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Bone adhesives offer distinct advantages over the use of screws to attached internal fixation plates (IFPs). As the chemical composition of bone is similar to dentine, it is possible that the types of monomers used to make dentine adhesives could be utilised to affix IFPs to bone. The ability to attach a bio-resorbable IFP to porcine bone was assessed for the monomer 10-methacryloyloxydecyl dihydrogen phosphate (MDP), used either as a homopolymer or a copolymer with urethane dimethacrylate (MDP + U). Additionally, the addition of a priming step (MDP + U + P) was evaluated. The chemical interactions of the monomers with bone were assessed using XRD and imaged using TEM, revealing the formation of nano-layered structures with the MDP primer, something we believe has not been reported on bone. In a 6-week artificial aging study both MDP + U and MDP + U + P demonstrated adequate shear bond strength to affix bio-resorbable IFPs. The cytotoxicity profiles of the adhesive formulations were determined using indirect and direct contact with MC3T3 cells, with indirect conditions suggesting the MDP + U + P is as cytocompatible as the resorbable IFP. The findings of this study suggest our newly developed adhesive has the potential to be used as a bone adhesive to affix bioresorbable IFPs. [ABSTRACT FROM AUTHOR]

The crystal structures of the ligand binding domain (LBD) of the rat vitamin D receptor (VDR) in ternary complexes with a synthetic LXXLL-containing peptide and four ligands are determined. The differences in the biological activity of the 2-carbon-modified analogues are not reflected in the three-dimensional structure of the LBD of the VDR.

This article describes a proof of concept study designed to evaluate the potential of an in vivo three-dimensional printing route to support minimally invasive repair of the musculoskeletal system. The study uses a photocurable material to additively manufacture in situ a model implant and demonstrates that this can be achieved effectively within a clinically relevant timescale. The approach has the potential to be applied with a wide range of light-curable materials and with development could be applied to create functionally gradient structures in vivo. [ABSTRACT FROM AUTHOR]

The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N10-formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido- d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed β-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a β-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate. [ABSTRACT FROM AUTHOR]

Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (Npro) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, Npro is no longer active as a protease. The released Npro suppresses the induction of the host's type-I interferon-α/β (IFN-α/β) response. Npro binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus Npro. Npro is structurally distinct from other known cysteine proteases and has a novel “clam shell” fold consisting of a protease domain and a zinc-binding domain. The unique fold of Npro allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of Npro involved in targeting IRF3 for proteasomal degradation provides insight into the nature of Npro's interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of Npro, and its role in subversion of host immune response. [ABSTRACT FROM AUTHOR]

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and 32P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group [ABSTRACT FROM AUTHOR]

High-throughput crystallography has reached a level of automation where complete computer-assisted robotic crystallization pipelines are capable of cocktail preparation, crystallization plate setup, and inspection and interpretation of results. While mounting of crystal pins, data collection and structure solution are highly automated, crystal harvesting and cryocooling remain formidable challenges towards full automation. To address the final frontier in achieving fully automated high-throughput crystallography, the prototype of an anthropomorphic six-axis universal micromanipulation robot (UMR) has been designed and tested; this UMR is capable of operator-assisted harvesting and cryoquenching of protein crystals as small as 10 µm from a variety of 96-well plates. The UMR is equipped with a versatile tool exchanger providing full operational flexibility. Trypsin crystals harvested and cryoquenched using the UMR have yielded a 1.5 Å structure demonstrating the feasibility of robotic protein crystal harvesting. [ABSTRACT FROM AUTHOR]

Describes the high-resolution x-ray structures of the different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta. Determination of the metal binding positions for metal-substituted enzyme; Replacement of metal with cadmium or manganese ions without loss of enzymatic activity.

Examines the molecular structure of methylmalonyl co-enzyme A (CoA) decarboxylase (MMCD) in the Escherichia coli. Evidence for an indirect involvement of MMCD in catalysis; Identification of MMCD active site region within one subunit of the hexameric region; Comparison and contrast with 4-chlorobenzoyl CoA dehalogenase.

Presents a study which examined rabbit muscle's pyruvate kinase which were cocrystallized. Indepth look at the physiological reaction of pyruvate kinase; Details on the binding of pyruvate in the presence of ATP; Methodology used to conduct the study; Results of the study.

Describes the three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3 which catalyzes the hydrolysis of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA. Crystallization and preparation of heavy atom derivatives; X-ray data collection and processing; Structural determination and least-square refinement.

Describes the three-dimensional structure of zinc containing phosphotriesterase with the bound substrate analog diethyl 4-methylbenzlphosphonate. Information on phosphotriesterase from pseudomonas diminuta; Refining of the structure by least-squares analysis to a nominal resolution of 2.1 A.

