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In 2011 NHS England commissioned a new national specialist MDT service for patients and families affected by Stickler syndrome. The Stickler syndromes form part of the spectrum of inherited vitreoretinopathies and are the most common cause of retinal detachment in childhood and the most common cause of familial retinal detachment. Now in its 10th year, the Stickler Highly Specialised Service (HSS) has assessed 1673 patients from 785 families. Using a combination of accurate phenotyping and molecular genetic analysis it is possible to identify the underlying genetic mutation in over 95% of cases including those with deep intronic mutations likely to be missed by conventional exome panel analysis and which require whole gene sequencing and supplementary functional analysis to confirm pathogenicity. The vast majority that presents to ophthalmologists will be from one of three autosomal dominant sub-groups with a high associated risk of retinal detachment but the diagnosis is often overlooked, especially in adults. In contrast to many other blinding retinal conditions, blindness through giant retinal tear detachment particularly in children is largely preventable provided these high-risk groups are identified and appropriate evidence-based prophylaxis offered. This article summarises ten selected briefcase histories from the national dataset with key learning points from each.

Fluvial erosion of cohesive soil is mediated by interactions between soil physical, biological, and chemical characteristics such as soil aggregate stability and extracellular polymeric substances (EPS). While labile organic matter (OM) stimulates microbial EPS production and significantly improves soil aggregate stability in agricultural soils, these interactions remain unexplored in streambank soils. The study goal was to quantify the impact of OM on aggregate stability, EPS, and fluvial erosion rates of cohesive streambank soil. Increasing amounts of 1‐mm sieved dry grass were incorporated at rates of 0, 1, and 4 g per 100 g of 2‐mm sieved silt‐loam soil (treatments T0, T1, and T4, respectively). Samples (eight replicates per treatment) were matured in a greenhouse for 50 days prior to flume erosion testing. EPS carbohydrates were significantly (p< 0.05) lower in T1 (324 ± 63 μg/g) compared to T0 (388 ± 37 μg/g) and T4 (376 ± 44 μg/g). EPS proteins were significantly higher in T1 (194 ± 15 μg/g) and T4 (223 ± 61 μg/g) compared to T0 (101 ± 20 μg/g) and positively correlated with mean weight diameter (MWD), a measure of soil stability against slaking. MWD was 16% and over 100% higher for T1 and T4, respectively, than for T0. Similarly, the average soil erodibility coefficient of T1 and T4 was 25% and 61% lower than the erodibility of T0; however, only the reduction for T4 was significant. The data presented here underscore the important role labile OM plays in improving soil physical stability and increasing the resistance of cohesive soil to fluvial erosion of streambanks. Floods can cause streambank erosion; however, organic matter can potentially increase the resistance of streambank soils to erosion by water. The organic material stimulates the release of “sticky” organic compounds by microorganisms called extracellular polymeric substances (EPS). While EPS have improved soil stability in agricultural soils, to date, no work has been done investigating the interactions between OM, EPS, and erosion resistance in streambank soils. Therefore, we aimed to determine if soils amended with OM would be more stable and resistant to erosion by water via an increase in EPS. Study results support our hypothesis that the added OM stimulated the production of EPS, leading to improved soil stability and reduced soil erosion rates. These results underscore the important role “fresh” organic matter plays in protecting streambanks from erosion, which is of particular importance for the management of vegetation along stream channels and for streambank stabilization. Soil aggregate stability and the protein component of extracellular polymeric substances were positively correlatedThe mean weight diameter of soils amended with labile organic matter increased by 16% to over 100%, compared to unamended soilsErodibility of organic‐amended soils was 25%–61% lower than for unamended soils Soil aggregate stability and the protein component of extracellular polymeric substances were positively correlated The mean weight diameter of soils amended with labile organic matter increased by 16% to over 100%, compared to unamended soils Erodibility of organic‐amended soils was 25%–61% lower than for unamended soils

