Bridge-building between communities: Imagining the future of biomedical autism research.

A paradigm shift in research culture is required to ease perceived tensions between autistic people and the biomedical research community. As a group of autistic and non-autistic scientists and stakeholders, we contend that through participatory research, we can reject a deficit-based conceptualization of autism while building a shared vision for a neurodiversity-affirmative biomedical research paradigm.

Do Material Discontinuities in Silica Affect Vibration Modes?

Structural properties of bioinorganic composites are of current interest in the areas of drug delivery, bone repair, and biomimetics. In such composite systems, structural analysis is enhanced when we combine methods of spectroscopy and simulation. Depending on size and shape, structural discontinuities of inorganic matter may modulate the optical response of a bound molecule. Using density functional theory, we explore the effects of a local field next to the surface of a silica cluster on frequencies of methyl stretching modes of associated methanols. Computation results predict that the electrostatic potential modulated by structural discontinuities of silica should not contribute to any systematic frequency shifts for normal modes of a guest molecule. Regardless of position, the methyl stretching modes of methanol demonstrate sensitivity only to the local chemistry of bonding with silanols, which may lead either to a low or high frequency shift for vibrations. In support, experimental studies of deuterated methanol at impurity levels in water show uniform broadening of resonances of carbon-deuterium stretching modes in the presence of both crystalline and amorphous silica nanoparticles. The significance of these findings is that the spectral responses of guest molecules on such surfaces should not be subject to bias introduced by edge effects.

Isolation of a wide range of minerals from a thermally treated plant: Equisetum arvense, a Mare's tale.

Silica is the second most abundant biomineral being exceeded in nature only by biogenic CaCO3. Many land plants (such as rice, cereals, cucumber, etc.) deposit silica in significant amounts to reinforce their tissues and as a systematic response to pathogen attack. One of the most ancient species of living vascular plants, Equisetum arvense is also able to take up and accumulate silica in all parts of the plant. Numerous methods have been developed for elimination of the organic material and/or metal ions present in plant material to isolate biogenic silica. However, depending on the chemical and/or physical treatment applied to branch or stem from Equisetum arvense; other mineral forms such glass-type materials (i.e. CaSiO3), salts (i.e. KCl) or luminescent materials can also be isolated from the plant material. In the current contribution, we show the chemical and/or thermal routes that lead to the formation of a number of different mineral types in addition to biogenic silica.

Functional material features of Bombyx mori silk light versus heavy chain proteins.

Bombyx mori (BM) silk fibroin is composed of two different subunits: heavy chain and light chain fibroin linked by a covalent disulfide bond. Current methods of separating the two silk fractions is complicated and produces inadequate quantities of the isolated components for the study of the individual light and heavy chain silks with respect to new materials. We report a simple method of separating silk fractions using formic acid. The formic acid treatment partially releases predominately the light chain fragment (soluble fraction) and then the soluble fraction and insoluble fractions can be converted into new materials. The regenerated original (total) silk fibroin and the separated fractions (soluble vs insoluble) had different molecular weights and showed distinctive pH stabilities against aggregation/precipitation based on particle charging. All silk fractions could be electrospun to give fiber mats with viscosity of the regenerated fractions being the controlling factor for successful electrospinning. The silk fractions could be mixed to give blends with different proportions of the two fractions to modify the diameter and uniformity of the electrospun fibers formed. The soluble fraction containing the light chain was able to modify the viscosity by thinning the insoluble fraction containing heavy chain fragments, perhaps analogous to its role in natural fiber formation where the light chain provides increased mobility and the heavy chain producing shear thickening effects. The simplicity of this new separation method should enable access to these different silk protein fractions and accelerate the identification of methods, modifications, and potential applications of these materials in biomedical and industrial applications.

Silk-silica composites from genetically engineered chimeric proteins: materials properties correlate with silica condensation rate and colloidal stability of the proteins in aqueous solution.

