Psychological Adjustment in Apert Syndrome: Parent and Young Person Perspectives.

To date, limited research has been carried out into the psychological impact of having a diagnosis of Apert syndrome (AS) and the life experiences of families living with this condition. The aim of the current study was to explore psychological adjustment to AS from the perspectives of young people, and their parents, with the broader goal of informing care, and support for this population.Four young people (2 male) aged 11 to 15 years and their mothers were interviewed in their homes using a semistructured interview guide and photo-elicitation methods. Transcripts were analyzed using Interpretive Phenomenological Analysis.Three superordinate themes were identified from the data: (1) Acceptance and Adjustment: A Cyclical Journey; (2) A Barrier to Adjustment: Navigating Treatment; and (3) Facilitating Adjustment: Social Support. Families described adjustment as a cyclical process, which was sensitive to change, particularly in the context of ongoing medical treatment. Families also utilized many resources, particularly in the form of social support, to adjust to the challenges of AS and build resilience.The findings of this study have important implications for the implementation of patient-centered care within designated craniofacial treatment centers, which should at a minimum include the provision of reliable information throughout the treatment pathway, additional support from health professionals at key times of transition, and the coordination of support across medical teams, and other key organizations in the child's life.

A Simple Method for Eye Protection During Prone Positioning for Craniofacial Surgery.

Surgery in the prone position risks vision loss due to a number of factors. Craniofacial surgery poses an even greater risk due to the anatomical and physiological makeup of these patients. Here, we describe a novel method of providing protection from direct pressure on the globe during prone positioning for craniofacial procedures and our protocol for improving safety and reducing the risk of postoperative vision loss.

Biallelic Mutation of SETX and Additional Likely "In Cis" SETX Sequence Change in Ataxia with Oculomotor Apraxia Type 2.

Ataxia with oculomotor apraxia type 2 (AOA2) is a slowly progressive, autosomal recessive disease characterized by the triad of ataxia, oculomotor apraxia, and sensorimotor neuropathy. The genetic basis of AOA2 is biallelic mutation of the SETX gene, resulting in reduced or absent senataxin, a DNA/RNA repair protein essential for genomic stability. In this case report, we described a case of AOA2 with two clear pathogenic SETX mutations, one of which is novel. We then discussed two further likely "in cis" SETX sequence changes (previously reported in the literature as pathogenic), and presented the case that they are likely benign polymorphisms.

Can we remove scar and fibrosis from adult human myocardium?

The pathological processes leading to heart failure are characterized by the formation of fibrosis and scar, yet the dynamics of scar production and removal are incompletely understood. Spontaneous disappearance of myocardial collagen is reported in infancy but doubted in adulthood where scar volume constitutes a better prognostic indicator than the conventional parameters of ventricular function. Whilst certain drugs are known to attenuate myocardial fibrosis evidence is emerging that stem cell therapy also has the potential to reduce scar size and improve myocardial viability. Both animal studies and clinical trials support the concept that, as in infancy, cellular processes can be triggered to remove collagen and regenerate injured myocardium. The molecular mechanisms likely involve anti-fibrotic cytokines growth factors and matrix-metalloproteinases. Autologous cardiac, bone-marrow and adipose tissue derived stem cells have each shown efficacy. Specific immune privileged mesenchymal stem cells and genetically modified immunomodulatory progenitor cells may in turn provide an allogenic source for the paracrine effects. Thus autologous and allogenic cells both have the potential through paracrine action to reduce scar volume, boost angiogenesis and improve ventricular morphology. The potential benefit of myocardial cell therapy for routine treatment of heart failure is an area that requires further study.

"Flipped classrooms" in training in maxillofacial surgery: preparation before the traditional didactic lecture?