Objective. We reviewed the literature focused on socioeconomic influences on teen childbearing and suggested directions for future research and practice related to this important indicator of teen sexual health.Methods. We conducted an electronic search of Medline, ERIC, PsychLit, and Sociological Abstracts databases for articles published from January 1995 to November 2011. Selected articles from peer-reviewed journals included original quantitative analyses addressing socioeconomic influences on first birth among teen women in the U.S. Articles were abstracted for key information, ranked for quality according to the U.S. Preventive Services Task Force guidelines, assessed for bias, and synthesized.Results. We selected articles with a range of observational study designs. Risk for bias varied across studies. All 12 studies that considered socioeconomic factors as influences on teen childbearing (vs. moderators or mediators of other effects) reported at least one statistically significant association relating low socioeconomic status, underemployment, low income, low education levels, neighborhood disadvantage, neighborhood physical disorder, or neighborhood-level income inequality to teen birth. Few reports included any associations contradicting this pattern.Conclusions. This review suggests that unfavorable socioeconomic conditions experienced at the community and family levels contribute to the high teen birth rate in the U.S. Future research into social determinants of sexual health should include multiple levels of measurement whenever possible Root causes of teen childbearing should be evaluated in various populations and contexts Interventions that address socioeconomic influences at multiple levels could positively affect large numbers of teens and help eliminate disparities in teen childbearing.

Background This study investigates the similarities and differences between epiretinal membranes in four clinically distinct types of vitreomaculopathy. We propose a hypothesis on the origin of the predominant cell type and its potential role in causing these conditions. Methods Epiretinal membranes (ERMs) surgically removed from a prospective, consecutive series of vitrectomies for macular pucker associated with an untreated peripheral horseshoe tear (MP), cellophane maculopathy (CM), stage 4 macular hole (MH) and vitreomacular traction syndrome (VMT) were examined by light microscopy and by immunocytochemistry (ICC) using antibodies marking type IV collagen, type II collagen, glial fibrillary acidic protein (GFAP), and low- and high-molecular-weight cytokeratin (MNF116). These specimens were compared with post-mortem control eyes with and without physiological posterior vitreous detachment (PVD). Light microscopy was carried out on 5-μm-thick sections cut from formalin-fixed, paraffin-embedded tissue blocks. Appropriate autoclave or enzyme pre-digestion steps were deployed to retrieve antigens for ICC. No patient had undergone previous vitreoretinal surgery or peripheral retinopexy. Results From a series of 38 patients, (13 CM, 8 MP, 16 MH and 1 VMT) a total of 20 specimens contained sufficient tissue for histology and immunocytochemistry. All specimens contained portions of inner limiting membrane (ILM) coated by GFAP-positive cells. Specimens from patients with MP and CM exhibited hyperconvolution of the ILM, which was not found in the specimens from patients with MH or VMT or in the control eyes. Hyperconvolution was associated with increased glial cell density, GFAP staining intensity and duplication of ILM basement membrane. Three cases of ERMs from the MP group contained, in addition, cytokeratin-positive cells. In the control group; post-mortem eyes with PVDs showed patchy staining of the posterior hyaloid membrane for GFAP and type 4 collagen. Post-mortem eyes with attached gel showed weak positivity of the ILM for type 4 collagen, and a monolayer of GFAP-positive cells lined the vitreous aspect of the ILM. Conclusions These results indicate that glial cells are fundamentally important in the formation of ERMs found in this group of vitreomaculopathies. The hyperconvolution and duplication of the ILM in CM and MP were striking and distinctive features and suggest a mechanism by which these membranes exert tractional forces on the retina. Post-mortem control eyes contained a similar (but more dispersed) population of GFAP-positive cells in the region of the ILM, suggesting the primary aetiology for CM and MP may originate within the ILM. ERMs from MP cases may, in addition, contain cytokeratin-positive cells, of probable RPE origin.

