Selective Metal Coordination in Antifouling Coatings

<p>Marine biofouling is the accumulation of biological material (e.g. microorganisms, soft- and hard-fouling organisms) on the surface of an object submerged in seawater, and it remains a worldwide problem for shipping industries. The fouling of ship hulls results in a reduction of speed and manoeuvrability due to frictional drag, as well as increased fuel consumption and accelerated corrosion, and the exorbitant expenses and losses of efficiency attributed to biofouling have prompted the development of antifouling coatings. Current antifouling paints use copper as a biocidal agent, but copper-based paints are increasingly being banned due to environmental concerns about the non-target effects of leached copper. This project aims to circumvent these concerns and tightening regulations via a revolutionary concept: the development of marine antifouling paints that incorporate Cu(II)-selective ligands to draw the biocidal ingredient (i.e. Cu(II)) from seawater. A multistage strategy emerged for the development of this technology. First, criteria were established for the project’s ideal ligand, and ligands were synthesised or selected based on these criteria. Second, the ligands were incorporated in coatings through covalent modification of the paint binder or additives. Third, methodology was developed and implemented to test each coating’s ability to coordinate and retain Cu(II), as well as its subsequent ability to prevent microfouling by marine bacteria. The suitability of two ligand classes was assessed: acylhydrazones and tetraaza macrocycles, specifically cyclen. Unlike the acylhydrazones, cyclen met the established criteria and was initially evaluated as a curing agent and/or surface-modifier in a two-pack epoxy system with resin Epikote™ 235. However, the Cu(II)-loading by these coatings was relatively low, being at most ~0.05% w/w, and the modification of silica, a common paint additive, with cyclen was explored as an alternative formulation route. The method for the functionalisation of silica with cyclen was optimised, and the maximum Cu(II)-loading achieved by the product was 2.60% w/w. The cyclen-functionalised silica was incorporated on the surface of an epoxy coating, and a bacterial adherence assay was developed to assess the cellular attachment of marine bacterium Vibrio harveyi to this coating, which was found to be undeterred. Yet, the development of the strategy and testing methodology by which the project’s goals may be achieved provides a solid foundation for future work.</p>

Selective Metal Coordination in Antifouling Coatings

<p>Marine biofouling is the accumulation of biological material (e.g. microorganisms, soft- and hard-fouling organisms) on the surface of an object submerged in seawater, and it remains a worldwide problem for shipping industries. The fouling of ship hulls results in a reduction of speed and manoeuvrability due to frictional drag, as well as increased fuel consumption and accelerated corrosion, and the exorbitant expenses and losses of efficiency attributed to biofouling have prompted the development of antifouling coatings. Current antifouling paints use copper as a biocidal agent, but copper-based paints are increasingly being banned due to environmental concerns about the non-target effects of leached copper. This project aims to circumvent these concerns and tightening regulations via a revolutionary concept: the development of marine antifouling paints that incorporate Cu(II)-selective ligands to draw the biocidal ingredient (i.e. Cu(II)) from seawater. A multistage strategy emerged for the development of this technology. First, criteria were established for the project’s ideal ligand, and ligands were synthesised or selected based on these criteria. Second, the ligands were incorporated in coatings through covalent modification of the paint binder or additives. Third, methodology was developed and implemented to test each coating’s ability to coordinate and retain Cu(II), as well as its subsequent ability to prevent microfouling by marine bacteria. The suitability of two ligand classes was assessed: acylhydrazones and tetraaza macrocycles, specifically cyclen. Unlike the acylhydrazones, cyclen met the established criteria and was initially evaluated as a curing agent and/or surface-modifier in a two-pack epoxy system with resin Epikote™ 235. However, the Cu(II)-loading by these coatings was relatively low, being at most ~0.05% w/w, and the modification of silica, a common paint additive, with cyclen was explored as an alternative formulation route. The method for the functionalisation of silica with cyclen was optimised, and the maximum Cu(II)-loading achieved by the product was 2.60% w/w. The cyclen-functionalised silica was incorporated on the surface of an epoxy coating, and a bacterial adherence assay was developed to assess the cellular attachment of marine bacterium Vibrio harveyi to this coating, which was found to be undeterred. Yet, the development of the strategy and testing methodology by which the project’s goals may be achieved provides a solid foundation for future work.</p>

