Formation of Antifouling Brushes on Various Substrates Using a Melanin-Inspired Initiator Film.

In this study, we developed a substrate-independent initiator film that can undergo surface-initiated polymerization to form an antifouling brush. Inspired by the melanogenesis found in nature, we synthesized a tyrosine-conjugated bromide initiator (Tyr-Br) that contains phenolic amine groups as the dormant coating precursor and α-bromoisobutyryl groups as the initiator. The resultant Tyr-Br was stable under ambient air conditions and underwent melanin-like oxidation only in the presence of tyrosinase to form an initiator film on various substrates. Subsequently, an antifouling polymer brush was formed using air-tolerant activators regenerated by electron transfer for atom transfer radical polymerization (ARGET ATRP) of zwitterionic carboxybetaine. The entire surface coating procedure, including the initiator layer formation and ARGET ATRP, occurred under aqueous conditions and did not require organic solvents or chemical oxidants. Therefore, antifouling polymer brushes can be feasibly formed not only on experimentally preferred substrates (e.g., Au, SiO2, and TiO2) but also on polymeric substrates such as poly(ethylene terephthalate) (PET), cyclic olefin copolymer (COC), and nylon.

Antifouling Surface Coating on Various Substrates by Inducing Tyrosinase-Mediated Oxidation of a Tyrosine-Conjugated Sulfobetaine Derivative.

Inspired by the melanogenesis occurring in nature, we report tyrosinase-mediated antifouling surface coating by synthesizing a tyrosine-conjugated sulfobetaine derivative (Tyr-SB). Synthetic Tyr-SB contains zwitterionic sulfobetaine and tyrosine, whose phenolic amine group acts as a dormant coating precursor. In contrast to catecholamine derivatives, tyrosine derivatives are stable against auto-oxidation and are enzymatically oxidized only in the presence of tyrosinase to initiate melanin-like oxidation. When the surface of interest was applied during the course of Tyr-SB oxidation, a superhydrophilic poly(Tyr-SB) film was coated on the surfaces, thereby showing antifouling performance against proteins or adherent cells. Because the oxidation of Tyr-SB occurred under mild aqueous conditions (pH 6-7) without the use of any chemical oxidants, such as sodium periodate or ammonium persulfate, we anticipate that the coating method described herein will serve as a biocompatible tool in the field of biosensors, cell surface engineering, and medical devices, whose interfaces differ in chemistry.

Effect of N-Methylation on Dopamine Surface Chemistry.

Dopamine (DA) surface chemistry has received significant attention because of its applicability in a wide range of research fields and the ability to graft functional molecules onto numerous solid surfaces. Various DA derivatives have been newly synthesized to identify key factors affecting the coating efficiency and to advance the coating system development. The oxidation of catechol into quinone followed by internal cyclization via the nucleophilic attack of primary amine is crucial for DA-based surface coating. Thus, it is expected that the amine group's nucleophilicity control directly affects the coating efficiency. However, it has not been systematically investigated, and most studies have been conducted with the focus on the transformation of amines into amides, despite such approaches decreasing the coating efficiency; the nitrogen in amides is less nucleophilic than that in free amines. In this study, we investigated the effect of N-alkylation on dopamine surface chemistry. N,N-Dimethyldopamine (DMDA) was newly synthesized, and the coating efficiency was systematically compared with DA and N-methyldopamine (MDA). DA N-monomethylation improved the coating rate by increasing the nitrogen nucleophilicity, whereas N,N-dimethylation dramatically decreased the DA surface coating property. In addition, MDA remained capable of universal surface coating and secondary reactions using the surface catechols. This study provides opportunities for developing coating materials with advanced functions and an improved coating rate.

Development of Universal and Clickable Film by Mimicking Melanogenesis: On-Demand Oxidation of Tyrosine-Based Azido Derivative by Tyrosinase.