Ameloblast-specific amelogenin gene expression is spatiotemporally regulated during tooth development. In a previous study, the CCAAT/enhancer-binding protein alpha (C/EBPalpha) was identified as a transcriptional activator of the mouse amelogenin gene in a cell type-specific manner. Here, Msx2 is shown to repress the promoter activity of amelogenin-promoter reporter constructs independent of its intrinsic DNA binding activity. In transient cotransfection assays, Msx2 and C/EBPalpha antagonize each other in regulating the expression of the mouse amelogenin gene. Electrophoresis mobility shift assays demonstrate that Msx2 interferes with the binding of C/EBPalpha to its cognate site in the mouse amelogenin minimal promoter, although Msx2 itself does not bind to the same promoter fragment. Protein-protein interaction between Msx2 and C/EBPalpha is identified with co-immunoprecipitation analyses. Functional antagonism between Msx2 and C/EBPalpha is also observed on the stably transfected 2.2-kilobase mouse amelogenin promoter in ameloblast-like LS8 cells. Furthermore, the carboxyl-terminal residues 183-267 of Msx2 are required for protein-protein interaction, whereas the amino-terminal residues 2-97 of Msx2 play a less critical role. Among three family members tested (C/EBPalpha, -beta, and -gamma), Msx2 preferentially interacts with C/EBPalpha. Taken together, these data indicate that protein-protein interaction rather than competition for overlapping binding sites results in the functional antagonism between Msx2 and C/EBPalpha in regulating the mouse amelogenin gene expression.

Amelogenin expression is ameloblast-specific and developmentally regulated at the temporal and spatial levels. In a previous transgenic mouse analysis, the expression pattern of the endogenous amelogenin gene was recapitulated by a reporter gene driven by a 2. 2-kilobase mouse amelogenin proximal promoter. To understand the molecular mechanisms underlying the spatiotemporal expression of the amelogenin gene during odontogenesis, the mouse amelogenin promoter was systematically analyzed in mouse ameloblast-like LS8 cells. Deletion analysis identified a minimal promoter (-70/+52) containing a CCAAT/enhancer-binding protein (C/EBP)-binding site upstream of the TATA box. In transient transfection assays, C/EBPalpha up-regulated the promoter activity in a dose-dependent manner. The C/EBP-binding site was necessary for both C/EBPalpha-mediated transactivation and basal promoter activity. Electrophoresis mobility shift assays demonstrated that C/EBPalpha bound to its cognate site in the amelogenin promoter and that the binding was specific. Endogenous C/EBPalpha was detected in LS8 cells, and overexpression of exogenous C/EBPalpha in LS8 cells was able to increase the expression level of the endogenous amelogenin protein. The activity of the amelogenin promoter in rat parotid Pa-4 cells and Madin-Darby canine kidney cells was minimal, ranging from 20 to 30% of the activity in ameloblast-like cells. Transient transfection experiments showed that C/EBPalpha transactivated the mouse amelogenin reporter gene in Pa-4 cells, but not in Madin-Darby canine kidney cells. Taken together, these data indicate that C/EBPalpha is a bona fide transcriptional activator of the mouse amelogenin gene in a cell type-specific manner.

The molecular mechanisms directing the highly restricted expression pattern of murine ameloblastin were characterized by cloning and functional analysis of the ameloblastin promoter. The transcription start site, mapped by primer extension, was located 19 base pairs (bp) 5' of the published cDNA. The promoter was analyzed in a mouse ameloblast-like cell line (LS8) and was compared with promoter activity in primary gingival fibroblasts and pulp fibroblasts. Sequential 5'-deletion mutants encompassing DNA sequences from -1616 to -781 bp exhibited high promoter activity in LS8 cells, whereas the promoter activity decreased to 50% of the full-length construct in the -781- and -477-bp regions. The -217-bp promoter region regained promoter activity that approached the activity of the full-length promoter construct, suggesting that both positive and negative cis-acting regions may be involved in ameloblastin transcriptional regulation. Activity of the ameloblastin promoter in gingival and pulp fibroblasts was minimal and ranged from 8 to 30% of the activity in ameloblast-like cells. Several DNA-protein complexes were formed between functionally important promoter fragments and nuclear extracts from LS8 cells. The inactivity of promoter constructs in pulp and gingival fibroblasts as well as the absence of similar DNA-protein complexes from these cells suggest that regulatory regions of the murine ameloblastin promoter may function in a cell-specific manner.

