression of genes associated with a wide range of cellular processes in mammalian cells. Sp1 can interact with a range of proteins, including other transcription factors, members of the transcription initiation complex and epigenetic regulators, enabling tight regulation of its target genes. In this review, we discuss the mechanisms involved in Sp1-mediated transcriptional regulation, as well as how a ubiquitous transcription factor can be involved in establishing a tissue-specific pattern of gene expression and mechanisms by which its activity may be regulated. We also consider the role of Sp1 in human diseases, such as cancer.

Applying an Established Exposure Response Prevention Protocol for Young People With Tourette Syndrome in an Intensive, Group Format: A Feasibility Study.

BACKGROUND: The motor and vocal tics that characterise Tourette syndrome are stigmatizing and impact on quality of life. Behavioural interventions such as Exposure Response Prevention or Comprehensive Behavioural Interventions for Tics are first line treatment for Tourette syndrome, but availability is limited. This study is the first to explore the impact of an established manualised Exposure Response Prevention treatment protocol, developed for individual therapy, but here uniquely delivered intensively, to a group. METHODS: A naturalistic study comprised of a consecutive series of children (N = 20), aged 8-16 years (M = 12, SD = 2.17) were offered Exposure Response Prevention in one of two groups, delivered in series within a specialist clinic. Young people received the equivalent of 12 sessions (matching the manualised individual protocol). RESULTS: The YGTSS and Giles de la Tourette Syndrome Quality of Life Scale for Children and Adolescents (Satisfaction Scale) showed significant improvement following treatment with moderate to large effect sizes. Thirty-five percent of children demonstrated a reliable improvement on the YGTSS Global Tic Severity score. CONCLUSIONS: These data suggest an established Exposure Response Prevention protocol can be delivered in an intensive, group setting with a positive clinical outcome. Replication in a randomized controlled trial is an important next step.

Improved understanding of non-epileptic seizures and reduced emergency health care usage following a single psychoeducational group for children and their parents.

RATIONALE: The aim of this study was to evaluate the effectiveness of a three-hour psychoeducation group in improving understanding of non-epileptic seizures (NES), health outcomes and quality of life in young people with NES. BACKGROUND: Multi-session psychoeducational groups for adults with NES have reported improved psychosocial functioning and reduced NES compared to those who do not receive psychoeducational interventions. To date there have been no studies in young people examining the effects of a single session of psychoeducation. METHOD: 15 young people with NES and their families attended a psychoeducation group within a specialist hospital following a multidisciplinary assessment. The group's effectiveness was evaluated in terms of perceptions of seizure controllability, seizure severity, the management of the condition and health-related quality of life measures. RESULTS: A significant decrease in accident and emergency (A&E) visits and ambulance call outs was observed following the psychoeducation group. Young people additionally reported increased knowledge of NES and ability to cope with the condition which was maintained at 6-week follow-up. Significant reduction in NES occurrence or quality of life was not observed. CONCLUSION: Significant reduction in A&E attendance and ambulance use was found following group psychoeducation and improvements in psychosocial functioning and knowledge about NES. Group psychoeducation has the potential to increase child and parental understanding of NES and reduce inappropriate healthcare usage.

High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules.

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of 'lead-like' small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N6,N6-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K(+)-ATP channel opener pinacidil and show that it probably promotes cell survival, by 'off-target' inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.

Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells.

hESCs (human embryonic stem cells) offer great potential for pharmaceutical research and development and, potentially, for therapeutic use. However, improvements in cell culture are urgently required to allow the scalable production of large numbers of cells that maintain pluripotency. Supplementing feeder-free conditions with either EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine] or readily synthesized analogues of this compound maintains hESC pluripotency in the absence of exogenous cytokines. When the hESC lines SA121 or SA461 were maintained in feeder-free conditions with EHNA they displayed no reduction in stem-cell-associated markers such as Nanog, Oct4 (octamer-binding protein 4) and SSEA4 (stage-specific embryonic antigen 4) when compared with cells maintained in full feeder-free conditions that included exogenously added bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but EHNA did not limit efficient spontaneous or directed differentiation following its removal. We conclude that EHNA or related compounds offers a viable alternative to exogenous cytokine addition in maintaining hESC cultures in a pluripotent state and might be a particularly useful replacement for bFGF for large-scale or GMP (good manufacturing practice)-compliant processes.

Fibronectin is a TH1-specific molecule in human subjects.