Describes the structure of kanamycin nucleotidyltransferase (KNTase) complexed wit both the nonhydrolyzable nucleotide analog AMPCPP and kanamycin. Suggestion that the putative mechanism of KNTase presented may possibly serve as a model for the more complex polymerases that catalyze nucleotidyl phosphate transfer.

Gives the results of the study conducted on unraveling the three-dimensional structure the binuclear metal center of Phosphotriesterase, an enzyme isolated from Pseudomonas diminuta. Importance of the binuclear metal center for the activity of the enzyme; Focus on the different apo and holo forms of the enzyme; Location of the binuclear metal center.

Reports the three-dimensional structure of phosphotriesterase, as determined by single crystal X-ray diffraction analysis to 2.1-A resolution. Crystals used in the study; Solving of the structure by multiple isomorphous replacement with two heavy-atom derivatives; Refinement of the structure to a crystallographic R factor of 18 percent.

Reports the crystallization, structure determination and least-squares refinement of the HiPIP (high-potential iron-sulfur proteins) isolated from `Ectothiorhodospira vacuolata'. Crystallization process; Molecular replacement procedures; Results and discussion.

Abstract: The hetL gene from the cyanobacterium Nostoc sp. PCC 7120 encodes a 237 amino acid protein (25.6kDa) containing 40 predicted tandem pentapeptide repeats. Nostoc sp. PCC 7120 is a filamentous cyanobacterium that forms heterocysts, specialized cells capable of fixing atmospheric N2 during nitrogen starvation in its aqueous environment. Under these conditions, heterocysts occur in a regular pattern of approximately one out of every 10–15 vegetative cells. Heterocyst differentiation is highly regulated involving hundreds of genes, one of which encodes PatS, thought to be an intercellular peptide signal made by developing heterocysts to inhibit heterocyst differentiation in neighboring vegetative cells, thus contributing to pattern formation and spacing of heterocysts along the filament. While overexpression of PatS suppresses heterocyst differentiation in Nostoc sp. PCC 7120, overexpression of HetL produces a multiple contiguous heterocyst phenotype with loss of the wild type heterocyst pattern, and strains containing extra copies of hetL allow heterocyst formation even in cells overexpressing PatS. Thus, HetL appears to interfere with heterocyst differentiation inhibition by PatS, however, the mechanism for HetL function remains unknown. As a first step towards exploring the mechanism for its biochemical function, the crystal structure of HetL has been solved at 2.0Å resolution using sulfur anomalous scattering. [Copyright &y& Elsevier]

Abstract: T-cell-restricted intracellular antigen-1 (TIA-1) regulates alternative pre-mRNA splicing in the nucleus, and mRNA translation in the cytoplasm, by recognizing uridine-rich sequences of RNAs. As a step towards understanding RNA recognition by this regulatory factor, the X-ray structure of the central RNA recognition motif (RRM2) of human TIA-1 is presented at 1.95Å resolution. Comparison with structurally homologous RRM-RNA complexes identifies residues at the RNA interfaces that are conserved in TIA-1–RRM2. The versatile capability of RNP motifs to interact with either proteins or RNA is reinforced by symmetry-related protein–protein interactions mediated by the RNP motifs of TIA-1–RRM2. Importantly, the TIA-1–RRM2 structure reveals the locations of mutations responsible for inhibiting nuclear import. In contrast with previous assumptions, the mutated residues are buried within the hydrophobic interior of the domain, where they would be likely to destabilize the RRM fold rather than directly inhibit RNA binding. [Copyright &y& Elsevier]

Abstract: We have successfully prepared E- and Z- isomers of 17–20 dehydro analogs of 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD). Both isomers bind to the recombinant rat vitamin D receptor (VDR) with high affinity. The Z-isomer (Vit-III 17–20Z) displays activity in vivo and in vitro that is similar to 2MD. The in vitro activity of the E-isomer (Vit-III 17–20E) is comparable to the natural hormone, though in vivo this analog is significantly less calcemic. Crystal structures of the rat VDR ligand binding domain complexed with the analogs demonstrate that the Vit-III 17–20Z analog is oriented almost identically to 2MD, with only minor differences induced by the planar configuration around the C17–C20 double bond. The Vit-III 17–20E analog is oriented in a conformation distinct from both 2MD and the natural hormone. The structural comparisons suggest that the position of C21 in the ligand binding site may be an important determinant of biological activity. [Copyright &y& Elsevier]

Advances in three-dimensional cell cultures offer new opportunities in biomedical research and drug development. However, there are still challenges to overcome, including the lack of reliability, repeatability and complexity of tissues obtained by these techniques. In this study, we describe a new bioprinting system called reactive jet impingement (ReJI) for the bioprinting of cell-laden hydrogels. Droplets of gel precursor solutions are jetted at one another such that they meet and react in mid-air before the gel droplets fall to the substrate. This technique offers a combination of deposition rate, cell density and cell viability which is not currently matched by any other bioprinting technique. The importance of cell density is demonstrated in the development of bone microtissues derived from immortalised human bone marrow stem cells. The cells were printed with high viability within a collagen-alginate-fibrin gel, and tissue specific gene expression shows significantly higher tissue maturation rates using the ability of the ReJI system to deposit gels with a high cell density.

