Home and neighbourhood built environment features in family-based treatment for childhood obesity.

BACKGROUND: Family-based behavioural weight loss treatment (FBT) for childhood obesity helps families develop strategies to facilitate healthy choices in their home and other environments (e.g. home neighbourhood). The current study examines how the home food environment, both pre-FBT and post-FBT, and the neighbourhoods in which families live are associated with child weight and weight-related outcomes in FBT. METHODS: Parent-child dyads (n = 181) completed a 16-session FBT programme and completed home environment, anthropometric and child dietary/activity assessments at pre-FBT and post-FBT. Parents reported on availability of food, electronics and physical activity equipment in the home. The neighbourhood food and recreation environments around each dyad's residence was characterized using existing data within a geographic information system. RESULTS: Families successfully made healthy home environment modifications during FBT. Regression models showed reducing RED (e.g. high-energy-dense and low-nutrient-dense) foods and electronics in the home during FBT had positive effects on child weight and weight-related outcomes. No neighbourhood food or recreation environment variables were significantly related to outcomes, although having a larger density of public recreation spaces was associated with increases in physical activity at the trend-level. CONCLUSIONS: Modifying the home environment, specifically reducing RED foods and electronics, may be particularly important for FBT success.

A physiologically relevant 3D collagen-based scaffold-neuroblastoma cell system exhibits chemosensitivity similar to orthotopic xenograft models.

3D scaffold-based in vitro cell culturing is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumours. The main challenge in treating neuroblastoma, a paediatric cancer of the sympathetic nervous system, is to combat tumour metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (Coll-GAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multi-functionality beyond this tumour type. STATEMENT OF SIGNIFICANCE: Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in our understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results. Here, we demonstrated the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.

Teaching ultrasound in tropical countries.

INTRODUCTION: This paper reports my experience as a teacher of clinical ultrasound (US) in an African hospital. While US in tropical countries has received some attention and a few papers - though possibly fewer than deserved by this issue-are available in the medical literature on this subject, very little has been done in terms of assessment of teaching. MATERIALS AND METHODS: Given the increasing number of groups, NGOs and volunteers that go to Africa and other resource limited settings to do this, I thought that sharing my experience with those who have walked or are thinking of walking the same path could be mutually beneficial. RESULTS: The first section of the article presents the situation where I've been working in the past 13 years, the second section details our teaching programme. DISCUSSION: This report describes the rationale for the implementation of ultrasound training programmes in rural areas of Africa and lessons learnt with 13 years experience from the UK with recommendations for the way forward.

Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product.

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called "residual" CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

Whole-mount in situ hybridization to mouse embryos.

The mouse is well-established as the major animal model for the study of mammalian development. Rapid progress in large-scale cDNA and also genomic sequencing projects is identifying new mouse genes at an unprecedented rate. As a first step toward understanding the function of these novel genes, it is important to determine their developmental expression pattern. Here we provide a reliable, sensitive method for whole-mount in situ hybridization using the mouse embryo.

Rapid generation of nested chromosomal deletions on mouse chromosome 2.

Nested chromosomal deletions are powerful genetic tools. They are particularly suited for identifying essential genes in development either directly or by screening induced mutations against a deletion. To apply this approach to the functional analysis of mouse chromosome 2, a strategy for the rapid generation of nested deletions with Cre recombinase was developed and tested. A loxP site was targeted to the Notch1 gene on chromosome 2. A targeted line was cotransfected with a second loxP site and a plasmid for transient expression of Cre. Independent random integrations of the second loxP site onto the targeted chromosome in direct repeat orientation created multiple nested deletions. By virtue of targeting in an F(1) hybrid embryonic stem cell line, F(1)(129S1xCast/Ei), the deletions could be verified and rapidly mapped. Ten deletions fell into seven size classes, with the largest extending six or seven centiMorgans. The cytology of the deletion chromosomes were determined by fluorescent in situ hybridization. Eight deletions were cytologically normal, but the two largest deletions had additional rearrangements. Three deletions, including the largest unrearranged deletion, have been transmitted through the germ line. Several endpoints also have been cloned by plasmid rescue. These experiments illustrate the means to rapidly create and map deletions anywhere in the mouse genome. They also demonstrate an improved method for generating nested deletions in embryonic stem cells.

Surface ectoderm is necessary for the morphogenesis of somites.