Tailoring Silicon Nitride Surface Chemistry for Facilitating Odontogenic Differentiation of Rat Dental Pulp Cells

Silicon nitride (Si3N4) can facilitate bone formation; hence, it is used as a biomaterial in orthopedics. Nevertheless, its usability for dentistry is unexplored. The aim of the present study was to investigate the effect of Si3N4 granules for the proliferation and odontogenic differentiation of rat dental pulp cells (rDPCs). Four different types of Si3N4 granules were prepared, which underwent different treatments to form pristine as-synthesized Si3N4, chemically treated Si3N4, thermally treated Si3N4, and Si3N4 sintered with 3 wt.% yttrium oxide (Y2O3). rDPCs were cultured on or around the Si3N4 granular beds. Compared with the other three types of Si3N4 granules, the sintered Si3N4 granules significantly promoted cellular attachment, upregulated the expression of odontogenic marker genes (Dentin Matrix Acidic Phosphoprotein 1 and Dentin Sialophosphoprotein) in the early phase, and enhanced the formation of mineralization nodules. Furthermore, the water contact angle of sintered Si3N4 was also greatly increased to 40°. These results suggest that the sintering process for Si3N4 with Y2O3 positively altered the surface properties of pristine as-synthesized Si3N4 granules, thereby facilitating the odontogenic differentiation of rDPCs. Thus, the introduction of a sintering treatment for Si3N4 granules is likely to facilitate their use in the clinical application of dentistry.

A pilot study on the use of 3D printers in veterinary medicine

Additive manufacturing (AM) is the process of joining materials to create layer-by-layer three-dimensional objects using a 3D printer from a digital model. The great advantage of Additive Manufacturing is to allow a freer design than traditional processes. The development of additive manufacturing processes has permitted to optimize the production of the customized product through the modeling of the geometry and the knowledge of the morphometric parameters of the body structures. 3D printing has revolutionized the field of Regenerative Medicine because, starting from computerized tomography (CT) images and using traditional materials such as plastic and metals, it can provide customized prostheses for each patient, which adapt perfectly to the needs of the subject and act as structures support. 3D printing allows you to print three-dimensional porous scaffolds with a precise shape and chemical composition suitable for medical and veterinary use. Some of these scaffolds are biodegradable and appear to be ideal for bone tissue engineering. In fact, they are able to simulate extracellular matrix properties that allow mechanical support, favoring mechanical interactions and providing a model for cellular attachment and in vivo stimulation of bone tissue formation.

Polyamines and eIF5A hypusination facilitate SREBP2 translation and cholesterol synthesis to enhance enterovirus attachment and infection

Metabolism is key to cellular processes that ultimately underly the ability of a virus to productively infect a cell. Polyamines are small metabolites that are vital for many host cell processes including cellular proliferation, transcription, and translation, and these molecules are also key in virus infection. Depletion of polyamines inhibits viral infection via diverse mechanisms, including by inhibiting polymerase activity, cellular attachment, and translation of viral proteins. The precise mechanisms underlying many of these phenotypes remain to be understood. We have shown previously that Coxsackievirus B3 requires polyamines for attachment and protease function; however, the mechanism behind this is unknown. Here, we report that polyamines' involvement in translation, through a process called hypusination, globally affects expression of cholesterol synthesis genes by supporting SREBP2 translation, the master transcriptional regulator of cholesterol synthesis genes. By measuring bulk cellular transcription, we found that polyamines enhance the expression of a wide variety of cholesterol synthesis genes, ultimately regulated by SREBP2. The net effect of polyamine depletion on cells negatively impacts CVB3 attachment and replication in polyamine depleted cells by depleting cellular cholesterol. Exogenous cholesterol rescues CVB3 binding and replication, and mutant CVB3 resistant to polyamine depletion exhibits resistance to cholesterol perturbation. This study provides a novel link between polyamine synthesis and cholesterol homeostasis that explains data seen in animals and provides a mechanism through which polyamines impact CVB3 infection.