The ability to culture cells in three-dimensions has many applications, from drug discovery to wound healing. 3D cell culture methods often require appropriate scaffolds that mimic the cellular environments of different tissue types. The choice of material from which these scaffolds are made is of paramount importance, as its properties will define the manner in which cells interact with the scaffold. Caf1 is a protein polymer that is secreted from its host organism, Yersinia pestis, to enable escape from phagocytosis. In vitro, cells adhere poorly to the protein unless adhesion motifs are specifically introduced. Caf1 is a good candidate biomaterial due to its definable bioactivity, economical production and its ability to form hydrogels, through the use of cross-linkers. In this study, the thermostability of Caf1 was tested over a range of chemical conditions, and an initial characterisation of its rheological properties conducted in order to assess the suitability of Caf1 as a biomedical material. The results show that Caf1 retains its high thermostability even in harsh conditions such as extremes of pH, high salt concentrations and the presence of detergents. In solution, the concentrated polymer behaves as a complex viscous liquid. Due to these properties, Caf1 polymers are compatible with 3D bioprinting technologies and could be made to form a stimuli-responsive biomaterial that can alter its macrorheological properties in response to external factors. Caf1 biomaterials could therefore prove useful as 3D cell scaffolds for use in cell culture and wound repair.

Temporary single-cell coating is a useful tool for cell processing, allowing manipulation of cells to prevent cell attachment and agglomeration, before re-establishing normal cell function. In this work, a speckled coating method using a known polycation [poly(l-lysine), PLL] is described to induce cell surface electrostatic charges on three different cell types, namely, two bone cancer cell lines and fibroblasts. The morphology of the PLL speckled coating on the cell surface, internalization and metabolization of the polymer, and prevention of cellular aggregations are reported. Polymer concentration was found to be the key parameter controlling both capsule morphology and cell health. This approach allows a temporary cell coating over the course of 1-2 h, with cells exhibiting phenotypically normal behavior after ingesting and metabolizing the polymer. The process offers a fast and efficient alternative to aid single-cell manipulation for bioprocessing applications. Preliminary work on the application of PLL speckled cell coating in enabling reliable bioprinting is also presented.

Pestiviruses express their genome as a single polypeptide that is subsequently cleaved into individual proteins by host- and virus-encoded proteases. The pestivirus N-terminal protease (N(pro)) is a cysteine autoprotease that cleaves between its own C-terminus and the N-terminus of the core protein. Due to its unique sequence and catalytic site, it forms its own cysteine protease family C53. After self-cleavage, N(pro) is no longer active as a protease. The released N(pro) suppresses the induction of the host's type-I interferon-α/β (IFN-α/β) response. N(pro) binds interferon regulatory factor-3 (IRF3), the key transcriptional activator of IFN-α/β genes, and promotes degradation of IRF3 by the proteasome, thus preventing induction of the IFN-α/β response to pestivirus infection. Here we report the crystal structures of pestivirus N(pro). N(pro) is structurally distinct from other known cysteine proteases and has a novel "clam shell" fold consisting of a protease domain and a zinc-binding domain. The unique fold of N(pro) allows auto-catalysis at its C-terminus and subsequently conceals the cleavage site in the active site of the protease. Although many viruses interfere with type I IFN induction by targeting the IRF3 pathway, little information is available regarding structure or mechanism of action of viral proteins that interact with IRF3. The distribution of amino acids on the surface of N(pro) involved in targeting IRF3 for proteasomal degradation provides insight into the nature of N(pro)'s interaction with IRF3. The structures thus establish the mechanism of auto-catalysis and subsequent auto-inhibition of trans-activity of N(pro), and its role in subversion of host immune response.

We have successfully prepared E- and Z- isomers of 17-20 dehydro analogs of 2-methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD). Both isomers bind to the recombinant rat vitamin D receptor (VDR) with high affinity. The Z-isomer (Vit-III 17-20Z) displays activity in vivo and in vitro that is similar to 2MD. The in vitro activity of the E-isomer (Vit-III 17-20E) is comparable to the natural hormone, though in vivo this analog is significantly less calcemic. Crystal structures of the rat VDR ligand binding domain complexed with the analogs demonstrate that the Vit-III 17-20Z analog is oriented almost identically to 2MD, with only minor differences induced by the planar configuration around the C17-C20 double bond. The Vit-III 17-20E analog is oriented in a conformation distinct from both 2MD and the natural hormone. The structural comparisons suggest that the position of C21 in the ligand binding site may be an important determinant of biological activity.