While virtual learning environments (VLE) can be used in medical education as stand-alone educational interventions, they can also be used in preparation for traditional "face-to-face" training sessions as part of a "flipped classroom" model. We sought to evaluate the introduction of this model in a single module on maxillofacial radiology from a course on trauma skills. Course delegates were randomised into two groups: one was given access to an e-learning resource (test group) and the other attended a traditional didactic lecture (control group). Knowledge and confidence were assessed before and after the course with a 20-question single-best-answer paper and a 10-situation 100mm visual analogue scale (VAS) paper, respectively. All participants were then given free access to the VLE for 30days and were invited to take part in an e-survey. Neither group showed improvements in the single-best-answer scores, but both groups showed comparable improvements in VAS (control: median (range) values improved from 40.8 (17.7-82.5) mm to 62.8 (35.3-88.7) mm, p=0.001; test group: from 47.7 (10.9-58.1) mm to 60.5 (32.4-75.6) mm, p=0.005). Half of the respondents stated that they preferred the "flipped classroom" approach, and 22/22 stated that they would be "likely" or "very likely" to use an e-learning resource with expanded content. The "flipped classroom" approach was well received and there were comparable improvements in confidence. As maxillofacial radiology lends itself to online instruction with its reliance on the recognition of patterns, and problem-based approach to learning, a piloted e-learning resource could be developed in this area.

Apoptosis gene profiling reveals spatio-temporal regulated expression of the p53/Mdm2 pathway during lens development.

Evidence is emerging for apoptosis gene expression in the lens during development. Therefore, here we used a filter array to assess expression of 243 apoptosis-related genes in the developing postnatal mouse lens using (33)P labelled cDNA synthesized from p7 and p14 mouse lenses. We demonstrated that 161 apoptosis-related genes were expressed at levels significantly above background and 20 genes were potentially significantly differentially expressed (P<0.05) by at least 2-fold between p7 and p14. We used RT-PCR to confirm expression of these genes in newborn, p7, p14 and 4 wk mouse lens cDNA samples. Expression of 19/20 of the genes examined was confirmed, while 5 genes (Huntingtin, Mdm2, Dffa, galectin-3 and Mcl-1) were confirmed as differentially regulated between p7 and p14. RT-PCR was also used to examine the expression of the chick homologues of the most-highly expressed and/or potentially differentially regulated genes in chick embryo lenses at E6-E16. The majority of genes expressed in the postnatal mouse lens were also expressed in the chick embryo lens. Western blotting confirmed developmentally regulated expression of Axl and Mcl-1 during mouse lens development and of Mdm2, Mdm4/X and p53 during mouse and chick lens development. Western blotting also revealed the presence of p53 and Mdm4/X splice variants and/or proteolytic cleavage products in the developing lens. Since Mdm2 is a regulator of the tumour suppressor gene p53, we chose to thoroughly investigate the spatio-temporal expression patterns of p53, Mdm2 and the functionally related Mdm4/X in mouse lens development at E12.5-E16.5 using immunocytochemistry. We also examined Mdm2 expression patterns during chick lens development at E6-E16 and Mdm4/X and p53 at E14. Expression of Mdm2, Mdm4/X and p53 was spatio-temporally regulated in various compartments of the developing lens in both mouse and chick, including lens epithelial and lens fibre cells, indicating potential roles for these factors in regulation of lens epithelial cell proliferation and/or lens fibre cell differentiation This study provides a thorough initial analysis of apoptosis gene expression in the postnatal mouse lens and provides a resource for further investigation of the roles in lens development of the apoptosis genes identified. Furthermore, building on the array studies, we present the first spatio-temporal analysis of expression of p53 pathway molecules (p53, Mdm2 and Mdm4/X) in both developing mouse and chick lenses, suggesting a potential role for the p53/Mdm2 pathway in lens development, which merits further functional analysis.

Gene expression profiles during early differentiation of mouse embryonic stem cells.