Tooth enamel biomineralization is mediated by enamel proteins synthesized by ameloblast cells. Two classes of proteins have been described: enamelins and amelogenins. In lower vertebrates the absence of amelogenins is believed to give rise to aprismatic enamel; however, rabbit teeth, which apparently do not synthesize amelogenins, form prismatic enamel. The present study was designed to characterize the enamel proteins present in rabbit tooth organs and to gain an insight into the process of biomineralization. Rabbit enamel extracellular-matrix proteins were isolated and characterized during sequential stages of rabbit tooth organogenesis. The biosynthesis of enamel proteins was analysed by metabolic ‘pulse-chase’ experiments as well as mRNA-translation studies in cell-free systems. Our results indicated that rabbit enamel extracellular matrix contains ‘amelogenin-like’ proteins. However, these proteins are not synthesized as typical amelogenins, as in other mammalian species, thus suggesting that they are the processing products of higher-molecular-mass precursors. An N-terminal amino acid sequence of 29 residues, considered characteristic of mammalian amelogenins, was present in the rabbit ‘amelogenin-like’ proteins. By using anti-peptide antibodies to this region, similar epitopes were detected in all nascent enamel proteins, including enamelins. These studies suggest that the N-terminal sequence might be characteristic of all enamel proteins, not only amelogenins.

There is increasing evidence that autocrine- and paracrine-acting growth factors participate in cell and tissue development, maintenance, and renewal. Recent advances in histochemical techniques have facilitated the localization of growth factor messenger RNAs or polypeptides in tissue sections. However, the spatial relationships between the sites of growth factor transcription, translation, and post-translational processing to functional bioactive peptides have been difficult to correlate because each method of detection requires separate tissue sections. We undertook the simultaneous detection of epidermal growth factor (EGF) transcripts and EGF epitopes by combining immunohistochemistry methods with in situ hybridization. Adult mouse submandibular gland was chosen as a representative model because it contains sites of EGF biosynthesis which may participate in mediating the development, maintenance, and renewal of the organ through autocrine or paracrine mechanism(s). Granular duct (GD) cells demonstrated the presence of both EGF transcripts and EGF peptides. In contrast, the interstitial cells lying adjacent to glandular epithelium also contained relatively high levels of EGF transcripts, although no mature EGF peptides were detected. The experimental approach of co-localization and the resulting data indicate previously unreported sites of EGF transcription in glandular interstitial cells, which may provide molecular information required for the morphogenesis and differentiation of adjacent glandular epithelium.

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

Summary: The interpretation of recent experimental evidence has lead to several discussions at international meetings which have included the suggestion that enamelins are not tissue-specific gene products but rather are similar to, or are identical to, albumin. Further complicating the issue has been the proposal by several investigators that albumin participates in the organization of hard tissues. The experimental strategy described in this study was to hybridize the mouse albumin gene cDNA clone, palb-3, to a Northern blot of secretory phase mouse mandibular first molar RNAs. This approach would permit the evaluation of the potential transcription of the albumin gene by differentiated ameloblasts as well as other cells of the odontogenic organ. The results of this approach indicated that odontogenic cells did not transcribe the albumin gene. Albumin polypeptides, therefore, cannot be synthesized by odontogenic cells and cannot be identical to odontogenic tissue-specific gene products. If albumin is incorporated into developing enamel matrix wherein it participates in undefined roles during enamel matrix organization, it cannot arise as a biosynthetic product of the odontogenic organ.

Mutations associated with genes of the EGF superfamily are implicated in facial malformations arising from abnormal development of the first branchial arch. EGF and EGF receptor (EGFr) transcripts are expressed in the mouse embryonic first branchial arch and derivatives from E9 through E15. EGF transcripts are localized to ectomesenchymal cells associated with precartilage, cartilage, bone and tooth-forming cells. EGF and EGFr proteins co-localize to the same cells suggesting an autocrine regulation. To test whether EGF effects the timing and positional information required for Meckel's cartilage (MC) and tooth development, we cultured E10 mandibular explants in serumless, chemically defined medium with either antisense or sense EGF oligodeoxynucleotides. Antisense inhibition of EGF expression produces bilaterally symmetrical Fusilli-form dysmorphogenesis of MC and decreases tooth bud size; these effects are reversed by the addition of exogenous EGF to the culture medium. Tyrphostin RG 50864, which inhibits EGF receptor kinase activity, inhibits EGF stimulation of tyrosine phosphorylation in a concentration-dependent manner and severely retards mandibular development yet increases tooth size. These findings support the hypothesis that endogenous EGF and EGF-like proteins provide signalling to regulate the size and shape both of cartilage and tooth formation during craniofacial morphogenesis.

