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Bluetongue virus (BTV) and African horse sickness virus (AHSV) are widespread arboviruses that cause important economic losses in the livestock and equine industries, respectively. In addition to these, another arthropod-transmitted orbivirus known as epizootic hemorrhagic disease virus (EHDV) entails a major threat as there is a conducive landscape that nurtures its emergence in non-endemic countries. To date, only vaccinations with live attenuated or inactivated vaccines permit the control of these three viral diseases, although important drawbacks, e.g., low safety profile and effectiveness, and lack of DIVA (differentiation of infected from vaccinated animals) properties, constrain their usage as prophylactic measures. Moreover, a substantial number of serotypes of BTV, AHSV and EHDV have been described, with poor induction of cross-protective immune responses among serotypes. In the context of next-generation vaccine development, antigen delivery systems based on nano- or microparticles have gathered significant attention during the last few decades. A diversity of technologies, such as virus-like particles or self-assembled protein complexes, have been implemented for vaccine design against these viruses. In this work, we offer a comprehensive review of the nano- and microparticulated vaccine candidates against these three relevant orbiviruses. Additionally, we also review an innovative technology for antigen delivery based on the avian reovirus nonstructural protein muNS and we explore the prospective functionality of the nonstructural protein NS1 nanotubules as a BTV-based delivery platform.

African horse sickness virus (AHSV) is an insect-borne pathogen that causes acute disease in horses and other equids. In an effort to improve the safety of currently available vaccines and to acquire new knowledge about the determinants of AHSV immunogenicity, new generation vaccines are being developed. In this work we have generated and tested a novel immunization approach comprised of nonstructural protein 1 (NS1) of AHSV serotype 4 (AHSV-4) incorporated into avian reovirus muNS protein microspheres (MS-NS1) and/or expressed using recombinant modified vaccinia virus Ankara vector (MVA-NS1). The protection conferred against AHSV by a homologous MS-NS1 or heterologous MS-NS1 and MVA-NS1 prime/boost was evaluated in IFNAR (-/-) mice. Our results indicate that immunization based on MS-NS1 and MVA-NS1 afforded complete protection against the infection with homologous AHSV-4. Moreover, priming with MS-NS1 and boost vaccination with MVA-NS1 (MS-MVA-NS1) triggered NS1 specific cytotoxic CD8 + T cells and prevented AHSV disease in IFNAR (-/-) mice after challenge with heterologous serotype AHSV-9. Cross-protective immune responses are highly important since AHS can be caused by nine different serotypes, which means that a universal polyvalent vaccination would need to induce protective immunity against all serotypes.

Employment of enzymes as biocatalysts offers immense benefits across diverse sectors in the context of green chemistry, biodegradability, and sustainability. When compared to free enzymes in solution, enzyme immobilization proposes an effective means of improving functional efficiency and operational stability. The advance of printable and functional materials utilized in additive manufacturing, coupled with the capability to produce bespoke geometries, has sparked great interest toward the 3-dimensional (3D) printing of immobilized enzymes. Printable biocatalysts represent a new generation of enzyme immobilization in a more customizable and adaptable manner, unleashing their potential functionalities for countless applications in industrial biotechnology. This review provides an overview of enzyme immobilization techniques and 3D printing technologies, followed by illustrations of the latest 3D printed enzyme-immobilized industrial and clinical applications. The unique advantages of harnessing 3D printing as an enzyme immobilization technique will be presented, alongside a discussion on its potential limitations. Finally, the future perspectives of integrating 3D printing with enzyme immobilization will be considered, highlighting the endless possibilities that are achievable in both research and industry

Non-canonical DNA structures, particularly 3-way junctions (3WJs) that are transiently formed during DNA replication, have recently emerged as promising chemotherapeutic targets. Here, we describe a new approach to target 3WJs that relies on the cooperative and sequence selective recognition of A/T-rich duplex DNA branches by three AT-Hook peptides attached to a three-fold symmetric and fluorogenic 1,3,5-tristyrylbenzene core