BACKGROUND: Understanding the mechanisms controlling stem cell differentiation is the key to future advances in tissue and organ regeneration. Embryonic stem (ES) cell differentiation can be triggered by embryoid body (EB) formation, which involves ES cell aggregation in suspension. EB growth in the absence of leukaemia inhibitory factor (LIF) leads EBs to mimic early embryonic development, giving rise to markers representative of endoderm, mesoderm and ectoderm. Here, we have used microarrays to investigate differences in gene expression between 3 undifferentiated ES cell lines, and also between undifferentiated ES cells and Day 1-4 EBs. RESULTS: An initial array study identified 4 gene expression changes between 3 undifferentiated ES cell lines. Tissue culture conditions for ES differentiation were then optimized to give the maximum range of gene expression and growth. -Undifferentiated ES cells and EBs cultured with and without LIF at each day for 4 days were subjected to microarray analysis. -Differential expression of 23 genes was identified. 13 of these were also differentially regulated in a separate array comparison between undifferentiated ES cells and compartments of very early embryos. A high degree of inter-replicate variability was noted when confirming array results. Using a panel of marker genes, RNA amplification and RT-PCR, we examined expression pattern variation between individual -D4-Lif EBs. We found that individual EBs selected from the same dish were highly variable in gene expression profile. CONCLUSION: ES cell lines derived from different mouse strains and carrying different genetic modifications are almost invariant in gene expression profile under conditions used to maintain pluripotency. Tissue culture conditions that give the widest range of gene expression and maximise EB growth involve the use of 20% serum and starting cell numbers of 1000 per EB. 23 genes of importance to early development have been identified; more than half of these are also identified using similar studies, thus validating our results. EBs cultured in the same dish vary widely in terms of their gene expression (and hence, undoubtedly, in their future differentiation potential). This may explain some of the inherent variability in differentiation protocols that use EBs.

Developmentally regulated expression of hemoglobin subunits in avascular tissues.

We investigated the spatio-temporal profile of hemoglobin subunit expression in developing avascular tissues. Significant up-regulation of hemoglobin subunits was identified in microarray experiments comparing blastocyst inner cell masses with undifferentiated embryonic stem (ES) cells. Hemoglobin expression changes were confirmed using embryoid bodies (derived from in vitro differentiation of ES cells) to model very early development at pre-vascular stages of embryogenesis; i.e. prior to hematopoiesis. We also demonstrate, using RT-PCR, Western blotting and immunocytochemistry, expression of adult and fetal mouse hemoglobin subunits in the avascular ocular lens at various stages of development and maturation. Hemoglobin proteins were expressed in lens epithelial cells (cytoplasmic) and cortical lens fiber cells (nuclear and cell-surface-associated); however, a sensitive heme assay demonstrated negligible levels of heme in the developing lens postnatally. Hemoglobin expression was also observed in the developing eye in corneal endothelium and retinal ganglion cells. Gut sections showed, in addition to erythrocytes, hemoglobin protein staining in rare, individual villus epithelial cells. These results suggest a paradigm shift: hemoglobin subunits are expressed in the avascular lens and cornea and in pre-hematopoietic embryos. It is likely, therefore, that hemoglobin subunits have novel developmental roles; the absence of the heme group from the lens would indicate that at least some of these functions may be independent of oxygen metabolism. The pattern of expression of hemoglobin subunits in the perinuclear region during lens fiber cell differentiation, when denucleation is taking place, may indicate involvement in the apoptosis-like signaling processes occurring in differentiating lens fiber cells.

Optimization of minuscule samples for use with cDNA microarrays.

The recent advent of microarray technology and RNA amplification allows us to compare the expression profiles of thousands of genes from small amounts of tissue or cells. We have compared and contrasted various methods of RNA preparation, RNA amplification, target labelling and array analysis in order to achieve a streamlined protocol for microarraying small samples. We have concluded that usage of the NIA 15K cDNA array set, in combination with RNA extraction using the Mini RNA Isolation kit (Zymo), amplification with the RiboAmp kit (Arcturus), followed by indirect labelling via the Atlas PowerScript Fluorescent Labelling kit (using a modified protocol), is optimal with a material derived from either very early stage mouse embryos or individually picked embryonic stem cell colonies. Normalisation using the analysis package Limma (Bioconductor) with data normalisation by print tip Loess, using the "normexp" function with an offset of 50 for background adjustment, and incorporating A-quantile between array normalisation was best with our results. Furthermore, RT-PCR confirmation of array results is achievable without amplification, thereby controlling for amplification bias. These methods will be of great utility in mapping the transcriptome of embryonic and other small samples.

Osteopenia in Sparc (osteonectin)-deficient mice: characterization of phenotypic determinants of femoral strength and changes in gene expression.

