Constructing molecularly imprinted membranes with instant noodles-like structure for selectively separating acteoside.

Acteoside (ACT) was the main bioactive components in phenylethanoid glycosides of Cistanche tubulosa. Currently, the development of an efficient method for selectively separating ACT was crucial. Consequently, yolk-shell magnetic mesoporous carbon (YSMMC) was synthesized as a nanofiller to prepare molecularly imprinted membranes (ACT-MIMs) with instant noodles-like structure for selectively separating ACT. The numerous YSMMC were moved to the upper surface of ACT-MIMs by magnetic guidance and constructed the instant noodles-like structure in ACT-MIMs. The instant noodle-like structure increased the surface roughness of ACT-MIMs, which was conducive to improving the effective imprinted interface, increasing the selectivity of ACT-MIMs. In addition, the instant noodle-like structure had dendritic interleaved pathways in ACT-MIMs. The dendritic interleaved pathways can intercept ACT through ACT-MIMs, enhancing the permselectivity of ACT-MIMs. The prepared YSMMC possessed the dendritic shell and interlayer cavity structure can provide a great accommodation space, improving the rebinding capacities of ACT-MIMs. The high permselectivity (14.49), remarkable selectivity (7.52), and excellent rebinding capacity (120.48 mg/g) were achieved for the prepared ACT-MIMs. Thus, the design of ACT-MIMs with the instant noodles-like structure were valuable for selectively separating of bioactive components.

Foot-and-mouth disease virus 3C protease cleaves NEMO to impair innate immune signaling.

Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (L(pro)) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C(pro), also inhibits IFN-α/ß response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C(pro) of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-κB and interferon-regulatory factor signaling pathways. 3C(pro) specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3C(pro) abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C(pro) and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.

The leader proteinase of foot-and-mouth disease virus negatively regulates the type I interferon pathway by acting as a viral deubiquitinase.

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is a papain-like proteinase that plays an important role in FMDV pathogenesis. Previously, it has been shown that L(pro) is involved in the inhibition of the type I interferon (IFN) response by FMDV. However, the underlying mechanisms remain unclear. Here we demonstrate that FMDV Lb(pro), a shorter form of L(pro), has deubiquitinating activity. Sequence alignment and structural bioinformatics analyses revealed that the catalytic residues (Cys51 and His148) are highly conserved in FMDV Lb(pro) of all seven serotypes and that the topology of FMDV Lb(pro) is remarkably similar to that of ubiquitin-specific protease 14 (USP14), a cellular deubiquitylation enzyme (DUB), and to that of severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a coronaviral DUB. Both purified Lb(pro) protein and in vivo ectopically expressed Lb(pro) removed ubiquitin (Ub) moieties from cellular substrates, acting on both lysine-48- and lysine-63-linked polyubiquitin chains. Furthermore, Lb(pro) significantly inhibited ubiquitination of retinoic acid-inducible gene I (RIG-I), TANK-binding kinase 1 (TBK1), TNF receptor-associated factor 6 (TRAF6), and TRAF3, key signaling molecules in activation of type I IFN response. Mutations in Lb(pro) that ablate the catalytic activity (C51A or D163N/D164N) or disrupt the SAP (for SAF-A/B, Acinus, and PIAS) domain (I83A/L86A) abrogated the DUB activity of Lb(pro) as well as its ability to block signaling to the IFN-ß promoter. Collectively, these results demonstrate that FMDV Lb(pro) possesses DUB activity in addition to serving as a viral proteinase and describe a novel mechanism evolved by FMDV to counteract host innate antiviral responses.

[Construction and functional characterization of a monocistronic replicon based on the HCV genotype 2a promotor].