Protein immobilization on solid supports offers a wide range of opportunities in the biotech industry. Its many possible applications in diverse fields cover from enzyme immobilization promoting an enhancement in the physicochemical properties, to vaccine candidate generation, reducing the associated production costs and improving the immunogenic performance of the vaccine candidate. The immobilization process implicates profound science in both substrate surface and proteins. Proteins are composed of amino acids polymerized jointly through the formation of peptide bonds. One or more polypeptide chains fold into a 3- dimensional (3-D) bioactive protein, with many exposed functional groups such as carboxyl, amine, hydroxyl, and sulfhydryl. Regarding the immobilization strategy, the conformation of the immobilized protein needs to be seriously considered. In the enzyme field, the active site is usually a groove or pocket where the target or substrate molecules bind and experience a chemical reaction. In order to function properly, this active site needs to be in specific and appropriate conformation and accessible to the target. The present study explores two fundamentally different methods for protein immobilization: the IC-Tagging methodology using a modified viral protein, and 3D printing thought the use of Stereolithography (SLA). We investigate the feasibility of these strategies and its efficiency in generating properly folded immobilized proteins for different applications in the field of biocatalysts and cancer immunotherapy.

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomes

Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic and wild ruminants. At the present, 27 different serotypes have been documented. Vaccination has been demonstrated as one of the most effective methods to avoid viral dissemination. To overcome the drawbacks associated with the use of inactivated and attenuated vaccines we engineered a new recombinant BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 that were incorporated into avian reovirus muNS-Mi microspheres (MS-VP2/VP7/NS1) and recombinant modified vaccinia virus Ankara (rMVA). The combination of these two antigen delivery systems in a heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies in IFNAR(−/−) mice. Furthermore, this immunization strategy increased the ratio of IgG2a/IgG1 in sera, indicating an induction of a Th1 response, and elicited a CD8 T cell response. Immunized mice were protected against lethal challenges with the homologous serotype 4 and the heterologous serotype 1 of BTV. All these results support the strategy based on microspheres in combination with rMVAs as a promising multiserotype vaccine candidate against BTV. © 2017 Elsevier B.V.

The generation of dendrimers is a powerful tool in the control of the size and biodistribution of polyion complexes (PIC). Using a combinatorial screening of six dendrimers (18–243 terminal groups) and five oppositely charged PEGylated copolymers, a dendrimer‐to‐PIC hierarchical transfer of structural information was revealed with PIC diameters that increased from 80 to 500 nm on decreasing the dendrimer generation. This rise in size, which was also accompanied by a micelle‐to‐vesicle transition, is interpreted according to a cone‐ to rod‐shaped progression in the architecture of the unit PIC (uPIC). This precise size tuning enabled dendritic PICs to act as nanorulers for controlled biodistribution. Overall, a domino‐like control of the size and biological properties of PIC that is not attainable with linear polymers is feasible through dendrimer generation

We have previously developed a methodology to produce protein microspheres (MS) that can be loaded with proteins of interest in living cells through their C or N-terminal tagging with the so-called IC-Tag. The IC-Tagging method has many applications ranging from the production of immobilized enzymes for industrial use to the production of subunit vaccines due to its intrinsic adjuvancy. Here we show the adaptation of the IC-Tagging to work inside the endoplasmic reticulum and bacteria, allowing us to produce properly modified viral glycoproteins. Additionally, we were able to express the Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), whose expression remained elusive to date possibly due to its toxicity when over-expressed. IGRP is an antigen of enormous pharmaceutical interest as it is specifically targeted during the autoimmune response taking place in both the Non-Obese Diabetic (NOD) mice and type 1 diabetes (T1D) patients leading to the destruction of insulin-producing beta cells

A set of Ru(II) metallopeptides containing the dppz ligand has been synthesized using SPPS methods. Fluorescence titration studies show that those metallopeptides featuring an octaarginine tail display a large binding preference for DNA G-quadruplex structures over those lacking it, and also that the interplay between the octoarginine functionalization and the ancillary ligand in the complex has an essential role in the recognition process. Furthermore, the oligoarginine metallopeptides are also efficiently internalized, causing cell death with signs of apoptosis