The paraxial mesoderm of the neck and trunk of mouse embryos undergoes extensive morphogenesis in forming somites. Paraxial mesoderm is divided into segments, it elongates along its anterior posterior axis, and its cells organize into epithelia. Experiments were performed to determine if these processes are autonomous to the mesoderm that gives rise to the somites. Presomitic mesoderm at the tailbud stage was cultured in the presence and absence of its adjacent tissues. Somite segmentation occurred in the absence of neural tube, notochord, gut and surface ectoderm, and occurred in posterior fragments in the absence of anterior presomitic mesoderm. Mesodermal expression of Dll1 and Notch1, genes with roles in segmentation, was largely independent of other tissues, consistent with autonomous segmentation. However, surface ectoderm was found to be necessary for elongation of the mesoderm along the anterior-posterior axis and for somite epithelialization. To determine if there is specificity in the interaction between ectoderm and mesoderm, ectoderm from different sources was recombined with presomitic mesoderm. Surface ectoderm from only certain parts of the embryo supported somite epithelialization and elongation. Somite epithelialization induced by ectoderm was correlated with expression of the basic-helix-loop-helix gene Paraxis in the mesoderm. This is consistent with the genetically defined requirement for Paraxis in somite epithelialization. However, trunk ectoderm was able to induce somite epithelialization in the absence of strong Paraxis expression. We conclude that somitogenesis consists of autonomous segmentation patterned by Notch signaling and nonautonomous induction of elongation and epithelialization by surface ectoderm.

Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.

BACKGROUND: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region. RESULTS: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM. CONCLUSIONS: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.

The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart.

In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA-binding proteins, the RRM (RNA Recognition Motif) family. The gene has been named hermes (for HEart, RRM Expressed Sequence). The hermes protein is 197-amino acids long and contains a single RRM domain. In situ hybridization analysis indicates that hermes is expressed at highest levels in the myocardium of the heart and to a lesser extent in the ganglion layer of the retina, the pronephros and epiphysis. Expression of hermes in each of these tissues begins at approximately the time of differentiation and is maintained throughout development. Analysis of the RNA expression of the hermes orthologues from chicken and mouse reveals that, like Xenopus, the most prominent tissue of expression is the developing heart. The sequence and expression pattern of hermes suggests a role in post-transcriptional regulation of heart development.

Expression pattern of two Frizzled-related genes, Frzb-1 and Sfrp-1, during mouse embryogenesis suggests a role for modulating action of Wnt family members.

Wnt proteins have been implicated in regulating growth and pattern formation in a variety of tissues during embryonic development. We previously identified Frzb-1, a gene which encodes a secreted protein with homology in the ligand binding domain to the Wnt receptor Frizzled, but lacking the domain encoding the putative seven transmembrane segments. Frzb-1 has recently been shown to bind to Wnt proteins in vitro, and to inhibit the activity of Xenopus Wnt-8 in vivo. We report now that mFrzb-1 and Wnt transcripts display both complementary and overlapping expression patterns at multiple sites throughout embryonic development. By Northern analysis, the expression of mFrzb-1 in the developing mouse embryo is greatest from 10.5 to 12.5 days postcoitum (dpc). In the early embryo, mFrzb-1 is expressed in the primitive streak, presomitic mesoderm, somites, and brain. Later, mFrzb-1 exhibits sharp boundaries of expression in the limb bud, branchial arches, facial mesenchyme, and in cartilaginous elements of the appendicular skeleton. We conclude from these experiments that Frzb-1 is expressed at a time and location to modulate the action of Wnt family members during development of the limbs and central nervous system.

Conservation of the Notch signalling pathway in mammalian neurogenesis.

The Notch pathway functions in multiple cell fate determination processes in invertebrate embryos, including the decision between the neuroblast and epidermoblast lineages in Drosophila. In the mouse, targeted mutation of the Notch pathway genes Notch1 and RBP-Jk has demonstrated a role for these genes in somite segmentation, but a function in neurogenesis and in cell fate decisions has not been shown. Here we show that these mutations lead to altered expression of the Notch signalling pathway homologues Hes-5, Mash-1 and Dll1, resulting in enhanced neurogenesis. Precocious neuronal differentiation is indicated by the expanded expression domains of Math4A, neuroD and NSCL-1. The RBP-Jk mutation has stronger effects on expression of these genes than does the Notch1 mutation, consistent with functional redundancy of Notch genes in neurogenesis. Our results demonstrate conservation of the Notch pathway and its regulatory mechanisms from fly to mouse, and support a role for the murine Notch signalling pathway in the regulation of neural stem cell differentiation.