Sparc null mutants have been generated independently via targeted mutations in exons 4 and 6. Previous studies have identified low-turnover osteopenia in the 129Sv/C57BL/6 exon 4 knockout. Since both Sparc null mutations result in complete absence of Sparc protein, similar phenotypic outcomes are likely. However, genetic background (strain) and/or linkage disequilibrium effects can influence phenotype. Different inactivating mutations should be tested in various mouse strains; similar phenotypic outcomes can then confidently be assigned to the mutated gene. We have evaluated the bone phenotype in the 129Sv/EvSparc(tm1cam) exon 6 knockout at 4 and 9 mo, using physical measurement, mechanical strength tests, and DXA scanning. We have also quantified bone marrow adiposity and circulating leptin levels to assess adipose tissue metabolism. 129Sv/EvSparc(tm1cam) null mice show decreased bone mineral density and bone mineral content and increased mechanical fragility of bone, in line with previous studies. Differences were also noted. Increased body weight and levels of bone marrow adiposity but decreased circulating leptin concentrations were identified at 4, but not 9 mo, and 129Sv/EvSparc(tm1cam) null mice also had shorter femurs. Molecular phenotyping was carried out using mouse HGMP NIA microarrays with cortical femur samples at various ages, using semiquantitative RT-PCR validation. We identified 429 genes highly expressed in normal bone. Six genes (Sparc, Zfp162, Bysl, E2F4, two ESTs) are differentially regulated in 129Sv/EvSparc(tm1cam) cortical femur vs. 129Sv/Ev controls. We confirm low-turnover osteopenia as a feature of the Sparc null phenotype, identifying the usefulness of this mouse as a model for human osteoporosis.

Altered collagen in tartrate-resistant acid phosphatase (TRAP)-deficient mice: a role for TRAP in bone collagen metabolism.

Tartrate-resistant acid phosphatase (TRAP) is an iron-containing protein that is highly expressed by osteoclasts, macrophages, and dendritic cells. The enzyme is secreted by osteoclasts during bone resorption, and serum TRAP activity correlates with resorptive activity in disorders of bone metabolism. TRAP is essential for normal skeletal development. In knockout mice lacking TRAP, bone shape and modeling is altered with increased mineral density. Here, we report the effect of TRAP on the biochemical and biomechanical properties of collagen, the major protein constituting the bone matrix, using these mice. Femurs from TRAP-/- and wild-type mice were used in these studies. The biomechanical properties were investigated using a three-point bending technique. Collagen synthesis was determined by measuring cross-link content using high-performance liquid chromatography and amino acid analysis. Collagen degradation was determined by measuring matrix metalloproteinase-2 (MMP-2) activity. The rates of collagen synthesis and degradation were significantly greater in bones from TRAP-/- mice compared with wild type. At 8 weeks, there was an increase in the intermediate cross-links but no significant difference in animals aged 6 months. There was a significant increase in mature cross-links at both ages. A significant increase in MMP-2 production both pro and active was observed. A significant increase in ultimate stress and Young's modulus of elasticity was needed to fracture the bones from mice deficient in TRAP. We conclude that both synthesis as well as degradation of collagen are increased when TRAP is absent in mice at 8 weeks and 6 months of age, showing that TRAP has an important role in the metabolism of collagen.

Deficiency of SPARC suppresses intestinal tumorigenesis in APCMin/+ mice.

BACKGROUND AND AIMS: SPARC (secreted protein acidic, rich in cysteine) is a matricellular protein that has been found to be activated in a number of human cancers. More recently, it has been shown to be upregulated in human gastric and colorectal cancer. We therefore wished to address the functional importance of SPARC upregulation to intestinal tumorigenesis in vivo. METHODS: SPARC upregulation was determined in intestinal adenomas of tumour-prone Apc(Min/+) mice at both the RNA and the protein level. To determine the functional importance of SPARC for intestinal tumorigenesis we then intercrossed Sparc knockout mice with Apc(Min/+) mice (n = 20). Intestinal enterocyte migration was examined using bromodeoxyuridine labelling studies. RESULTS: Levels of murine Sparc and several related proteins were upregulated in adenomas arising in Apc(Min/+) mice. A deficiency of Sparc strongly suppressed adenoma formation in Apc(Min/+) mice (p>or=0.0001). Importantly, a deficiency of Sparc also accelerated enterocyte migration (p = 0.01), as perturbed slow epithelial migration may underpin adenoma formation in the intestine. CONCLUSIONS: These data implicate Sparc in both cell migration and tumour formation, and identify Sparc as a potential therapeutic target for colorectal cancer.