La metodología del IC-Tagging se basa en el uso de una proteína, muNS-Mi, que por sí sola es capaz de formar estructuras esféricas en las células (microesferas, MS) que son fácilmente purificables. Además podemos etiquetar cualquier proteína con un tag denominado IC con el cual provocamos que se modifique la localización de forma que las proteínas etiquetadas quedan incluidas en las microesferas. En este estudio se utilizó este sistema para el desarrollo de un sensor de retrotranslocación de proteínas del retículo endoplasmático (RE). Para esto adaptamos el reensamblaje de fragmentos de EGFP al sistema IC-Tagging donde los resultados obtenidos mostraron que sería un sensor poco eficaz. También adaptamos el sistema al interior del retículo endoplasmático para intentar el desarrollo de vacunas contra virus envueltos, produciendo y purificando MS formadas en el interior del RE cargadas con glicoproteínas. Para intentar expresar proteínas difíciles, adaptamos el sistema para expresar y purificar proteínas en bacterias. Por último intentamos resolver la estructura de la proteína muNS-Mi y descubrir como las proteínas etiquetadas se distribuyen en las mismas.

Vaccination is critical for controlling the spread of bluetongue virus (BTV). The inactivated BTV vaccines that are now being used in Europe are effective in preventing outbreaks of BTV but secondary effects associated to repetitive inoculation of aluminum-containing adjuvants and the need to develop safer, cross-reactive, and more efficacious vaccines with differential diagnostic capability have re-stimulated the interest in developing improved vaccination strategies against BTV. We have engineered a subunit BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 incorporated into avian reovirus (ARV) muNS-Mi microspheres (MS-VP2/MS-VP7/MS-NS1). IFNAR(-/-) mice immunized with MS-VP2/MS-VP7/MS-NS1 without adjuvant generated significant levels of neutralizing antibodies specific to BTV-4. In addition, vaccination stimulated specific T cell responses, predominantly CD4+, against the virus. Immunized mice were fully protected against a homologous challenge with a lethal dose of BTV-4 and partially cross-protected against a heterologous challenge with a lethal dose of BTV-1. These results support MS-VP2/MS-VP7/MS-NS1 as a promising subunit vaccine candidate against multiple serotypes of BTV as well as the use of microspheres as an alternative delivery method with potent intrinsic adjuvant activity. © 2014 Elsevier B.V. All rights reserved.

Polyion complex (PIC) micelles incorporating PEG-dendritic copolymers display an unprecedented stability towards ionic strength that is amplified via hydrophobic interactions. The tridimensional orientation of peripheral hydrophobic linkers between charged groups and the globular/rigid dendritic scaffold maximizes this stabilization compared to PIC micelles from linear polymers. As a result, micelles stable at concentrations higher than 3 M NaCl are obtained, which represents the highest saline concentration attained with PIC micelles. Advantages of this stabilizing dendritic effect have been taken for the design of a robust, pH-sensitive micelle for the controlled intracellular release of the anticancer drug doxorubicin. This micelle displays a slightly higher toxicity, and distinctive mechanisms of cell uptake and intracellular trafficking relative to the free drug. The preparation of mixed PIC micelles by combining differently functionalized PEG-dendritic block copolymers has allowed the fine-tuning of their stability, paving the way towards the facile modulation of properties like biodegradability, drug loading, or the response to external stimuli

Bluetongue virus (BTV) is the causative agent of bluetongue disease (BT), which affects domestic and wild ruminants. At the present, 27 different serotypes have been documented. Vaccination has been demonstrated as one of the most effective methods to avoid viral dissemination. To overcome the drawbacks associated with the use of inactivated and attenuated vaccines we engineered a new recombinant BTV vaccine candidate based on proteins VP2, VP7, and NS1 of BTV-4 that were incorporated into avian reovirus muNS-Mi microspheres (MS-VP2/VP7/NS1) and recombinant modified vaccinia virus Ankara (rMVA). The combination of these two antigen delivery systems in a heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies in IFNAR(−/−) mice. Furthermore, this immunization strategy increased the ratio of IgG2a/IgG1 in sera, indicating an induction of a Th1 response, and elicited a CD8 T cell response. Immunized mice were protected against lethal challenges with the homologous serotype 4 and the heterologous serotype 1 of BTV. All these results support the strategy based on microspheres in combination with rMVAs as a promising multiserotype vaccine candidate against BTV

We have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cells

A series of oligoarginine peptide derivatives containing cyclometallated iridium(III) units display remarkable cytotoxicity, comparable to that of cisplatin. In vitro studies with unilamellar vesicles support a membrane-disrupting mechanism of action