To construct a hepatitis C virus (HCV) genotype 2a monocistronic replicon and investigate its replication capabilities in the human hepatocarcinoma cell lines, Huh7.5 and Huh7.1, in order to determine its potential as a molecular tool for future in vitro studies of HCV replication and selection studies for putative anti-HCV drugs. Site-directed mutagenesis was used to delete the Core-E1-E2-p7-NS2 fragment (about 3090 bp) from plasmid pJ6JFH1BlaRL. The resultant trianglepJ6JFH1BlaRL plasmid was digested with AgeI and AvrII to release the cDNA fragment (hereafter, referred to as fragment L) containing partial 5'-untranslated region (UTR), the first 12 amino acid (aa) of HCV Core coding sequence, full-length coding sequences for the blasticidin-resistance gene, Renilla luciferase, foot-and-mouth disease virus (FMDV) 2a antiprotease and ubiquitin, and partial coding sequence for HCV NS3. To generate the monocistronic replicon, pSGRmJFH1BlaRL, fragment L was ligated into the pSGR-JFH1 vector that had been digested with AgeI and AvrII to remove the partial 5'-UTR, the first 19 aa of HCV Core coding sequence, the full-length coding sequence for the neomycin phosphotransferase II gene, the internal ribosomal entry site from encephalomyocarditis virus, and partial HCV NS3 coding sequence. A replication-defective mutant replicon, pSGRmJFH1BlaRL/GND, was constructed by a similar procedure using the pSGR-JFH1/GND vector. Fragment L was confirmed in both constructs by sequencing. Replicon RNAs were prepared from XbaI-linearized plasmid DNA templates with Invitrogen's T7 MEGAscript kit, and were purified by DNase I treatment and LiCl precipitation. RNAs were quanti?ed by optical density, and the quality and concentration were con?rmed by agarose gel electrophoresis. Replicon RNAs were transfected into Huh7.5 and Huh7.1 cells using Invitrogen's DMRIE-C transfection reagent at a ratio of 5 mug of lipid to 1mug of RNA. Time course assay of Renilla luciferase activity indicated the replicon's replication function. The pSGRmJFH1BlaRL monocistronic replicon and pSGRmJFH1BlaRL/GND replication-defective mutant replicon were successfully constructed. The pSGRmJFH1BlaRL replicon was replication-proficient in Huh7.5 and Huh7.1 cells, with replication peaking at 72 hours post-transfection and decreasing after 96 hours. No replication was detected at any time point post-transfection for the defective mutant replicon. A monocistronic replicon of HCV genotype 2a was constructed and shown to be replication-proficient in human hepatocarcinoma cell lines.

Corrigendum: Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms.

[This corrects the article DOI: 10.3389/fimmu.2018.01142.].

The Chromosome-Based Rubber Tree Genome Provides New Insights into Spurge Genome Evolution and Rubber Biosynthesis.

The rubber tree, Hevea brasiliensis, produces natural rubber that serves as an essential industrial raw material. Here, we present a high-quality reference genome for a rubber tree cultivar GT1 using single-molecule real-time sequencing (SMRT) and Hi-C technologies to anchor the ∼1.47-Gb genome assembly into 18 pseudochromosomes. The chromosome-based genome analysis enabled us to establish a model of spurge chromosome evolution, since the common paleopolyploid event occurred before the split of Hevea and Manihot. We show recent and rapid bursts of the three Hevea-specific LTR-retrotransposon families during the last 10 million years, leading to the massive expansion by ∼65.88% (∼970 Mbp) of the whole rubber tree genome since the divergence from Manihot. We identify large-scale expansion of genes associated with whole rubber biosynthesis processes, such as basal metabolic processes, ethylene biosynthesis, and the activation of polysaccharide and glycoprotein lectin, which are important properties for latex production. A map of genomic variation between the cultivated and wild rubber trees was obtained, which contains ∼15.7 million high-quality single-nucleotide polymorphisms. We identified hundreds of candidate domestication genes with drastically lowered genomic diversity in the cultivated but not wild rubber trees despite a relatively short domestication history of rubber tree, some of which are involved in rubber biosynthesis. This genome assembly represents key resources for future rubber tree research and breeding, providing novel targets for improving plant biotic and abiotic tolerance and rubber production.