BACKGROUND: T(H)1 cell-mediated immunity is essential for host defense against a variety of intracellular pathogens, such as mycobacteria, salmonella, and Leishmania species. A major T(H)1-mediated effector mechanism involves the IFN-gamma-induced killing of the pathogen by infected macrophages. OBJECTIVES: The range of known T(H)1-specific effector molecules is limited, especially in human subjects. We sought to identify novel effector molecules that might be involved in T(H)1-mediated pathogen clearance. METHODS: We performed microarray-based analysis of human T(H)1 and T(H)2 cells to identify T(H)1-specific molecules. These analyses identified the extracellular matrix molecule fibronectin as a highly expressed T(H)1-specific molecule. We examined the expression of fibronectin in a variety of human cell types by using real-time RT-PCR, ELISA, and Western blotting. We also studied the role of fibronectin in modulating monocyte phenotype using in vitro culture. RESULTS: We show that human T(H)1 cells constitutively express and secrete fibronectin after in vitro differentiation from naive precursors. Furthermore, we demonstrate that ex vivo human T(H)1 cells selectively express fibronectin when compared with T(H)2 cells. The predominant isoform of fibronectin expressed by T(H)1 cells contains additional domains of the protein responsible for alpha4beta1 integrin binding and activation of Toll-like receptor 4. We show that treatment of monocytes with T(H)1 cell-derived fibronectin induces expression of the proinflammatory cytokine IL-6 while inhibiting IL-10 expression. CONCLUSIONS: Because fibronectin also plays a major role in the attachment and opsonization of numerous intracellular pathogens, we propose that it might be a critical molecule produced by T(H)1 cells involved in pathogen eradication.

Childhood rituals and executive functions.

Repetitive and ritualistic behaviours (RRBs) are a feature of both typical and atypical development. While the cognitive correlates of these behaviours have been investigated in some neurodevelopmental conditions these links remain largely unexplored in typical development. The current study examined the relationship between RRBs and executive functions (EF) in a sample of typically developing children aged between 37 and 107 months. Results showed that cognitive flexibility, and not response inhibition or generativity, was most strongly associated with the frequency of RRBs in this sample. In younger children (<67.5 months) cognitive flexibility was significantly associated with 'Repetitive Behaviours' but in older children (>67.5 months) cognitive flexibility was associated with both 'Just Right' and 'Repetitive Behaviour', suggesting that the association between EF and RRBs may become stronger with age in typically developing children.

Robust hematopoietic specification requires the ubiquitous Sp1 and Sp3 transcription factors.

BACKGROUND: Both tissue-specific and ubiquitously expressed transcription factors, such as Sp-family members, are required for correct development. However, the molecular details of how ubiquitous factors are involved in programming tissue-specific chromatin and thus participate in developmental processes are still unclear. We previously showed that embryonic stem cells lacking Sp1 DNA-binding activity (Sp1ΔDBD/ΔDBD cells) are able to differentiate into early blood progenitors despite the inability of Sp1 to bind chromatin without its DNA-binding domain. However, gene expression during differentiation becomes progressively deregulated, and terminal differentiation is severely compromised. RESULTS: Here, we studied the cooperation of Sp1 with its closest paralogue Sp3 in hematopoietic development and demonstrate that Sp1 and Sp3 binding sites largely overlap. The complete absence of either Sp1 or Sp3 or the presence of the Sp1 DNA-binding mutant has only a minor effect on the pattern of distal accessible chromatin sites and their transcription factor binding motif content, suggesting that these mutations do not affect tissue-specific chromatin programming. Sp3 cooperates with Sp1ΔDBD/ΔDBD to enable hematopoiesis, but is unable to do so in the complete absence of Sp1. Using single-cell gene expression analysis, we show that the lack of Sp1 DNA binding leads to a distortion of cell fate decision timing, indicating that stable chromatin binding of Sp1 is required to maintain robust differentiation trajectories. CONCLUSIONS: Our findings highlight the essential contribution of ubiquitous factors such as Sp1 to blood cell development. In contrast to tissue-specific transcription factors which are required to direct specific cell fates, loss of Sp1 leads to a widespread deregulation in timing and coordination of differentiation trajectories during hematopoietic specification.

Control of IL-4 expression in T helper 1 and 2 cells.

The mechanism of differentiation of naïve T cells to a variety of effector lineages, but particularly to T helper type 1 (Th1) and Th2 cells, has been the subject of intense scrutiny over the past two decades. Studies have revealed that the expression of cytokines, receptors, signalling molecules, transcription factors, DNA methylating enzymes and histone-modifying enzymes is altered during the process and has been shown to play a co-ordinated role to facilitate expression of the cytokines interleukin-4 (IL-4), IL-5 and IL-13 in Th2 cells, or interferon-gamma in Th1 cells. Regulation of IL-4 expression has been of particular interest for two main reasons: first because IL-4 acts as a growth factor for Th2 cells, and second because of its role in the induction of immunoglobulin class switching to immunoglobulin E, which plays a critical role in mediating allergic responses. Study of the pathways that promote this tissue-restricted expression of IL-4 may highlight potential areas for therapeutic intervention.