A new method is reported herein for screening the biological activity of functional polymers across a consistent degree of polymerization and in situ, that is, under aqueous conditions and without purification/isolation of candidate polymers. In brief, the chemical functionality of a poly(acryloyl hydrazide) scaffold was activated under aqueous conditions using readily available aldehydes to obtain amphiphilic polymers. The transport activity of the resulting polymers can be evaluated in situ using model membranes and living cells without the need for tedious isolation and purification steps. This technology allowed the rapid identification of a supramolecular polymeric vector with excellent efficiency and reproducibility for the delivery of siRNA into human cells (HeLa-EGFP). The reported method constitutes a blueprint for the high-throughput screening and future discovery of new polymeric functional materials with important biological applications

Los reovirus aviares son virus sin envoltura lipídica y genoma de ARN bicatenario que infectan la aves, causando enfermedades como la artritis infecciosa o el síndrome de malabsorción. Durante su infección entre las proteínas virales expresadas la proteína no estructural muNS juega un papel clave llevando a cabo la función de formar y organizar los viroplasmas y atraer a los mismos parte de las proteínas estructurales que formarán los viriones de la progenie. Durante la infección del virus homólogo de mamíferos (MRV) se expresan dos isoformas que se diferencian en el extremo amino de su secuencia, donde se encuentran secuencias responsables de la interacción con otras proteínas virales. En este trabajo demostramos que durante la infección de células aviares (CEF) por el reovirus aviar (ARV) también se expresan dos isoformas de muNS diferentes en su extremo amino pero, a diferencia del MRV, donde un mecanismo de iniciación alternativo de la traducción es el responsable, en el caso de los ARV éstas se generan por un procesamiento proteolítico de la proteína muNS en el aminoácido que ocupa la posición 154. De esta manera en realidad son 3 las isoformas que se pueden detectar de muNS en células infectadas, muNS entera y los dos productos muNSN y MUNSC. Esta modificación de la proteína muNS de reovirus aviar conlleva la pérdida de la interacción con las proteínas estructurales lambdaA y lambdaC y la no estructural sigmaNS. Asimismo, este procesamiento es llevado a cabo por una caspasa de tipo 3 activada como consecuencia del desencadenamiento de la apoptosis activada por la infección viral. En este trabajo también demostramos que la activación de la apoptosis le sirve al virus para aumentar su diseminación en células en cultivo. Por otra parte, también demostramos que la proteína muNS de origen aviar sufre fosforilaciónes múltiples en residuos de serina principalmente así como en treoninas también.

Os reovirus aviarios son virus sen envoltura lipídica e xenoma de ARN bicatenario que infectan a aves, causando enfermidades coma a artrite infecciosa ou a síndrome de malabsorción. Neste traballo estudouse a resposta inmune innata que este virus desencadea en dous tipos celulares do seu hospedador natural, o polo, e comparouse coa xerada por outros virus coma o virus vaccinia e o virus da estomatite vesicular. O reovirus aviario é o único dos virus estudados capaz de inducir a produción de interferón (IFN) e aumentar os niveis da proteína quinasa R (PKR) en cultivos primarios de fibroblastos embrionarios de polo (CEF), mentres que é incapaz de facelo na liña celular DF1 derivada destes, o que podería depender de diferenzas nos niveis dos receptores de recoñecemento de patrón (PRRs) ou a unha menor eficiencia de infección destas células. A indución de IFN en CEF infectadas co reovirus aviario depende da decapsidación do virus pero non da expresión dos seus xenes. O reovirus aviario é resistente ao IFN e é capaz de impedir a activación de PKR e a fosforilación de eIF2, ao contrario que os outros dous virus estudados. Esta capacidade para impedir a activación de PKR depende da capacidade da proteína A para unir ARN bicatenario. Por outra banda, observouse que o pretratamento con IFN de células DF1 aumenta a cantidade de células infectadas co reovirus aviario mediante un mecanismo independente da activación das caspasas. Finalmente, estudáronse algúns posibles mecanismos da inhibición da síntese das proteínas celulares durante a infección con reovirus aviario.