Introducing technology into the public STD clinic.

Review of the literature on public health services shows that virtually no information is available on how the state-supported networks of STD clinics now function, what the possibilities are for achieving efficiency in service delivery, or the implications of local, state, and federal funding and staffing changes. This article describes models of STD services now offered and thus allows one to project impending changes in the public health STD clinic system. The description includes a brief recount of how the imposition of HIV testing and counseling has taxed clinic resources and has sharpened the need for more efficient, technology-supported management. The status of federal staff is also summarized, with consideration of how decreases in staff will affect partner notification, the cornerstone of traditional STD-clinic-based services. Data on clinic function and staffing trends frame suggestions for the placement of computer technology in the system.

Mice lacking all isoforms of retinoic acid receptor beta develop normally and are susceptible to the teratogenic effects of retinoic acid.

Retinoic acids (RA) are vitamin A derivatives essential for normal embryonic development and viability of vertebrates. The RA signal is mediated by two distinct classes of receptors, RA receptors (RARs) and retinoid X receptors (RXRs). The RAR family is composed of three genes: RAR alpha, beta, and gamma. The expression of RAR beta gene is spatially and temporally restricted in certain structures in the developing embryo, suggesting that RAR beta could play specific roles during morphogenesis. Four isoforms of the RAR beta gene (beta 1-beta 4) are generated by differential usage of promoters and alternative splicing. It has recently been demonstrated that the RAR beta 2 isoform is dispensable for normal development. To ascertain the function of all RAR beta isoforms in vivo, we have generated a mutation that disrupts all isoforms of the RAR beta gene in the mouse by gene targeting in embryonic stem cells. Mice homozygous for the mutation are viable and fertile with no externally apparent abnormalities. During development, 1/11 RAR beta mutant embryos showed fusion of the ninth and tenth cranial ganglia on both sides of the hindbrain. However, no obvious alterations in the spatial pattern of expression of Hoxb-1, Hoxb-4 and Hoxb-5 were observed in day 9.5 p.c. embryos. The RAR beta null mutation did not alter the pattern or extent of the limb and craniofacial malformations induced by RA excess, suggesting that RAR beta may not be mandatory to mediate the observed teratological effects of RA in these structures. These experiments demonstrate that RAR beta isoforms are not absolutely required for embryonic development and provide additional support to the concept of functional redundancy among members of the RAR family.

Retinoic acid and pattern formation in vertebrates.

Retinoic acid has spectacular teratogenic effects on the development of vertebrate embryos, but whether this has significance for the normal mechanisms of development remains an open question. Recent results from the analyses of Hox gene regulation and the phenotypes of mice mutant for the components of the retinoid signaling pathway suggest that retinoic acid is involved in the genesis of pattern in vivo.

Notch1 is required for the coordinate segmentation of somites.

Members of the Notch family of transmembrane receptors mediate a number of developmental decisions in invertebrates. In order to study Notch function in a vertebrate organism, we have mutated the Notch1 gene of the mouse. Notch1 gene function is required for embryonic survival in the second half of gestation. In the first half of gestation, we have found no effect of the mutation on the normal programs of neurogenesis, myogenesis or apoptosis. We conclude that Notch1 function is not essential for these processes, at least in early postimplantation development. However, we have found that somitogenesis is delayed and disorganized in Notch1 mutant embryos. We propose that Notch1 normally coordinates the process of somitogenesis, and we provide a model of how this might occur.

Positive and negative signals from mesoderm regulate the expression of mouse Otx2 in ectoderm explants.

Otx2, a mouse homolog of the Drosophila orthodenticle gene, is first widely expressed in the epiblast but becomes progressively restricted to the anterior third of the embryo by the headfold stage. This progressive restriction correlates with the anterior migration of mesoderm in the embryo, suggesting that interactions with mesoderm may be involved in setting up the anterior domain of Otx2 expression in vivo. Using explant-recombination assays, we have obtained evidence that a positive signal from anterior mesendoderm is required to stabilize expression of Otx2 in vivo, whereas a negative signal from the later-forming posterior mesendoderm represses Otx2 expression in the posterior part of the embryo. We have also found that exogenous retinoic acid can mimic the effect of this negative signal and reduces the anterior domain of Otx2 expression.