A tyrosine-based azido derivative (TBAD) that permits both substrate-independent surface coating and clickable film functionalization by mimicking natural melanogenesis is synthesized here. In contrast to catechol derivatives, which are generally susceptible to oxidation by air under ambient conditions, the monophenol-based TBAD remains stable under alkaline and neutral conditions and is activated to oxidized quinone in situ by tyrosinase to initiate melanin-like polymerization. The resulting poly(TBAD) film can be formed on various substrates including noble metals, metal oxides, and synthetic polymers, which can undergo click reaction with terminal alkyne moieties on the entire surface or a specific region through Cu(I)-catalyzed azide-alkyne cycloaddition. The enzyme-mediated coating can rapidly form thin films (≈10 nm) and produce a uniform film morphology, which are important aspects in surface chemistry. This on-demand, clickable coating may become a significant tool for bioconjugation, soft lithography, and labeling techniques.

Development of Stimulus-Responsive Degradable Film via Codeposition of Dopamine and Cystamine.

Herein, we report a degradable film that can be coated on various substrates by the codeposition of dopamine and cystamine. The thickness of the resulting film (pDC) varies depending on the initial ratio of dopamine/cystamine dissolved in a solution; the thickest film (ca. 60 nm) is obtained under optimized codeposition conditions. Selective degradation of pDC occurs in the presence of tris(2-carboxyethyl)phosphine (TCEP), the reaction kinetics of which are highly dependent on the TCEP concentration. For further application as a drug-delivery platform, doxorubicin can be loaded within the pDC film, which is released actively under film degradation in response to TCEP. We expect that the developed pDC film will be a useful tool for developing drug delivery cargo, antibacterial surface, and cell surface coating for various biomedical applications.

Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants.

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

Principles and Recommendations for Standardizing the Use of the Next-Generation Sequencing Variant File in Clinical Settings.

A national workgroup convened by the Centers for Disease Control and Prevention identified principles and made recommendations for standardizing the description of sequence data contained within the variant file generated during the course of clinical next-generation sequence analysis for diagnosing human heritable conditions. The specifications for variant files were initially developed to be flexible with regard to content representation to support a variety of research applications. This flexibility permits variation with regard to how sequence findings are described and this depends, in part, on the conventions used. For clinical laboratory testing, this poses a problem because these differences can compromise the capability to compare sequence findings among laboratories to confirm results and to query databases to identify clinically relevant variants. To provide for a more consistent representation of sequence findings described within variant files, the workgroup made several recommendations that considered alignment to a common reference sequence, variant caller settings, use of genomic coordinates, and gene and variant naming conventions. These recommendations were considered with regard to the existing variant file specifications presently used in the clinical setting. Adoption of these recommendations is anticipated to reduce the potential for ambiguity in describing sequence findings and facilitate the sharing of genomic data among clinical laboratories and other entities.

Poxviruses Encode a Reticulon-Like Protein that Promotes Membrane Curvature.

Poxviruses are enveloped DNA viruses that replicate within the cytoplasm. The first viral structures are crescents and spherical particles, with a lipoprotein membrane bilayer, that are thought to be derived from the ER. We determined that A17, a conserved viral transmembrane protein essential for crescent formation, forms homo-oligomers and shares topological features with cellular reticulon-like proteins. The latter cell proteins promote membrane curvature and contribute to the tubular structure of the ER. When the purified A17 protein was incorporated into liposomes, 25 nm diameter vesicles and tubules formed at low and high A17 concentrations, respectively. In addition, intracellular expression of A17 in the absence of other viral structural proteins transformed the ER into aggregated three-dimensional (3D) tubular networks. We suggest that A17 is a viral reticulon-like protein that contributes to curvature during biogenesis of the poxvirus membrane.

Analysis of viral membranes formed in cells infected by a vaccinia virus L2-deletion mutant suggests their origin from the endoplasmic reticulum.

