Top 10 priorities for future infertility research: an international consensus development study.

Study Question: Can the priorities for future research in infertility be identified?

Summary Answer: The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction, and ethics, access, and organization of care for people with fertility problems were identified.

What Is Known Already: Many fundamental questions regarding the prevention, management, and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems.

Top 10 priorities for future infertility research: an international consensus development study† ‡.

Study Question: Can the priorities for future research in infertility be identified?

Summary Answer: The top 10 research priorities for the four areas of male infertility, female and unexplained infertility, medically assisted reproduction and ethics, access and organization of care for people with fertility problems were identified.

What Is Known Already: Many fundamental questions regarding the prevention, management and consequences of infertility remain unanswered. This is a barrier to improving the care received by those people with fertility problems.

Elongated growth of octacalcium phosphate crystals in recombinant amelogenin gels under controlled ionic flow.

Amelogenin proteins constitute the primary structural entity of the extracellular protein framework of the developing enamel matrix. Recent data on the interactions of amelogenin with calcium phosphate crystals support the hypothesis that amelogenins control the oriented and elongated growth of enamel carbonate apatite crystals. To exploit further the molecular mechanisms involved in amelogenin-calcium phosphate mineral interactions, we conducted in vitro experiments to examine the effect of amelogenin on synthetic octacalcium phosphate (OCP) crystals.

Progressive accretion of amelogenin molecules during nanospheres assembly revealed by atomic force microscopy.

Amelogenin proteins, the principal components of the developing dental enamel matrix, self-assemble to form nanosphere structures that are believed to function as structural components directly involved in the matrix mediated enamel biomineralization. The self-assembly behavior of a recombinant murine amelogenin (rM179) was investigated by atomic force microscopy (AFM) for further understanding the roles of amelogenin proteins in dental enamel biomineralization. Recombinant rM179 amelogenin was dissolved in a pH 7.

Amelogenin-cytokeratin 14 interaction in ameloblasts during enamel formation.

The enamel protein amelogenin binds to the GlcNAc-mimicking peptide (GMp) (Ravindranath, R. M. H.

The changing face of dental education: the impact of PBL.

The past decade has seen increasing demands for reform of dental education that would produce a graduate better equipped to work in the rapidly changing world of the twenty-first century. Among the most notable curriculum changes implemented in dental schools is a move toward Problem-Based Learning (PBL). PBL, in some form, has been a feature of medical education for several decades, but has only recently been introduced into dental schools.

Controlled proteolysis of amelogenins reveals exposure of both carboxy- and amino-terminal regions.

The matrix-mediated enamel biomineralization involves secretion of the enamel specific amelogenin proteins that through self-assembly into nanosphere structures provide the framework within which the initial enamel crystallites are formed. During enamel mineralization, amelogenin proteins are processed by tooth-specific proteinases. The aim of this study was to explore the factors that affect the activity of enamel proteases to process amelogenins.

Dose-dependent modulation of octacalcium phosphate crystal habit by amelogenins.

In vitro studies on interactions between amelogenins and calcium phosphate crystals are critical for elucidating biomineralization mechanisms of tooth enamel. This work was aimed at investigating the effects of native porcine amelogenins on octacalcium phosphate (OCP) crystal growth in a gelatin gel. We prepared OCP mineral discs by circulating calcium and phosphate solutions on the opposite ends of the gels loaded with 0-2% amelogenin for one week.

Does amelogenin nanosphere assembly proceed through intermediary-sized structures?

We have proposed that these nanosphere structures are functionally involved in the organization and control of initial enamel biomineralization at the ultrastructural level. Based on the observed nanosphere hydrodynamic radii (18-20 nm diameter) computation suggests these structures to be compounded of some 100 amelogenin monomers, raising the question as to the possible molecular mechanism for the assembly of such structures? Based on recent dynamic light scattering experiments using the recombinant murine amelogenin M179, and employing a newer size distribution algorithm we now report that the size distribution data for M179 are better described by a bimodal distribution model, than the monomodal distribution as previously described. We suggest that amelogenin nanosphere assembly proceeds through intermediate structures (perhaps represented in vivo by "stippled material") of some 4-5 nm hydrodynamic radius, and computed to comprise 4-6 amelogenin monomers.

The pH dependent amelogenin solubility and its biological significance.

Amelogenins are a group of extracellular enamel matrix proteins which are believed to be involved in the regulation of the size and habit of enamel crystals. The aim of this study was to compare the solubility properties of several amelogenins in various pH (4.0-9.

Effect of apatite crystals on the activity of amelogenin degrading enzymes in vitro.

The objective of the present study was to determine the effect of apatite crystals on the activity of amelogenin degrading enzymes in vitro. Current experimental data, together with previous reports support the view that among the different proteinases present in the enamel extracellular matrix, serine proteinase(s) are responsible for the massive degradation of amelogenins during the maturation stage. For our in-vitro experiments we used the recombinant amelogenin M179 as substrate and a "65%-satd.

Effects of amelogenin on the transforming surface microstructures of Bioglass in a calcifying solution.