The aim of the study was to determine the extent and mechanism of influence on silica condensation that is presented by a range of known silicifying recombinant chimeras (R5: SSKKSGSYSGSKGSKRRIL; A1: SGSKGSKRRIL; and Si4-1: MSPHPHPRHHHT and repeats thereof) attached at the N-terminus end of a 15-mer repeat of the 32 amino acid consensus sequence of the major ampullate dragline Spindroin 1 (Masp1) Nephila clavipes spider silk sequence ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](15)X). The influence of the silk/chimera ratio was explored through the adjustment of the type and number of silicifying domains (denoted X above), and the results were compared with their non-chimeric counterparts and the silk from Bombyx mori. The effect of pH (3-9) on reactivity was also explored. Optimum conditions for rate and control of silica deposition were determined, and the solution properties of the silks were explored to determine their mode(s) of action. For the silica-silk-chimera materials formed there is a relationship between the solution properties of the chimeric proteins (ability to carry charge), the pH of reaction, and the solid state materials that are generated. The region of colloidal instability correlates with the pH range observed for morphological control and coincides with the pH range for the highest silica condensation rates. With this information it should be possible to predict how chimeric or chemically modified proteins will affect structure and morphology of materials produced under controlled conditions and extend the range of composite materials for a wide spectrum of uses in the biomedical and technology fields.

Bioinspired silicification of silica-binding peptide-silk protein chimeras: comparison of chemically and genetically produced proteins.

Novel protein chimeras constituted of "silk" and a silica-binding peptide (KSLSRHDHIHHH) were synthesized by genetic or chemical approaches and their influence on silica-silk based chimera composite formation evaluated. Genetic chimeras were constructed from 6 or 15 repeats of the 32 amino acid consensus sequence of Nephila clavipes spider silk ([SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQG](n)) to which one silica binding peptide was fused at the N terminus. For the chemical chimera, 28 equiv of the silica binding peptide were chemically coupled to natural Bombyx mori silk after modification of tyrosine groups by diazonium coupling and EDC/NHS activation of all acid groups. After silica formation under mild, biomaterial-compatible conditions, the effect of peptide addition on the properties of the silk and chimeric silk-silica composite materials was explored. The composite biomaterial properties could be related to the extent of silica condensation and to the higher number of silica binding sites in the chemical chimera as compared with the genetically derived variants. In all cases, the structure of the protein/chimera in solution dictated the type of composite structure that formed with the silica deposition process having little effect on the secondary structural composition of the silk-based materials. Similarly to our study of genetic silk based chimeras containing the R5 peptide (SSKKSGSYSGSKGSKRRIL), the role of the chimeras (genetic and chemical) used in the present study resided more in aggregation and scaffolding than in the catalysis of condensation. The variables of peptide identity, silk construct (number of consensus repeats or silk source), and approach to synthesis (genetic or chemical) can be used to "tune" the properties of the composite materials formed and is a general approach that can be used to prepare a range of materials for biomedical and sensor-based applications.

The discovery of potent and long-acting oral factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs.

The discovery and evaluation of potent and long-acting oral sulfonamidopyrrolidin-2-one factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs are described. Unexpected selectivity issues versus tissue plasminogen activator in the former series were addressed in the later, delivering a robust candidate for progression towards clinical studies.

From biosilicification to tailored materials: optimizing hydrophobic domains and resistance to protonation of polyamines.

Considerable research has been directed toward identifying the mechanisms involved in biosilicification to understand and possibly mimic the process for the production of superior silica-based materials while simultaneously minimizing pollution and energy costs. Molecules isolated from diatoms and, most recently sponges, thought to be key to this process contain polyamines with a propylamine backbone and variable levels of methylation. In a chemical approach to understanding the role of amine (especially propylamine) structures in silicification we have explored three key structural features: (i) the degree of polymerization, (ii) the level of amine methylation, and (iii) the size of the amine chain spacers. In this article, we show that there are two factors critical to their function: the ability of the amines to produce microemulsions and the presence of charged and uncharged amine groups within a molecule, with the latter feature helping to catalyze silicic acid condensation by a proton donor/acceptor mechanism. The understanding of amine-silicate interactions obtained from this study has enabled the controlled preparation of hollow and nonporous siliceous materials under mild conditions (circumneutral pH, room temperature, and in all aqueous systems) possibly compatible with the conditions used by biosystems. The "rules" identified from our study were further used predictively to modulate the activity of a given amine. We believe that the outcomes of the present contribution will form the basis for an approach to controlling the growth of inorganic materials by using tailor-made organic molecules.

Physical properties of root cementum: part 15. Analysis of elemental composition by using proton-induced x-ray and gamma-ray emissions in orthodontically induced root resorption craters of rat molar cementum after exposure to systemic fluoride.