Combinatorial F-G Immunogens as Nipah and Respiratory Syncytial Virus Vaccine Candidates

Nipah virus (NiV) and respiratory syncytial virus (RSV) possess two surface glycoproteins involved in cellular attachment and membrane fusion, both of which are potential targets for vaccines. The majority of vaccine development is focused on the attachment (G) protein of NiV, which is the immunodominant target. In contrast, the fusion (F) protein of RSV is the main target in vaccine development. Despite this, neutralising epitopes have been described in NiV F and RSV G, making them alternate targets for vaccine design. Through rational design, we have developed a vaccine strategy applicable to phylogenetically divergent NiV and RSV that comprises both the F and G proteins (FxG). In a mouse immunization model, we found that NiV FxG elicited an improved immune response capable of neutralising pseudotyped NiV and a NiV mutant that is able to escape neutralisation by two known F-specific antibodies. RSV FxG elicited an immune response against both F and G and was able to neutralise RSV; however, this was inferior to the immune response of F alone. Despite this, RSV FxG elicited a response against a known protective epitope within G that is conserved across RSV A and B subgroups, which may provide additional protection in vivo. We conclude that inclusion of F and G antigens within a single design provides a streamlined subunit vaccine strategy against both emerging and established pathogens, with the potential for broader protection against NiV.

Rapid Fabrication of MgNH4PO4·H2O/SrHPO4 Porous Composite Scaffolds with Improved Radiopacity via 3D Printing Process

Although bone repair scaffolds are required to possess high radiopacity to be distinguished from natural bone tissues in clinical applications, the intrinsic radiopacity of them is usually insufficient. For improving the radiopacity, combining X-ray contrast agents with bone repair scaffolds is an effective method. In the present research, MgNH4PO4·H2O/SrHPO4 3D porous composite scaffolds with improved radiopacity were fabricated via the 3D printing technique. Here, SrHPO4 was firstly used as a radiopaque agent to improve the radiopacity of magnesium phosphate scaffolds. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive spectroscopy (EDS) were used to characterize the phases, morphologies, and element compositions of the 3D porous composite scaffolds. The radiography image showed that greater SrHPO4 contents corresponded to higher radiopacity. When the SrHPO4 content reached 9.34%, the radiopacity of the composite scaffolds was equal to that of a 6.8 mm Al ladder. The porosity and in vitro degradation of the porous composite scaffolds were studied in detail. The results show that magnesium phosphate scaffolds with various Sr contents could sustainably degrade and release the Mg, Sr, and P elements during the experiment period of 28 days. In addition, the cytotoxicity on MC3T3-E1 osteoblast precursor cells was evaluated, and the results show that the porous composite scaffolds with a SrHPO4 content of 9.34% possessed superior cytocompatibility compared to that of the pure MgNH4PO4·H2O scaffolds when the extract concentration was 0.1 g/mL. Cell adhesion experiments showed that all of the scaffolds could support MC3T3-E1 cellular attachment well. This research indicates that MgNH4PO4·H2O/SrHPO4 porous composite scaffolds have potential applications in the bone repair fields.