Foot-and-Mouth Disease Virus Counteracts on Internal Ribosome Entry Site Suppression by G3BP1 and Inhibits G3BP1-Mediated Stress Granule Assembly via Post-Translational Mechanisms.

Foot-and-mouth disease (FMD) is a highly contagious, severe viral illness notifiable to the World Organization for Animal Health. The causative agent, FMD virus (FMDV), replicates rapidly and efficiently inhibits host translation and the innate immune response for it has developed multiple tactics to evade host defenses and takes over gene expression machinery in the host cell. Here, we report a systemic analysis of the proteome and phosphoproteome of FMDV-infected cells. Bioinformatics analysis suggested that FMDV infection shuts off host cap-dependent translation, but leaves intact internal ribosome entry site (IRES)-mediated translation for viral proteins. Interestingly, several FMDV IRES-transacting factors, including G3BP stress granule assembly factor 1 (G3BP1), were dephosphorylated during FMDV infection. Ectopic expression of G3BP1 inhibited FMDV IRES activity, promoted assembly of stress granules, and activated innate immune responses, collectively suppressing FMDV replication. To counteract these host protective responses, FMDV-induced dephosphorylation of G3BP1, compromising its inhibitory effect on viral IRES. In addition, FMDV also proteolytically cleaved G3BP1 by its 3C protease (3Cpro). G3BP1 was cleaved at glutamic acid-284 (E284) by FMDV 3Cpro, and this cleavage completely lost the abilities of G3BP1 to activate innate immunity and to inhibit FMDV replication. Together, these data provide new insights into the post-translational mechanisms by which FMDV limits host stress and antiviral responses and indicate that G3BP1 dephosphorylation and its proteolysis by viral protease are important factors in the failure of host defense against FMDV infection.

Microporous nano-MgO/diatomite ceramic membrane with high positive surface charge for tetracycline removal.

A novel microporous nano-MgO/diatomite ceramic membrane with high positive surface charge was prepared, including synthesis of precursor colloid, dip-coating and thermal decomposition. Combined SEM, EDS, XRD and XPS studies show the nano-MgO is irregularly distributed on the membrane surface or pore walls and forms a positively charged nano coating. And the nano-MgO coating is firmly attached to the diatomite membrane via SiO chemical bond. Thus the nano-MgO/diatomite membrane behaves strong electropositivity with the isoelectric point of 10.8. Preliminary filtration tests indicate that the as-prepared nano-MgO/diatomite membrane could remove approximately 99.7% of tetracycline in water through electrostatic adsorption effect. The desirable electrostatic property enables the nano-MgO/diatomite membrane to be a candidate for removal of organic pollutants from water. And it is convinced that there will be a great application prospect of charged ceramic membrane in water treatment field.

Multi­transgenic minipig models exhibiting potential for hepatic insulin resistance and pancreatic apoptosis.

There are currently no multi­transgenic minipig models of diabetes for the regulation of multiple genes involved in its pathogenesis. The foot and mouth disease virus 2A (F2A)­mediated polycistronic system possesses several advantages, and the present study developed a novel multi­transgenic minipig model associated with diabetes using this system. The tissue­specific polycistronic system used in the present study consisted of two expression cassettes, separated by an insulator: (i) 11­ß­hydroxysteroid dehydrogenase 1 (11ß­HSD1), driven by the porcine liver­specific apolipoprotein E promoter; (ii) human islet amyloid polypeptide (hIAPP) and C/EBP homologous protein (CHOP), linked to the furin digested site and F­2A, driven by the porcine pancreas­specific insulin promoter. In the present study, porcine fetal fibroblasts were transfected with this vector. Following somatic cell nuclear transfer using 10 cell clones and the transplantation of 1,459 embryos in total, three Landrace x Yorkshire surrogates became pregnant and delivered three Wuzhishan piglets. Genomic polymerase chain reaction (PCR) demonstrated that the piglets were multi­transgenic. Reverse transcription­quantitative PCR confirmed that 11ß­HSD1 transcription was upregulated in the targeted liver. Similarly, hIAPP and CHOP were expressed at high levels, compared with the control (P<0.05 and P<0.01) in the pancreas, consistent with the western blotting and immunohistochemistry results. The primary results also showed that overexpression of 11ß­HSD1 in the liver increased the liver fat lipid parameters; and the levels of hIAPP and CHOP in the pancreatic islet cells, leading to delayed ß­cell development and apoptosis. This novel tissue­specific polycistronic system offers a promising starting point for efficiently mimicking multigenic metabolic disease.