Assembly of the poxvirus immature virion (IV) membrane is a poorly understood event that occurs within the cytoplasm. At least eight viral proteins participate in formation of the viral membrane. Of these, A14, A17, and D13 are structural components whereas A6, A11, F10, H7, and L2 participate in membrane biogenesis. L2, the object of this study, is conserved in all chordopoxviruses, expressed early in infection, and associated with the endoplasmic reticulum (ER) throughout the cell and at the edges of crescent-shaped IV precursors. Previous studies with an inducible L2 mutant revealed abortive formation of the crescent membrane. However, possible low-level L2 synthesis under nonpermissive conditions led to ambiguity in interpretation. Here, we constructed a cell line that expresses L2, which allowed the creation of an L2-deletion mutant. In noncomplementing cells, replication was aborted prior to formation of mature virions and two types of aberrant structures were recognized. One consisted of short crescents, at the surface of dense masses of viroplasm, which were labeled with antibodies to the A11, A14, A17, and D13 proteins. The other structure consisted of "empty" IV-like membranes, also labeled with antibodies to the viral proteins, which appeared to be derived from adjacent calnexin-containing ER. A subset of 25 proteins examined, exemplified by components of the entry-fusion complex, were greatly diminished in amount. The primary role of L2 may be to recruit ER and modulate its transformation to viral membranes in juxtaposition with the viroplasm, simultaneously preventing the degradation of viral proteins dependent on viral membranes for stability.

Kinetics and intracellular location of intramolecular disulfide bond formation mediated by the cytoplasmic redox system encoded by vaccinia virus.

Poxviruses encode a redox system for intramolecular disulfide bond formation in cytoplasmic domains of viral proteins. Our objectives were to determine the kinetics and intracellular location of disulfide bond formation. The vaccinia virus L1 myristoylated membrane protein, used as an example, has three intramolecular disulfide bonds. Reduced and disulfide-bonded forms of L1 were distinguished by electrophoretic mobility and reactivity with monoclonal and polyclonal antibodies. Because disulfide bonds formed during 5 min pulse labeling with radioactive amino acids, a protocol was devised in which dithiothreitol was present at this step. Disulfide bond formation was detected by 2 min after removal of reducing agent and was nearly complete in 10 min. When the penultimate glycine residue was mutated to prevent myristoylation, L1 was mistargeted to the endoplasmic reticulum and disulfide bond formation failed to occur. These data suggested that viral membrane association was required for oxidation of L1, providing specificity for the process.

Assembly and disassembly of the capsid-like external scaffold of immature virions during vaccinia virus morphogenesis.

Infectious poxvirus particles are unusual in that they are brick shaped and lack symmetry. Nevertheless, an external honeycomb lattice comprised of a capsid-like protein dictates the spherical shape and size of immature poxvirus particles. In the case of vaccinia virus, trimers of 63-kDa D13 polypeptides form the building blocks of the lattice. In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the immature virion (IV) membrane to form the lattice structure and how this scaffold is removed during the subsequent stage of morphogenesis. Interaction of D13 with the A17 membrane protein was demonstrated by immunoaffinity purification and Western blot analysis. In addition, the results of immunogold electron microscopy indicated a close association of A17 and D13 in crescents, as well as in vesicular structures when crescent formation was prevented. Further studies indicated that binding of A17 to D13 was abrogated by truncation of the N-terminal segment of A17. The N-terminal region of A17 was also required for the formation of crescent and IV structures. Disassembly of the D13 scaffold correlated with the processing of A17 by the I7 protease. When I7 expression was repressed, D13 was retained on aberrant virus particles. Furthermore, the morphogenesis of IVs to mature virions was blocked by mutation of the N-terminal but not the C-terminal cleavage site on A17. Taken together, these data indicate that A17 and D13 interactions regulate the assembly and disassembly of the IV scaffold.

Vaccinia virus l1 protein is required for cell entry and membrane fusion.