The Co-operation of RUNX1 with LDB1, CDK9 and BRD4 Drives Transcription Factor Complex Relocation During Haematopoietic Specification.

Haematopoietic cells arise from endothelial cells within the dorsal aorta of the embryo via a process called the endothelial-haematopoietic transition (EHT). This process crucially depends on the transcription factor RUNX1 which rapidly activates the expression of genes essential for haematopoietic development. Using an inducible version of RUNX1 in a mouse embryonic stem cell differentiation model we showed that prior to the EHT, haematopoietic genes are primed by the binding of the transcription factor FLI1. Once expressed, RUNX1 relocates FLI1 towards its binding sites. However, the nature of the transcription factor assemblies recruited by RUNX1 to reshape the chromatin landscape and initiate mRNA synthesis are unclear. Here, we performed genome-wide analyses of RUNX1-dependent binding of factors associated with transcription elongation to address this question. We demonstrate that RUNX1 induction moves FLI1 from distal ETS/GATA sites to RUNX1/ETS sites and recruits the basal transcription factors CDK9, BRD4, the Mediator complex and the looping factor LDB1. Our study explains how the expression of a single transcription factor can drive rapid and replication independent transitions in cellular shape which are widely observed in development and disease.

Dynamic Gene Regulatory Networks Drive Hematopoietic Specification and Differentiation.

Metazoan development involves the successive activation and silencing of specific gene expression programs and is driven by tissue-specific transcription factors programming the chromatin landscape. To understand how this process executes an entire developmental pathway, we generated global gene expression, chromatin accessibility, histone modification, and transcription factor binding data from purified embryonic stem cell-derived cells representing six sequential stages of hematopoietic specification and differentiation. Our data reveal the nature of regulatory elements driving differential gene expression and inform how transcription factor binding impacts on promoter activity. We present a dynamic core regulatory network model for hematopoietic specification and demonstrate its utility for the design of reprogramming experiments. Functional studies motivated by our genome-wide data uncovered a stage-specific role for TEAD/YAP factors in mammalian hematopoietic specification. Our study presents a powerful resource for studying hematopoiesis and demonstrates how such data advance our understanding of mammalian development.

Small Molecule Inhibitor of CBFß-RUNX Binding for RUNX Transcription Factor Driven Cancers.

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFß binding partner. CBFß enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFß are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFß and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers.

Developmental-stage-dependent transcriptional response to leukaemic oncogene expression.

Acute myeloid leukaemia (AML) is characterized by a block in myeloid differentiation the stage of which is dependent on the nature of the transforming oncogene and the developmental stage of the oncogenic hit. This is also true for the t(8;21) translocation that gives rise to the RUNX1-ETO fusion protein and initiates the most common form of human AML. Here we study the differentiation of mouse embryonic stem cells expressing an inducible RUNX1-ETO gene into blood cells as a model, combined with genome-wide analyses of transcription factor binding and gene expression. RUNX1-ETO interferes with both the activating and repressive function of its normal counterpart, RUNX1, at early and late stages of blood cell development. However, the response of the transcriptional network to RUNX1-ETO expression is developmental stage specific, highlighting the molecular mechanisms determining specific target cell expansion after an oncogenic hit.

Toward specifying pervasive developmental disorder-not otherwise specified.

Pervasive developmental disorder-not otherwise specified (PDD-NOS) is the most common and least satisfactory of the PDD diagnoses. It is not formally operationalized, which limits its reliability and has hampered attempts to assess its validity. We aimed, first, to improve the reliability and replicability of PDD-NOS by operationalizing its DSM-IV-TR description and, second, to test its validity through comparison with autistic disorder (AD) and Asperger's disorder (AsD). In a sample of 256 young people (mean age = 9.1 years) we used Developmental, Diagnostic and Dimensional (3Di) algorithmic analysis to classify DSM-IV-TR AD (n = 97), AsD (n = 93) and PDD-NOS (n = 66). Groups were compared on independent measures of core PDD symptomatology, associated autistic features, and intelligence. Contrary to the assumption that PDD-NOS is heterogeneous, almost all (97%) of those with PDD-NOS had one distinct symptom pattern, namely impairments in social reciprocity and communication, without significant repetitive and stereotyped behaviors (RSB). Compared to AD and AsD, they had comparably severe but more circumscribed social communication difficulties, with fewer non-social features of autism, such as sensory, feeding and visuo-spatial problems. These individuals appear to have a distinct variant of autism that does not merely sit at the less severe end of the same continuum of symptoms. The current draft guidelines for DSM-V, which mandate the presence of RSBs for any PDD diagnosis, would exclude such people from the autistic spectrum.