TRACP Influences Th1 pathways by affecting dendritic cell function.

UNLABELLED: TRACP, a marker of osteoclasts, is also expressed by cells of the immune system. We identified a novel function for TRACP in the dendritic cell. DCs from TRACP knockout mice have impaired maturation and trigger reduced Th1 responses in vivo. We postulate that TRACP has an important role in the presentation of antigens to T cells. INTRODUCTION: TRACP is highly expressed by osteoclasts, activated macrophages, and dendritic cells (DCs). Knockout mice lacking TRACP have an intrinsic defect in osteoclastic resorption and macrophages that display abnormal immunomodulatory responses and cytokine secretion profiles. Our aim in this study was to investigate the significance of TRACP in the inductive phase of the immune response by examining dendritic cells from TRACP(-/-) mice. MATERIALS AND METHODS: Maturational state and function of leukocyte subsets in mice was assessed by flow cytometry. The ability of the immune system to respond to nonspecific activation and to specific antigen was assessed by delayed type hypersensitivity and the presence of isotype-specific serum antibody in vivo and T-cell proliferation and cytokine production in vitro. RESULTS: The ability of lipopolysaccharide (LPS) to upregulate MHC II and CD80 in DCs from TRACP(-/-) mice was reduced compared with wildtype mice, although production of IL-10 by DCs from TRACP-deficient animals was increased. T- and B-cell responses not involving antigen presentation (anti-CD3, TNP-ficoll) were normal in TRACP(-/-) mice, but responses to T-dependent antigens were impaired. Specifically, TRACP(-/-) mice had defective delayed hypersensitivity responses to picryl chloride and reduced proliferative responses to ovalbumin compared with wildtype mice. In response to ovalbumin, but not anti-CD3, T cells from TRACP(-/-) mice produced less interferon-gamma (IFN-gamma), but there was no difference in IL-4 production: TRACP(-/-) mice also produced less ovalbumin (OVA)-specific IgG2a after immunization. CONCLUSIONS: The finding that DCs from TRACP(-/-) mice have impaired maturation and defective Th1 responses shows that TRACP is important for polarizing responses in naïve T cells to antigen-presented dendritic cells.

Gene expression changes during cataract progression in Sparc null mice: differential regulation of mouse globins in the lens.

PURPOSE: Sparc/osteonectin is a hydroxyapatite, calcium and, collagen binding protein, implicated in tissue morphogenesis, cell proliferation, and repair. Sparc null mice develop sub-cortical posterior cataract with eventual rupture of the lens. We wished to correlate genotype with phenotype in these mice via analysis of gene expression pattern changes leading to disease. METHODS: We carried out microarray analysis of adult lenses from Sparctm1cam knockout mice on two strain backgrounds of varying phenotypic severity at two time points, 4 and 9 months. Labelled cDNA from Sparctm1cam knockout and age, strain, and sex matched control lenses was hybridized with HGMP NIA 15,000 clone set arrays. Differential expression was confirmed using semi-quantitative RT-PCR. RESULTS: We have confirmed differential expression of 54 genes. Most notably, 5 of the mouse globin genes, Hbb-b1, Hbb-b2, Hba, Hba-x, and Hbb-y and an EST, C79876, were significantly downregulated in 9-month old Sparc null mice from two genetic backgrounds at different stages of disease. Another downregulated gene, EraF, is involved in folding of globin proteins. Immune response components, including various members of the complement cascade, were upregulated in lenses with advanced cataract. CONCLUSIONS: Five mouse globins show persistent downregulation as a result of Sparc loss. We speculate as to possible roles of this phenomenon on pathogenesis of cataract in these mice. Other confirmed genes allow extension of previous models of cataract development in Sparc null mice.

Expression profiling and gene discovery in the mouse lens.

