Colorimetric Aerogel Gas Sensor with High Sensitivity and Stability.

The detection of formic acid vapor in the usage environment is extremely important for human health and safety. The utilization of metal-organic frameworks (MOFs) for the detection of gaseous molecules is an attractive strategy. However, the rational design and construction of MOF-based gas sensors with high sensitivity and mechanical stability remain a significant challenge. In this study, a simple approach is reported to fabricate colorimetric aerogel sensors assembled from MOF particles via ice template-assisted methods. As the aerogel sensor with staggered lamellae structures significantly provides a high air-volume intake of flowing gas, it generates a sufficient probability of contact reactions for highly mobile target molecules. Additionally, it enhances the mechanical stability by providing stress resistance between the staggered lamellae structures. Compared to conventional film sensors for the detection of formic acid molecules, aerogel sensors exhibit an 8-fold lower limit of detection, 15-fold better sensitivity at low concentrations, 34-fold faster response time, and higher stability. This approach shows great potential for rapid and real-time detection of target molecules as well as superior performance in the structural construction of various gas-sensitive materials.

In-vitro and in-vivo evaluation and anti-colitis activity of esculetin-loaded nanostructured lipid carrier decorated with DSPE-MPEG2000.

OBJECTIVE: Encapsulation of esculetin into DSPE-MPEG2000 carrier was performed to improve its water solubility and oral bioavailability, as well as enhance its anti-inflammatory effect on a mouse model of ulcerative colitis that was induced with dextran sulphate sodium (DSS). METHODS: We determined the in-vitro and in-vivo high-performance liquid chromatographic (HPLC) analysis method of esculetin; Esculetin-loaded nanostructure lipid carrier (Esc-NLC) was prepared using a thin-film dispersion method, wherein a particle size analyser was used to measure the particle size (PS) and zeta potential (ZP) of the Esc-NLC, while a transmission electron microscope (TEM) was employed to observe its morphology. Also, HPLC was used to measure its drug loading (DL), encapsulation efficiency (EE) and the in-vitro release of the preparation, as well as investigate the pharmacokinetic parameters. In addition, its anti-colitis effect was evaluated via histopathological examination of HE-stained sections and detection of the concentrations of tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1 beta (ß), and IL-6 in serum with ELISA kits. RESULTS: The PS of Esc-NLC was 102.29 ± 0.63 nm with relative standard deviation (RSD) of 1.08% (with poly-dispersity index-PDI of 0.197 ± 0.023), while the ZP was -15.67 ± 1.39 mV with RSD of 1.24%. Solubility of esculetin was improved coupled with prolonged release time. Its pharmacokinetic parameters were compared with that of free esculetin, wherein the maximum concentration of the drug in plasma was increased by 5.5 times. Of note, bioavailability of the drug was increased by 1.7 times, while the half-life was prolonged by 2.4 times. In the anti-colitis efficacy experiment, the mice in Esc and Esc-NLC groups exhibited significantly reduced levels of TNF-α, IL-1ß, and IL-6 in their sera comparable to the DSS group. Colon histopathological examination revealed that mice with ulcerative colitis in both Esc and Esc-NLC groups displayed improved inflammation, amid the Esc-NLC groups having the best prophylactic treatment effect. CONCLUSION: Esc-NLC could ameliorate DSS-induced ulcerative colitis by improving bioavailability, prolonging drug release time and regulating cytokine release. This observation confirmed the potential of Esc-NLC to reduce inflammation in ulcerative colitis, albeit the need for follow-up research to verify the application of this strategy to clinical treatment of ulcerative colitis.

Toxicity of avermectin to Eriocheir sinensis and the isolation of a avermectin-degrading bacterium, Ochrobactrum sp. AVM-2.

Avermectin is widely used in the prevention and treatment of parasites diseases in aquaculture. However, the residual avermectin has a serious impact on the growth and quality of aquatic animals including Eriocheir sinensis. This study shows that the LC50 of avermectin to E. sinensis for 24, 48, 72 and 96 h was 21.88, 13.40, 9.11 and 7.10 mg/L, respectively. After avermectin stress, the activities of superoxide dismutase (SOD), catalase (CAT) and phenol oxidase (PO) in the hepatopancreas of E. sinensis increased and reached the peak on the 6th day. The content of malondialdehyde (MDA) accumulated with the increase of exposure time and concentration of avermectin. After 15 days of avermectin exposure, hepatopancreas was damaged seriously. These results indicated that avermectin had toxicity to E. sinensis. In order to solve the pollution problem caused by residual avermectin, a degrading bacterium AVM-2 was separated from the sediment of E. sinensis breeding pond. The strain was confirmed to be Ochrobactrum sp by morphology observation, physiological and biochemical identification and 16 S rDNA sequences analysis. When the pH value was 7, the temperature was 30 â„ƒ, the concentration of substrate was low, the quantity of inoculation was high, Ochrobactrum sp. AVM-2 had better degradation effect on avermectin. When the addition of Ochrobactrum sp. AVM-2 was 2.34 × 108 CFU/L, the residual avermectin in muscle and hepatopancreatine significantly decreased, and the degradation rate was about 66%. In summary, Ochrobactrum sp. AVM-2 could be used to solve the residual problem of avermectin and ensure the food safety of E. sinensis.

Preparation, in vitro and in vivo evaluation of pinocembrin-loaded TPGS modified liposomes with enhanced bioavailability and antihyperglycemic activity.