INTRODUCTION: Root resorption resulting from orthodontic treatment is an unpredictable adverse effect. Literature examining the potential protective influence of tooth cementum minerals against orthodontically induced inflammatory root resorption has been sparse. Fluorine could have a role in minimizing the extent and severity of resorptive lesions. The purpose of this study was to examine the elemental content of tooth cementum in orthodontically induced inflammatory root resorption lesions and the effect of systemic fluoride. METHODS: Twenty 7-week-old Wistar rats were divided into 2 groups of 10 and exposed to systemic fluoride (100 ppm) or nonfluoridated drinking water for 2 weeks. Orthodontic tooth movement was implemented with a nickel-titanitum closing coil with a force of 100 g. The molars were then extracted, dissected, and prepared for cross-sectioning through the largest mesial midroot crater. The samples were mounted and scanned by using the Commonwealth Scientific and Industrial Research Organisation and the Australian Research Council's National Key for Geochemical Evolution and Metallogeny of Continents Nuclear Microprobe (Melbourne, Victoria, Australia). Analysis of variance (ANOVA) was used for statistical comparison of the elements and to determine the effect of fluoride, and unaffected tooth structure compared with root resorption craters. The Student t test was used to compare root crater lengths and depths of the fluoride vs no-fluoride groups. RESULTS: Root resorption lesions of the group exposed to fluoride were significantly reduced in length and depth (P <0.01). The mineral content of the root resorption craters of the fluoride group had higher concentrations of fluorine and zinc (P <0.01). There was less calcium in the craters of the no-fluoride group compared with the fluoride group (P <0.05). CONCLUSIONS: Cementum quality (influenced by systemic fluoride exposure) might impact the extent of orthodontically induced resorptive defects.

A closed-form expression of the positional uncertainty for 3D point clouds.

We present a novel closed-form expression of positional uncertainty measured by a near-monostatic and time-of-flight laser range finder with consideration of its measurement uncertainties. An explicit form of the angular variance of the estimated surface normal vector is also derived. This expression is useful for the precise estimation of the surface normal vector and the outlier detection for finding correspondence in order to register multiple three-dimensional point clouds. Two practical algorithms using these expressions are presented: a method for finding optimal local neighbourhood size which minimizes the variance of the estimated normal vector and a resampling method of point clouds.

Traditional materials from new sources - conflicts in analytical methods for calcium carbonate.

Calcium carbonate (E170) is a common food and pharmaceutical additive/ingredient. In addition to a source of calcium, the carbonate has uses including as a colour, acidity regulator and bulking agent. Globally, a range of regulatory agencies and pharmacopoeia control the analyses and specification of additives in food, supplements, pharmaceutical substances and excipients. Accordingly, a range of specifications and analyses exist for calcium carbonate depending on the application and market of the product. In this contribution, we analyse calcium carbonates from geological, synthetic and biogenic sources, focussing on acid insoluble impurities, a test required by current monographs. Analysis of calcium carbonate from different origins may require modification of existing tests to comply with regulatory bodies, due to the variation of impurities specific to the source of the material. We suggest an analytical approach involving centrifugation that improves analytical efficiency (up to 85% time reduction), especially for calcium carbonate of biological origin.

A robust spectroscopic method for the determination of protein conformational composition - Application to the annealing of silk.

The physical and mechanical properties of structural proteins such as silk fibroin can be modified by controlled conformational change, which is regularly monitored by Fourier transform infrared spectroscopy by peak fitting of the amide I band envelope. Although many variables affecting peak shape are well established, there is no fixed methodology to compare and follow secondary structural differences without significant operator input especially where low frequency spectral noise is a problem. The aim of this contribution is to establish a method for such analyses to be carried at high levels of autonomy to prevent subjective or erroneous fitting. A range of approaches was trialled with optimal peak parameters selected based on overall goodness of fit and reproducibility of fit of replicate sample spectra. The method was successfully tested against reference proteins having contrasting ß content and the rationale for parameter selection is presented. Further, we applied this method to measure the effect of conformational change on the energy of the amide I band of silk fibroin during annealing. Energy changes were ca. 400 kJ mol-1 of fibroin. To confirm that this energy change was a consequence of increased hydrogen bonding we used a Thioflavin T staining method typically used to identify ß aggregate type structures in amyloid plaques. We propose that the approach described herein can aid in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity. STATEMENT OF SIGNIFICANCE: The physical and mechanical properties of proteins including silk fibroin can be modified by controlled structural change, which is regularly monitored by Fourier transform infrared spectroscopy (FTIR) by peak fitting of the amide I band. Currently there is no fixed methodology to compare and follow secondary structural differences without significant operator input leading to subjectivity and error. This contribution establishes a method for such analyses to be carried at high levels of autonomy applicable to a wide range of proteins and the conformational changes have been quantified as a single energy change output, which clearly shows the progression of the annealing process used. We propose that the approach can help in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity.