Genetic and biochemical studies have provided evidence for an entry/fusion complex (EFC) comprised of at least eight viral proteins (A16, A21, A28, G3, G9, H2, J5, and L5) that together with an associated protein (F9) participates in entry of vaccinia virus (VACV) into cells. The genes encoding these proteins are conserved in all poxviruses, are expressed late in infection, and are components of the mature virion membrane but are not required for viral morphogenesis. In addition, all but one component has intramolecular disulfides that are formed by the poxvirus cytoplasmic redox system. The L1 protein has each of the characteristics enumerated above except that it has been reported to be essential for virus assembly. To further investigate the role of L1, we constructed a recombinant VACV (vL1Ri) that inducibly expresses L1. In the absence of inducer, L1 synthesis was repressed and vL1Ri was unable to form plaques or produce infectious progeny. Unexpectedly, assembly and morphogenesis appeared normal and the noninfectious virus particles were indistinguishable from wild-type VACV as determined by transmission electron microscopy and analysis of the component polypeptides. Notably, the L1-deficient virions were able to attach to cells but the cores failed to penetrate into the cytoplasm. In addition, cells infected with vL1Ri in the absence of inducer did not form syncytia following brief low-pH treatment even though extracellular virus was produced. Coimmunoprecipitation experiments demonstrated that L1 interacted with the EFC and indirectly with F9, suggesting that L1 is an additional component of the viral entry apparatus.

Neutralizing antibody and protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein.

A secreted, glycosylated polypeptide containing amino acids 14 to 762 of the SARS coronavirus (SARS-CoV) spike protein and a polyhistidine tag was expressed in recombinant baculovirus-infected insect cells. Mice received the affinity-purified protein with either a saponin (QS21) or a Ribi (MPL + TDM) adjuvant subcutaneously and were challenged intranasally with SARS-CoV. Both regimens induced binding and neutralizing antibodies and protection against SARS-CoV intranasal infection. However, the best results were obtained with QS21 and protein, which provided the highest antibody as well as complete protection of the upper and lower respiratory tract.

Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice.

The spike protein (S), a membrane component of severe acute respiratory syndrome coronavirus (SARS-CoV) is anticipated to be an important component of candidate vaccines. We constructed recombinant forms of the highly attenuated modified vaccinia virus Ankara (MVA) containing the gene encoding full-length SARS-CoV S with and without a C-terminal epitope tag called MVA/S-HA and MVA/S, respectively. Cells infected with MVA/Sor MVA/S-HA synthesized a 200-kDa protein, which was recognized by antibody raised against a synthetic peptide of SARS-CoV S or the epitope tag in Western blot analyses. Further studies indicated that S was N-glycosylated and migrated in SDS polyacrylamide gels with an apparent mass of approximately 160 kDa after treatment with peptide N-glycosidase F. The acquisition of resistance to endoglycosidase H indicated trafficking of S to the medial Golgi compartment, and confocal microscopy showed that S was transported to the cell surface. Intranasal or intramuscular inoculations of BALB/c mice with MVA/S produced serum antibodies that recognized the SARS S in ELISA and neutralized SARS-CoV in vitro. Moreover, MVA/S administered by either route elicited protective immunity, as shown by reduced titers of SARS-CoV in the upper and lower respiratory tracts of mice after challenge. Passive transfer of serum from mice immunized with MVA/S to naïve mice also reduced the replication of SARS-CoV in the respiratory tract after challenge, demonstrating a role for antibody to S in protection. The attenuated nature of MVA and the ability of MVA/S to induce neutralizing antibody that protects mice support further development of this candidate vaccine.

Recombinant dengue virus type 2 envelope/hepatitis B surface antigen hybrid protein expressed in Pichia pastoris can function as a bivalent immunogen.

A truncated version of the dengue virus type 2 envelope protein (Den2E) encoding the first 395 amino acid (aa) residues, and Den2E fused in-frame with the full-length 226-aa hepatitis B surface antigen (Den2E-HBsAg) protein were expressed in the methylotrophic yeast, Pichia pastoris. Both the recombinant proteins showed evidence of the capacity to form high molecular weight aggregates. Electron microscopic analysis of the purified proteins showed that while Den2E displayed an amorphous morphology, Den2E-HBsAg existed as well-structured virus-like particles (VLPs). Using immuno-gold electron microscopy, these VLPs were demonstrated to contain both components of the Den2E-HBsAg hybrid protein. Seroanalysis showed that the hybrid VLPs could function in vivo as bivalent immunogens, which could elicit immune responses directed against both components of the hybrid protein, as evidenced by ELISA, immunoprecipitation and immunofluorescence data.