Three-Dimensional Culture of Rhipicephalus (Boophilus) microplus BmVIII-SCC Cells on Multiple Synthetic Scaffold Systems and in Rotating Bioreactors

Tick cell culture facilitates research on the biology of ticks and their role as vectors of pathogens that affect humans, domestic animals, and wildlife. Because two-dimensional cell culture doesn’t promote the development of multicellular tissue-like composites, we hypothesized that culturing tick cells in a three-dimensional (3-D) configuration would form spheroids or tissue-like organoids. In this study, the cell line BmVIII-SCC obtained from the cattle fever tick, Rhipicephalus (Boophilus) microplus (Canestrini, 1888), was cultured in different synthetic scaffold systems. Growth of the tick cells on macrogelatinous beads in rotating continuous culture system bioreactors enabled cellular attachment, organization, and development into spheroid-like aggregates, with evidence of tight cellular junctions between adjacent cells and secretion of an extracellular matrix. At least three cell morphologies were identified within the aggregates: fibroblast-like cells, small endothelial-like cells, and larger cells exhibiting multiple cytoplasmic endosomes and granular vesicles. These observations suggest that BmVIII-SCC cells adapted to 3-D culture retain pluripotency. Additional studies involving genomic analyses are needed to determine if BmVIII-SCC cells in 3-D culture mimic tick organs. Applications of 3-D culture to cattle fever tick research are discussed.

Receptor switching in newly evolved adeno-associated viruses

Adeno-associated viruses utilize different glycans and the AAV receptor (AAVR) for cellular attachment and entry. Directed evolution has yielded new AAV variants; however, structure-function correlates underlying their improved transduction are generally overlooked. Here, we report that infectious cycling of structurally diverse AAV surface loop libraries yields functionally distinct variants. Newly evolved variants show enhanced cellular binding, uptake and transduction; but through distinct mechanisms. Using glycan-based and genome-wide CRISPR knockout screens, we discover that one AAV variant acquires the ability to recognize sulfated glycosaminoglycans, while another displays receptor switching from AAVR to Integrin β1 (ITGB1). A previously evolved variant, AAVhum.8, preferentially utilizes the ITGB1 receptor over AAVR. Visualization of the AAVhum.8 capsid by cryo-EM at 2.49Å resolution localizes the newly acquired integrin recognition motif adjacent to the AAVR footprint. These observations underscore the new finding that distinct AAV surface epitopes can be evolved to exploit different cellular receptors for enhanced transduction. Importance Understanding how viruses interact with host cells through cell surface receptors is central to discovery and development of antiviral therapeutics, vaccines and gene transfer vectors. Here, we demonstrate that distinct epitopes on the surface of adeno-associated viruses can be evolved by infectious cycling to recognize different cell surface carbohydrates and glycoprotein receptors and solve the 3D structure of one such newly evolved AAV capsid, which provides a roadmap for designing viruses with improved attributes for gene therapy applications.

Thbs1 induces lethal cardiac atrophy through PERK-ATF4 regulated autophagy

The thrombospondin (Thbs) family of secreted matricellular proteins are stress- and injury-induced mediators of cellular attachment dynamics and extracellular matrix protein production. Here we show that Thbs1, but not Thbs2, Thbs3 or Thbs4, induces lethal cardiac atrophy when overexpressed. Mechanistically, Thbs1 binds and activates the endoplasmic reticulum stress effector PERK, inducing its downstream transcription factor ATF4 and causing lethal autophagy-mediated cardiac atrophy. Antithetically, Thbs1−/− mice develop greater cardiac hypertrophy with pressure overload stimulation and show reduced fasting-induced atrophy. Deletion of Thbs1 effectors/receptors, including ATF6α, CD36 or CD47 does not diminish Thbs1-dependent cardiac atrophy. However, deletion of the gene encoding PERK in Thbs1 transgenic mice blunts the induction of ATF4 and autophagy, and largely corrects the lethal cardiac atrophy. Finally, overexpression of PERK or ATF4 using AAV9 gene-transfer similarly promotes cardiac atrophy and lethality. Hence, we identified Thbs1-mediated PERK-eIF2α-ATF4-induced autophagy as a critical regulator of cardiomyocyte size in the stressed heart.