Summary: To experimentally examine the participation of amelogenins in controlled mineral-phase maturation of mammalian enamel, the identification of the individual proteins and their corresponding gene(s) is required. For this purpose, cDNAs were constructed from polyadenylated RNA from 2-day postnatal murine teeth, molecularly cloned into lambda-gt11 expression vectors and transfected intoE. coli. The cDNA library was screened for amelogenin gene(s) by using either antibody or nucleic acid probes. An amelogenin cDNA clone encoding 79 carboxy-terminal amino acid residues and 100 nucleotides of the 3′ noncoding sequence was demonstrated to contain a major antigenic site for amelogenin protein by immunostaining of specific amelogenin proteins from total extracted enamel protein blots using clonal epitope selected antibody. This is the first report linking amelogenin epitope(s) to a defined DNA sequence, and consequently a defined portion of the amino acid sequence for amelogenins. Secondary structure analysis, based on the relative average linear hydropathy of the amino acid sequence of amelogenin, predicted epitopes in the amino terminus of the molecule rather than the carboxy terminus. Our present data suggest that the carboxy terminus of the amelogenins is sufficiently externalized to be an antigenic domain. These data may be useful in subsequent structural analysis of amelogenin proteins and enhancing our understanding of their physicochemical participation in biomineralization.

The coordinate growth of the brain and skull is achieved through a series of interactions between the developing brain, the growing bones of the skull, and the fibrous joints, or sutures, that unite the bones. These interactions couple the expansion of the brain to the growth of the bony plates at the sutures. Craniosynostosis, the premature fusion of the bones of the skull, is a common birth defect (1 in 3000 live births) that disrupts coordinate growth and often results in profoundly abnormal skull shape. Individuals affected with Boston-type craniosynostosis, an autosomal dominant disorder, bear a mutated copy of MSX2, a homeobox gene thought to function in tissue interactions. Here we show that expression of the mouse counterpart of this mutant gene in the developing skulls of transgenic mice causes craniosynostosis and ectopic cranial bone. These mice provide a transgenic model of craniosynostosis as well as a point of entry into the molecular mechanisms that coordinate the growth of the brain and skull.

Understanding the cellular and molecular events that regulate the formation of enamel is a major driving force in efforts to characterize critical events during amelogenesis. It is anticipated that through such an understanding, improvements in prevention, diagnosis and treatment-intervention into heritable and acquired diseases of enamel could be achieved. While knowledge of the precise role of an enamel-specific protein in directing the formation of inorganic crystallites remains refractory, progress has been made with other aspects of amelogenesis that can be brought to bear on the subject One such area of progress has been with the identification of an ameloblast-lineage specific amelogenin gene promoter. This promoter can be used to direct the expression of enamel-specific proteins, as well as the expression of proteins foreign to amelogenesis, into the enamel extracellular matrix where their effect on biomineralization can be ascertained in a prospective manner. The resulting enamel from such animals can be examined by morphologic and biochemical modalities in order to identify the effect of the transgene protein on enamel crystallite formation and subsequent biomineralization. This manuscript outlines such a strategy with the potential for enhancing our understanding of amelogenesis.

The determination of the biochemical phenotype of tooth epithelium requires specification by the dental mesenchyme. This is a general feature of epithelial-mesenchymal interaction in a number of different epidermal organ systems (e.g., salivary gland, mammary gland, feather, skin, and hair morphogenesis). To investigate these developmental processes, we have identified a cDNA clone representing the major group of gene products associated with enamel extracellular matrix formation. The mRNAs for mouse amelogenins, representing approximately equal to 90% of the total enamel proteins, have been isolated and partially characterized by specific immunoprecipitation. The poly(A)-containing RNAs were used for the synthesis and cloning of the mouse amelogenin cDNA. Recombinant plasmids containing amelogenin cDNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analyses. A cloned sequence was used to identify the expression of amelogenins during tooth development. The mouse cDNA sequence hybridized to genomic mouse and human DNAs. This amelogenin cDNA probe now enables molecular investigations of a number of classical problems in developmental biology.

Isolated motor third, fourth or sixth nerve palsies are a well recognized complication following botulinum toxin injection for hemifacial spasm or essential blepharospasm. A case of hemifacial spasm secondary to giant aneurysm of the internal carotid artery is reported, stressing the importance of further investigation if such neurological sequelae develop.