Development of a highly sensitive TaqMan method based on multi-probe strategy: its application in ASFV detection.

The establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/µl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.

Protein kinase Cdc7 supports viral replication by phosphorylating Avibirnavirus VP3 protein.

IMPORTANCE: The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.

African swine fever virus protein p17 promotes mitophagy by facilitating the interaction of SQSTM1 with TOMM70.

Viruses have developed different strategies to hijack mitophagy to facilitate their replication. However, whether and how African swine fever virus (ASFV) regulates mitophagy are largely unknown. Here, we found that the ASFV-encoded p17 induced mitophagy. Coimmunoprecipitation/mass spectrometry assays identified translocase of outer mitochondrial membrane 70 (TOMM70) as the protein that interacted with p17. The binding of TOMM70 to p17 promoted the binding of the mitophagy receptor SQSTM1 to TOMM70, led to engulfment of mitochondria by autophagosomes, and consequently decreased the number of mitochondria. Consistently, the levels of TOMM70 and TOMM20 decreased substantially after p17 expression or ASFV infection. Furthermore, p17-mediated mitophagy resulted in the degradation of mitochondrial antiviral signalling proteins and inhibited the production of IFN-α, IL-6 and TNFα. Overall, our findings suggest that ASFV p17 regulates innate immunity by inducing mitophagy via the interaction of SQSTM1 with TOMM70.

Metal-printing tunable interlayer waveguide coupler using low-loss fluorinated polycarbonate.

Tunable three-dimensional (3D) integrated optical waveguide chips with optical interconnection function are beneficial to expand the application of optical devices in a 3D integrated photonic module. Here, we propose a thermo-optic (TO) tunable interlayer waveguide coupler based on the metal-printing technique. Low-loss fluorinated polycarbonate (AF-Ali-PC MA) and poly (methyl methacrylate-glycidyl methacrylate) [P(MMA-co-GMA)] are synthesized as waveguide core and cladding layer, respectively. The thermal stability and optical adsorption characteristics of AF-Ali-PC MA are analyzed. Optical signal transmission features of the interlayer coupling waveguides are simulated. The optical response properties and fabrication process flows of a dynamic multilayer waveguide chip can be greatly improved by the metal-printing technique. The on-off time of the TO interlayer coupling chip is obtained as 250 µs, and the electrical power consumption is measured as 7.6 mW. To the best of our knowledge, this is the first time that a TO tunable interlayer waveguide coupler is achieved by an efficient metal-printing method, which is suitable for large-scale photonic integrated circuit (PIC) systems and multilayer optical interconnection (OXC) networks.

Interlayer directional coupling thermo-optic waveguide switches based on functionalized epoxy-crosslinking polymers.

In this study, interlayer directional coupling (DC) thermo-optic (TO) waveguide switches were designed and fabricated using functionalized epoxy-crosslinking polymers. Fluorinated SU-8 (FSU-8) with a photo-initiating epoxy-crosslinking network was self-synthesized as a waveguide core material. A copolymer of methyl methacrylate and glycidyl methacrylate P(MMA-co-GMA) with a thermo-initiating epoxy crosslinking structure was self-synthesized as a waveguide cladding material. Compared with commercial pure SU-8 and PMMA, FSU-8 exhibited a lower absorption loss and P(MMA-co-GMA) exhibited a higher thermal stability. Using epoxy-crosslinking functionalized polymers, the structure of the waveguides and electrode heaters were optimized, and the performance parameters of the interlayer DC TO switches were simulated. At a signal wavelength of 1550 nm, the insertion loss, extinction ratio, and power consumption of the actual interlayer devices were measured as 6.7 dB, 15.6 dB, and 9 mW, respectively. The rising and falling response times of the TO switches were obtained as 631.6 µs and 362 µs, respectively. The self-leveling ability and solvent resistance characteristic of the epoxy-crosslinking network for FSU-8 and P(MMA-co-GMA) may guarantee the realization of interlayer DC TO waveguide switches. The proposed technique will be suitable for photonic integrated waveguide chips with multilayer stacking dynamic optical information interactions.

TRAF6 autophagic degradation by avibirnavirus VP3 inhibits antiviral innate immunity via blocking NFKB/NF-κB activation.