PURPOSE: Defects in the development and physiology of the lens can result in cataracts (opacification of the lens), which are currently treatable only by surgical removal. The lens is also an excellent system for understanding fundamental biological processes such as cellular differentiation and ageing. Here, microarrays have been used to gain insights into global patterns of gene expression in the mouse lens. Lens gene expression compared to non-lens tissues has been investigated in order to identify genes preferentially expressed in the lens and lenses of different ages have been compared to identify differentially regulated genes. METHODS: Genes expressed in the lens were identified using mouse GeneFilters microarrays (GF400; ResGen). Each array comprises 5,184 mouse cDNAs representing sequence-verified known genes and uncharacterized ESTs spotted onto a nylon membrane. Target RNA (33P labeled) from lens and non-lens samples was hybridized to the arrays. The proportion of genes involved in various biological processes was investigated using Onto-Express to search for GeneOntology terms associated with them. Differential gene expression was investigated using K-means clustering analysis. Expression of known and uncharacterized genes selected from the arrays was investigated further using semi-quantitative RT-PCR. RESULTS: 1,668 genes were expressed in one or more of newborn, 7 day old, and adult mouse lenses at levels significantly above background. Raw data and bioinformatics data relating to these genes have been published herein. There were 543 (33%) known genes, 124 (7%) had some similarity to known genes, 400 (24%) were functionally uncharacterized, and the remaining 601 (36%) genes were novel (matching only existing ESTs). Onto-Express identified genes involved in various biological processes including several categories containing greater numbers of genes than would be expected by chance, such as transcription regulation and G-protein coupled receptor signaling genes. Semi-quantitative RT-PCR confirmed preferential expression of several genes in the lens compared to non-lens tissues and genes exhibiting significantly higher expression in the 7 day lens compared to either adult or newborn lenses. Expression in the lens of 10 genes involved in apoptosis was also confirmed and, intriguingly, expression of hemoglobin isoforms (Hba-a1, Hba-X, Hbb-b1, Hbb-b2, and Hbb-Y) was confirmed using isotype specific primers. Finally, we confirmed the expression in the lens of all additional novel, uncharacterized and known genes tested. CONCLUSIONS: The present work has provided insights into global patterns of gene expression in the lens and the expression of a significant number of genes has been confirmed using semi-quantitative RT-PCR. Genes preferentially expressed in the lens compared to non-lens tissues have been identified as well as genes differentially expressed between lenses at different ages. Gene expression profiling and gene discovery in the lens are essential prerequisites for future functional studies aimed at gaining insights into the potential roles of these genes in lens development, maturation, physiology, and pathogenesis (using targeted mutagenesis in mice, for instance).

Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Since approximately 5% of all mutant CF alleles are stop mutations, it can be calculated that approximately 10% of CF patients carry a premature stop mutation in at least one copy of the CFTR gene. Certain ethnic groups, such as the Ashkenazi Jewish population, carry a much higher percentage of CF stop mutations. Consequently, a therapeutic strategy aimed at suppressing this class of mutation would be highly desirable for the treatment of this common genetic disease. We have shown previously that aminoglycoside antibiotics can suppress premature stop mutations in the CFTR gene in a bronchial epithelial cell line [Nat Med (1997) 3:1280]. To address whether aminoglycosides can suppress a CFTR premature stop mutation in an animal model, we constructed a transgenic mouse with a null mutation in the endogenous CFTR locus (Cftr-/-) that also expressed a human CFTR-G542X cDNA under control of the intestinal fatty acid binding protein promoter. We then investigated whether the daily administration of the aminoglycoside antibiotics gentamicin or tobramycin could restore the expression of a detectable level of CFTR protein. Immunofluorescence staining of intestinal tissues from Cftr-/- hCFTR-G542X mice revealed that gentamicin treatment resulted in the appearance of hCFTR protein at the apical surface of the glands of treated mice. Weaker staining was also observed in the intestinal glands following tobramycin treatment. Short-circuit current measurements made on intestinal tissues from these mice demonstrated that a significant number of positive cAMP-stimulated transepithelial chloride current measurements could be observed following gentamicin treatment (P=0.008) and a near significant number following tobramycin treatment (P=0.052). When taken together, these results indicate that gentamicin, and to a lesser extent tobramycin, can restore the synthesis of functional hCFTR protein by suppressing the hCFTR-G542X premature stop mutation in vivo.