Topographies of a bioactive glass (45S5 type Bioglass(R)) during 0-4 h of immersion in a supersaturated calcifying solution (SCS) and the SCS containing recombinant porcine amelogenin rP172 (SCS(rP172)) were observed by atomic force microscopy. Other techniques including X-ray diffraction, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, and transmission electron microscopy were used for some complementary microstructural investigations. The smooth Bioglass surface changed to be very rough after 0.

The enamel protein amelogenin binds to the N-acetyl-D-glucosamine-mimicking peptide motif of cytokeratins.

Amelogenins bind to GlcNAc of the dentine-enamel matrix proteins (Ravindranath, R. M. H.

Self-assembly properties of recombinant engineered amelogenin proteins analyzed by dynamic light scattering and atomic force microscopy.

Dynamic light scattering (DLS) analysis together with atomic force microscopy (AFM) imaging was applied to investigate the supramolecular self-assembly properties of a series of recombinant amelogenins. The overall objective was to ascertain the contribution of certain structural motifs in amelogenin to protein-protein interactions during the self-assembly process. Mouse amelogenins lacking either amino- or carboxy-terminal domains believed to be involved in self-assembly and amelogenins having single or double amino acid mutations identical to those found in cases of amelogenesis imperfecta were analyzed.

Modulation of apatite crystal growth on Bioglass by recombinant amelogenin.

The effects of a recombinant mouse amelogenin (rM179) on the growth of apatite crystals nucleated on a bioactive glass (45S5 type Bioglass) surface were investigated with a view to gaining a better understanding of the role of amelogenin protein in tooth enamel formation and of its potential application in the design of novel enamel-like biomaterials. Bioglass discs were incubated in phosphate-buffered saline (PBS) to preform a calcium phosphate surface layer and subsequently immersed in blank, bovine serum albumin (BSA)- and rM179-containing supersaturated calcification solutions (SCS(B), SCS(BSA) and SCSrM179), respectively. Calcium phosphate layers formed on all the treated samples and were characterized to be apatite by X-ray diffraction and Fourier transmission infrared spectrophotometry.

Effects of recombinant amelogenin on hydroxyapatite formation in vitro.

To determine the role of amelogenin in the mineralization of dental enamel, the effects of the recombinant mouse amelogenin rM179 on in vitro hydroxyapatite formation have been studied. In a steady-state agarose gel assay for hydroxyapatite nucleation, rM179 lacked significant activity at concentrations up to 300 microgram/ml. In an autotitration assay for inhibition of de novo hydroxyapatite formation, rM179 had no significant activity at concentrations up to 30 microgram/ml.

The structural biology of the developing dental enamel matrix.

The biomineralization of the dental enamel matrix with a carbonated hydroxyapatite mineral generates one of the most remarkable examples of a vertebrate mineralized tissue. Recent advances in the molecular biology of ameloblast gene products have now revealed the primary structures of the principal proteins involved in this extracellular mineralizing system, amelogenins, tuftelins, ameloblastins, enamelins, and proteinases, but details of their secondary, tertiary, and quaternary structures, their interactions with other matrix and or cell surface proteins, and their functional role in dental enamel matrix mineralization are still largely unknown. This paper reviews our current knowledge of these molecules, the probable molecular structure of the enamel matrix, and the functional role of these extracellular matrix proteins.

Microstructures of an amelogenin gel matrix.

The thermo-reversible transition (clear <--> opaque) of the amelogenin gel matrix, which has been known for some three decades, has now been clarified by microstructural investigations. A mixed amelogenin preparation extracted from porcine developing enamel matrix (containing "25K," 7.4%; "23K," 10.

Characterization of recombinant pig enamelysin activity and cleavage of recombinant pig and mouse amelogenins.

Enamelysin (MMP-20) is a tooth-specific matrix metalloproteinase that is initially expressed by ameloblasts and odontoblasts immediately prior to the onset of dentin mineralization, and continues to be expressed throughout the secretory stage of amelogenesis. During the secretory stage, enamel proteins are secreted and rapidly cleaved into a large number of relatively stable cleavage products. Multiple proteinases are present in the developing enamel matrix, and the precise role of enamelysin in the processing of enamel proteins is unknown.

Tyrosyl motif in amelogenins binds N-acetyl-D-glucosamine.

Ameloblasts secrete amelogenins on the pre-existing enamel matrix glycoproteins at the dentine-enamel junction. The hypothesis that amelogenins may interact with enamel matrix glycoproteins is tested by hemagglutination of purified, native (porcine) and recombinant murine amelogenins (rM179 and rM166) and hemagglutination inhibition with sugars. Amelogenin agglutination of murine erythrocytes was specifically inhibited by N-acetylglucosamine (GlcNAc), chitobiose, and chitotetraose and by ovalbumin with terminal GlcNAc.

Temperature and pH-dependent supramolecular self-assembly of amelogenin molecules: a dynamic light-scattering analysis.

Evidence for the molecular self-assembly of amelogenin proteins to form quasi-spherical particles ("nanospheres") in solution, both in vitro and in vivo, has recently been documented. A particle-size distribution analysis of dynamic light-scattering data was undertaken to investigate the influence of temperature on this molecular self-assembly process at three different pH's. The long-term objective was to correlate these observations to the unusual physiochemical characteristics of the protein, to improve understanding of the molecular mechanisms involved in the generation of amelogenin "nanospheres" and understanding of their putative relation to amelogenin function in vivo.