A flexible and versatile synthetic approach for the construction of water-stable DNA-binding chiral peptide helicates based on the solid phase peptide synthesis (SPPS) methodology is reported

Avian reovirus non-structural protein muNS is partially cleaved in infected chicken embryo fibroblast cells to produce a 55-kDa carboxyterminal protein, termed muNSC, and a 17-kDa aminoterminal polypeptide, designated muNSN. In this study we demonstrate that muNS processing is catalyzed by a caspase 3-like protease activated during the course of avian reovirus infection. The cleavage site was mapped by site directed mutagenesis between residues Asp-154 and Ala-155 of the muNS sequence. Although muNS and muNSC, but not muNSN, are able to form inclusions when expressed individually in transfected cells, only muNS is able to recruit specific ARV proteins to these structures. Furthermore, muNSC associates with ARV factories more weakly than muNS, sigmaNS and lambdaA. Finally, the inhibition of caspase activity in ARV-infected cells does not diminish ARV gene expression and replication, but drastically reduces muNS processing and the release and dissemination of progeny viral particles

We report the discovery of a fluorogenic dye, N1,N3-di(2-aminidonaphthalen-6-yl) propane-1,3-diamine, MitoBlue, which selectively stains functional mitochondria while displaying low toxicity, bright blue emission, and high resistance to photobleaching. Additionally, we show that a biotin-labeled MitoBlue derivative can be used as a handle for the delivery of streptavidin-tagged species to the mitochondria

Bisbenzamidines are an important family of minor groove DNA-binding agents. We present a one-step synthesis of aromatic aza-bisbenzamidines that allows straightforward and versatile access to a large number of these molecules. One of them, the azide-aza-bisbenzamidine 13, can be readily modified via click-chemistry with a variety of functionalities that can, therefore, be delivered to the vicinity of an A/T-rich DNA minor groove. This strategy, therefore, provides a simple means for triggering site selective, DNA-promoted biochemical and physicochemical processes

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells

Chemical derivatization of nucleic stains such as ethidium bromide or DAPI with tailored, photoresponsive caging groups, allows for “on demand” spatiotemporal control of their in vivo nucleic acid binding, as well as for improving their cellular uptake. This effect was particularly noteworthy for a nitro-veratryloxycarbonyl-caged derivative of ethidium bromide that, in contrast with the parent stain, is effectively internalized into living cells. The activation strategy works in light-accessible, therapeutically relevant settings, such as human retinas, and can even be applied for the release of active compounds in the eyes of living mice

Background: Characterization of protein-protein interactions is essential for understanding cellular functions. Although there are many published methods to analyze protein-protein interactions, most of them present serious limitations. In a different study we have characterized a novel avian reovirus muNS-based protein tagging and inclusion targeting method, and demonstrated its validity to purify free an immobilized protein. Methodology/Principal Findings: Here we present a method to identify protein-protein interactions inside living eukaryotic cells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477–542), it not only got integrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to these structures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimeras that form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturing IC-tagged proteins, and that the proteins targeted to nuclear inclusions are able to interact with their known ligands. Conclusions/Significance: Our protein redistribution method does not present the architectural requirement of reconstructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus of living cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration.

Background: Avian reoviruses replicate in viral factories, which are dense cytoplasmic compartments estabilished by protein-protein interactions. The non-structural protein muNS forms the factory scaffold that attracts other viral components in a controlled fashion. To create such a three-dimensional network, muNS uses several different selfinteracting domains. Methodology/Principal Findings: In this study we have devised a strategy to identify muNS regions containing selfinteracting domains, based on the capacity of muNS-derived inclusions to recruit muNS fragments. The results revealed that the muNS region consisting of residues 477–542 was recruited with the best efficiency, and this raised the idea of using this fragment as a molecular tag for delivering foreign proteins to muNS inclusions. By combining such tagging system with our previously established method for purifying muNS inclusions from baculovirus-infected insect cells, we have developed a novel protein purification protocol. Conclusions/Significance: We show that our tagging and inclusion-targeting system can be a simple, versatile and efficient method for immobilizing and purifying active proteins expressed in baculovirus-infected cells. We also demonstrate that muNS inclusions can simultaneously recruit several tagged proteins, a finding which may be used to generate protein complexes and create multiepitope particulate material for immunization purposes.