Biological evaluation of the copper/low-density polyethylene nanocomposite intrauterine device.

Devices and materials intended for clinical applications as medical and implant devices should be evaluated to determine their biocompatibility in physiological systems. This article presents results from cytotoxicity assay of L929 mouse fibroblasts culture, tests for skin irritation, intracutaneous reactivity and sensitization, and material implantation tests for the novel copper/low-density polyethylene nanocomposite intrauterine device (nano-Cu/LDPE IUD) with potential for future clinical utilization. Cytotoxicity test in vitro was conducted to evaluate the change in morphology, growth and proliferation of cultured L929 mouse fibroblasts, which in vivo examination for skin irritation (n = 6) and intracutaneous reactivity (n = 6) were carried out to explore the irritant behavior in New Zealand White rabbits. Skin sensitization was implemented to evaluate the potential skin sensitizing in Hartley guinea pigs (n = 35). The materials were implanted into the spinal muscle of rabbits (n = 9). The cytotoxicity grade of the nano-Cu/LDPE IUD was 0-1, suggested that the composite was nontoxic or mildly cytotoxic; no irritation reaction and skin sensitization were identified in any animals of specific extracts prepared from the material under test; similarly to the control sides, the inflammatory reaction was observed in the rabbits living tissue of the implanted material in intramuscular implantation assay. They indicated that the novel composite intrauterine device presented potential for this type of application because they meet the requirements of the standard practices recommended for evaluating the biological reactivity. The nano-Cu/LDPE IUD has good biocompatibility, which is biologically safe for the clinical research as a novel contraceptive device.

[Sodium hypochlorite disinfection on effluent of MBR in municipal wastewater treatment process].

Sodium hypochlorite (NaClO) used in wastewater disinfection was assessed by examining its performances in lab fed by the effluent from a MBR treatment plant. The influence of sodium hypochlorite initial concentrations (0.5-3.0 mg/L) on the presence of indicator microorganisms (total coliforms, fecal coliforms), disinfection by-products (DBPs) concentrations and the acute toxicity were evaluated. Results indicate the total coliforms and the fecal coliform were 1500-2400 and 10-40 CFU/L, which is difficult to meet the present reclaimed water quality standards. A chlorine dose of 2.0 mg/L and contact for 1 h could achieve a 3 lg indicator bacteria reduction in MBR effluent samples. THMs (trihalomethanes) analysis indicated that concentrations of THMs increase with the raise of the active chlorine dose. After adding sodium hypochlorite 1 h the concentrations of trihalomethanes (THMs) were 16.22, 7.35 microg/L respectively and chloroform (TCM) accounted for 87% of THMs, the haloacetic acids (HAAs) was involved trichloroacetic acid (TCAA) 2.01 microg/L, dichloroacetic acid (DCAA) 1.58 microg/L and under the national limits. Luminescence bacteria acute toxicity analysis showed that the chlorinated effluent has higher inhibition rate (48%) in comparison to the control with a chlorine dosage of 3.0 mg/L. The results which could provide theoretical basis to production show that NaClO disinfection not only can inactivate microbe with the DBPs and acute toxicity of the effluent under the safety limits, but also meet the requirement of health and safety.