Ubiquitination is an important reversible post-translational modification. Many viruses hijack the host ubiquitin system to enhance self-replication. In the present study, we found that Avibirnavirus VP3 protein was ubiquitinated during infection and supported virus replication by ubiquitination. Mass spectrometry and mutation analysis showed that VP3 was ubiquitinated at residues K73, K135, K158, K193, and K219. Virus rescue showed that ubiquitination at sites K73, K193, and K219 on VP3 could enhance the replication abilities of infectious bursal disease virus (IBDV), and that K135 was essential for virus survival. Binding of the zinc finger domain of TRAF6 (TNF receptor associated factor 6) to VP3 mediated K11- and K33-linked ubiquitination of VP3, which promoted its nuclear accumulation to facilitate virus replication. Additionally, VP3 could inhibit TRAF6-mediated NFKB/NF-κB (nuclear factor kappa B) activation and IFNB/IFN-ß (interferon beta) production to evade host innate immunity by inducing TRAF6 autophagic degradation in an SQSTM1/p62 (sequestosome 1)-dependent manner. Our findings demonstrated a macroautophagic/autophagic mechanism by which Avibirnavirus protein VP3 blocked NFKB-mediated IFNB production by targeting TRAF6 during virus infection, and provided a potential drug target for virus infection control.Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; Cas9: CRISPR-associated protein 9; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GST: glutathione S-transferase; IBDV: infectious bursal disease virus; IF: indirect immunofluorescence; IFNB/IFN-ß: interferon beta; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MS: mass spectrometry; NFKB/NF-κB: nuclear factor kappa B; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; pAb: polyclonal antibody; PRRs: pattern recognition receptors; RNF125: ring finger protein 125; RNF135/Riplet: ring finger protein 135; SQSTM1/p62: sequestosome 1; TAX1BP1: tax1 binding protein1; TCID50: 50% tissue culture infective dose; TRAF3: TNF receptor associated factor 3; TRAF6: TNF receptor associated factor 6; TRIM25: tripartite motif containing 25; Ub: ubiquitin; Wort: wortmannin; WT: wild type.

Dual Cross-Linked Hydrogels with Injectable, Self-Healing, and Antibacterial Properties Based on the Chemical and Physical Cross-Linking.

Injectable hydrogels have become a promising material for biomedical engineering applications, but microbial infection remains a common challenge in their application. In this study, we presented an injectable antibacterial hydrogel with self-healing property based on a dual cross-linking network structure of dynamic benzoxaborole-sugar and quadruple hydrogen bonds of the 2-ureido-4-pyrimidone (UPy) moieties at physiological pH. Dynamic rheological experiments demonstrated the gelatinous behavior of the double cross-linking network (storage modulus G' > loss modulus G″), and the modulus showed frequency-dependent behavior. The noncovalent interactions of UPy units in the polymer segment endowed the injectable hydrogels with good mechanical strength. By varying the solid contents, UPy units, as well as the pH, the mechanical properties of hydrogels could be controlled. Additionally, the hydrogels exhibited not only excellent self-healing and injectable properties but also pH and sugar dual-responsiveness. Moreover, the hydrogels could effectively inhibit the growth of both Escherichia coli and Staphylococcus aureus while exhibiting low toxicity. 3D cell encapsulation experiment results also demonstrated the potential use of these hydrogels as cell culture scaffolds. Taken together, the injectability, self-healing, and antimicrobial properties of the prepared hydrogels showed great promise for translational medicine, such as cell and tissue engineering applications.

Bottom-metal-printed thermo-optic waveguide switches based on low-loss fluorinated polycarbonate materials.

In this work, thermo-optic (TO) waveguide switches are designed and fabricated based on the bottom-metal-printed technique. Low-loss fluorinated polycarbonate (AF-Z-PC MA) and polymethyl methacrylate (PMMA) are used as core and cladding materials, respectively. The thermal stability and optical absorption characteristics of AF-Z-PC MA are analyzed. The optical and thermal field distributions of the TO switch are simulated. The insertion loss and extinction ratio of the device are found to be 4.5 dB and 19.8 dB, respectively, at a wavelength of 1550 nm. The on-off time of the switching chip is 80 µs. The electrical power consumption is approximately 8.8 mW. The proposed low-loss fluorinated polymer TO waveguide switch realized by bottom-metal-printed fabrication technology is suitable for large-scale integrated photonic circuit systems.