Quantifying the efficiency of Hydroxyapatite Mineralising Peptides.

We present a non-destructive analytical calibration tool to allow quantitative assessment of individual calcium phosphates such as hydroxyapatite (HAP) from mixtures including brushite. Many experimental approaches are used to evaluate the mineralising capabilities of biomolecules including peptides. However, it is difficult to quantitatively compare the efficacy of peptides in the promotion of mineralisation when inseparable mixtures of different minerals are produced. To address this challenge, a series of hydroxyapatite and brushite mixtures were produced as a percent/weight (0-100%) from pure components and multiple (N = 10) XRD patterns were collected for each mixture. A linear relationship between the ratio of selected peak heights and the molar ratio was found. Using this method, the mineralising capabilities of three known hydroxyapatite binding peptides, CaP(S) STLPIPHEFSRE, CaP(V) VTKHLNQISQSY and CaP(H) SVSVGMKPSPRP, was compared. All three directed mineralisation towards hydroxyapatite in a peptide concentration dependent manner. CaP(V) was most effective at inducing hydroxyapatite formation at higher reagent levels (Ca2+ = 200 mM), as also seen with peptide-silk chimeric materials, whereas CaP(S) was most effective when lower concentrations of calcium (20 mM) and phosphate were used. The approach can be extended to investigate HAP mineralisation in the presence of any number of mineralisation promoters or inhibitors.

Osteoinductive recombinant silk fusion proteins for bone regeneration.

Protein polymers provide a unique opportunity for tunable designs of material systems due to the genetic basis of sequence control. To address the challenge of biomineralization interfaces with protein based materials, we genetically engineered spider silks to design organic-inorganic hybrid systems. The spider silk inspired domain (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT)15 served as an organic scaffold to control material stability and to allow multiple modes of processing, whereas the hydroxyapatite binding domain VTKHLNQISQSY (VTK), provided control over osteogenesis. The VTK domain was fused either to the N-, C- or both terminals of the spider silk domain to understand the effect of position on material properties and mineralization. The addition of the VTK domain to silk did not affect the physical properties of the silk recombinant constructs, but it had a critical role in the induction of biomineralization. When the VTK domain was placed on both the C- and N-termini the formation of crystalline hydroxyapatite was significantly increased. In addition, all of the recombinant proteins in film format supported the growth and proliferation of human mesenchymal stem cells (hMSCs). Importantly, the presence of the VTK domain enhanced osteoinductive properties up to 3-fold compared to the control (silk alone without VTK). Therefore, silk-VTK fusion proteins have been shown suitable for mineralization and functionalization for specific biomedical applications. STATEMENT OF SIGNIFICANCE: Organic-inorganic interfaces are integral to biomaterial functions in many areas of repair and regeneration. Several protein polymers have been investigated for this purpose. Despite their success the limited options to fine-tune their material properties, degradation patterns and functionalize them for each specific biomedical application limits their application. Various studies have shown that the biological performance of such proteins can be improved by genetic engineering. The present study provides data relating protein design parameters and functional outcome quantified by biomineralization and human mesenchymal stem cell differentiation. As such, it helps the design of osteoinductive recombinant biomaterials for bone regeneration.

A new stepwise synthesis of a family of propylamines derived from diatom silaffins and their activity in silicification.

A new method for the stepwise synthesis of propylamines containing fragments of N-methyl propylamine as found in diatom bioextracts is presented and their activity in silicic acid condensation is described.

Influence of silk-silica fusion protein design on silica condensation in vitro and cellular calcification.

Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterial integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on the biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGGQG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have a significant effect on wettability and surface energies, while the C-terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cell (hMSC) differentiation were demonstrated for both variants of the fusion proteins.

Putrescine homologues control silica morphogenesis by electrostatic interactions and the hydrophobic effect.

A systematic model study on the role(s) of putrescine homologues on silicification is presented and it is proposed that electrostatic forces between additive and silicic acid, and the hydrophobic behaviour of the additives are both important in silicification.

Control of silicification by genetically engineered fusion proteins: silk-silica binding peptides.

In the present study, an artificial spider silk gene, 6mer, derived from the consensus sequence of Nephila clavipes dragline silk gene, was fused with different silica-binding peptides (SiBPs), A1, A3 and R5, to study the impact of the fusion protein sequence chemistry on silica formation and the ability to generate a silk-silica composite in two different bioinspired silicification systems: solution-solution and solution-solid. Condensed silica nanoscale particles (600-800 nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras, revealing that the molecular weight of the silk domain correlated to the sizes of the condensed silica particles in the solution system. In addition, the chimeras (6mer-A1/A3/R5) produced smaller condensed silica particles than the control (6mer), revealing that the silica particle size formed in the solution system is controlled by the size of protein assemblies in solution. In the solution-solid interface system, silicification reactions were performed on the surface of films fabricated from the recombinant silk proteins and chimeras and then treated to induce ß-sheet formation. A higher density of condensed silica formed on the films containing the lowest ß-sheet content while the films with the highest ß-sheet content precipitated the lowest density of silica, revealing an inverse correlation between the ß-sheet secondary structure and the silica content formed on the films. Intriguingly, the 6mer-A3 showed the highest rate of silica condensation but the lowest density of silica deposition on the films, compared with 6mer-A1 and -R5, revealing antagonistic crosstalk between the silk and the SiBP domains in terms of protein assembly. These findings offer a path forward in the tailoring of biopolymer-silica composites for biomaterial related needs.

An overview of the fundamentals of the chemistry of silica with relevance to biosilicification and technological advances.

Biomineral formation is widespread in nature, and occurs in bacteria, single-celled protists, plants, invertebrates, and vertebrates. Minerals formed in the biological environment often show unusual physical properties (e.g. strength, degree of hydration) and often have structures that exhibit order on many length scales. Biosilica, found in single-celled organisms through to higher plants and primitive animals (sponges), is formed from an environment that is undersaturated with respect to silicon, and under conditions of approximately neutral pH and relatively low temperatures of 4-40 °C compared to those used industrially. Formation of the mineral may occur intracellularly or extracellularly, and specific biochemical locations for mineral deposition that include lipids, proteins and carbohydrates are known. In most cases, the formation of the mineral phase is linked to cellular processes, an understanding of which could lead to the design of new materials for biomedical, optical and other applications. In this contribution, we describe the aqueous chemistry of silica, from uncondensed monomers through to colloidal particles and 3D structures, that is relevant to the environment from which the biomineral forms. We then describe the chemistry of silica formation from alkoxides such as tetraethoxysilane, as this and other silanes have been used to study the chemistry of silica formation using silicatein, and such precursors are often used in the preparation of silicas for technological applications. The focus of this article is on the methods, experimental and computational, by which the process of silica formation can be studied, with an emphasis on speciation.

A solution study of silica condensation and speciation with relevance to in vitro investigations of biosilicification.

Requiring mild synthesis conditions and possessing a high level of organization and functionality, biosilicas constitute a source of wonder and inspiration for both materials scientists and biologists. In order to understand how such biomaterials are formed and to apply this knowledge to the generation of novel bioinspired materials, a detailed study of the materials, as formed under biologically relevant conditions, is required. In this contribution, data from a detailed study of silica speciation and condensation using a model bioinspired silica precursor (silicon catechol complex, SCC) is presented. The silicon complex quickly and controllably dissociates under neutral pH conditions to well-defined, metastable solutions of orthosilicic acid. The formation of silicomolybdous (blue) complexes was used to monitor and study different stages of silicic acid condensation. In parallel, the rates of silicomolybdic (yellow) complex formation, with mathematical modeling of the species present, was used to follow the solution speciation of polysilicic acids. The results obtained from the two assays correlate well. Monomeric silicic acid, trimeric silicic acids, and different classes of oligomeric polysilicic acids and silica nuclei can be identified and their periods of stability during the early stages of silica condensation measured. For experiments performed at a range of temperatures (273-323 K), an activation energy of 77 kJ.mol(-1) was obtained for the formation of trimers. The activation energies for the forward and reverse condensation reactions for addition of monomers to polysilicic acids (273-293 +/- 1 K) were 55.0 and 58.6 kJ.mol(-1), respectively. For temperatures above 293 K, these energies were reduced to 6.1 and 7.3 kJ.mol(-1), indicating a probable change in the prevailing condensation mechanism. The impact of pH on the rates of condensation were measured. There was a direct correlation between the apparent third-order rate constant for trimer formation and pH (4.7-6.9 +/- 0.1) while values for the reversible first-order rates reached a plateau at circumneutral pH. These different behaviors are discussed with reference to the generally accepted mechanism for silica condensation in which anionic silicate solution species are central to the condensation process. The results presented in this paper support the use of precursors such as silicon catecholate complexes in the study of biosilicification in vitro. Further detailed experimentation is needed to increase our understanding of specific biomolecule silica interactions that ultimately generate the complex, finely detailed siliceous structures we observe in the world around us.