PURPOSE: To prepare polyethylene glycol succinate-vitamin E modified pinocembrin (PCB)-loaded liposomes (PCBT-liposomes) and evaluate PCBT-liposomal pharmacokinetics and antihyperglycemic activity. SIGNIFICANCE: The novel PCBT-liposomes demonstrated a promising application prospect as a nano drug carrier for future research. METHODS: Thin film dispersion was used to prepare PCBT-liposomes. We measured a series of characterization, followed by in vitro cumulative release, in vivo pharmacokinetic study, and antihyperglycemic activity evaluation. RESULTS: PCBT-liposomes displayed spherical and bilayered nanoparticles with mean particle size (roughly 92 nm), negative zeta potential (about -26.650 mV), high drug encapsulation efficiency (87.32 ± 1.34%) and good storage (at 4 or 25 °C) stability during 48 h after hydration. The cumulative release rate of PCBT-liposomes was markedly higher than free PCB in four different pH media. In vivo investigation showed that PCBT-liposomes could obviously improve oral bioavailability of PCB by 1.96 times, whereas the Cmax, MRT0-t, and T1/2 of PCBT-liposomes were roughly 1.700 ± 0.139 µg·mL-1, 12.695 ± 1.647 h, and 14.244 h, respectively. In terms of biochemical analysis, aspartate amino-transferase (AST), alanine amino-transferase (ALT), interleukin-1 (IL-1), and tumor necrosis factor-α (TNF-α) concentrations in serum of diabetic mice were respectively decreased 28.28%, 17.23%, 17.77%, and 8.08% after PCBT-liposomal treatment. CONCLUSION: These results show PCBT-liposomal preparation as an excellent nano-carrier which has the potential to improve water solubility, bioavailability, and antihyperglycemic activity of PCB, amid broadening the application of PCB in the clinical settings.

Pinocembrin polymeric micellar drug delivery system: preparation, characterisation and anti-hyperuricemic activity evaluation.

Aim: Hydrophobic pinocembrin (PCB) was incorporated into a new nano-drug delivery system to enhance solubility, bioavailability and anti-hyperuricemic activity of the drug.Methods: We fabricated PCB loaded polymeric micelles (PCB-FPM) by thin film dispersion method and appropriately determined their physical characteristics. The oral relative bioavailability and anti-hyperuricemic activity of PCB-FPM and free PCB were observed.Results: The optimum particle size of the micelles was 19.90 ± 0.93 nm. PCB-FPM exhibited great stability within 18 days, coupled with lower cytotoxicity and higher biocompatibility. Moreover, the percent cumulative release of PCB-FPM was much higher than free PCB in the dissolution media. The oral bioavailability of PCB-FPM was increased by 2.61 times compared with free PCB. Uric acid (UA) level of rats was reduced in PCB-FPM group (200 mg/kg) by 78.82% comparable to the model control.Conclusion: PCB-FPM may become an ideal strategy to increase oral in-vivo availability and anti-hyperuricemic activity of PCB.

Hyperoside-loaded TPGs/mPEG-PDLLA self-assembled polymeric micelles: preparation, characterization and in vitro/in vivo evaluation.

Hyperoside (Hyp) self-assembled polymeric micelles (Hyp-PMs) were purposely developed to enhance aqueous solubility, in vivo availability and anti-oxidative effect of Hyp. In preparing Hyp-PMs, we employed the thin film dispersion method with the micelles consisting of TPGs and mPEG2000-PDLLA3000. The particle size, polydispersity index and zeta potential of Hyp-PMs were 67.42 ± 1.44 nm, 0.229 ± 0.015 and -18.67 ± 0.576 mV, respectively, coupled with high encapsulation efficiency （EE）of 90.63 ± 1.45% and drug loading (DL) of 6.97 ± 1.56%. Furthermore, the value of critical micelle concentration (CMC) was quite low, which indicated good stability and improved self-assembly ability of Hyp-PMs. Also, trend of in vitro Hyp release from Hyp-PMs demonstrated enhanced solubility of Hyp. Similarly, in comparison with free Hyp, oral bioavailability of Hyp-PMs was improved (about 8 folds) whilst half-life of Hyp-PMs was extended (about 3 folds). In vitro anti-oxidative effect showed obvious strong scavenging DPPH capability of Hyp-PMs, which may be attributed to its smaller size and better solubility. Altogether, Hyp-PMs may serve as a possible strategy to potentially enhance aqueous solubility, bioavailability and anti-oxidative effect of Hyp, which may play a key role in Hyp application in the pharmaceutical industries.

Preparation of Pinocembrin-Loaded F127/MPEG-PDLLA Polymer Micelles and Anti-Osteoporotic Activity.

Pinocembrin (PCB) is 5,7-dihydroxyl flavanone and has multiple pharmacological activities, namely, anti-inflammation, anti-osteoporotic, and so on. However, low water solubility and bioavailability have hindered its application. Herein, we aimed to increase its bioavailability through preparation of F127/MPEG-PDLLA polymer micelles (PCB-M). We characterized the micelles through appropriate attributes such as analysis of particle size (PS), polydispersity (PDI), transmission electron microscopic (TEM) image, stability test, and evaluation of in vitro release of drug. After physical characterization, the respective PS, PDI, and entrapment efficiency (EE) of PCB-M were estimated to be 27.63 ± 0.17 nm, 0.055 ± 0.02, and 90.53 ± 0.01%. Fluorescence probe method was employed to measure critical micelle concentration (CMC) of PCB-M, we observed CMC was low, thereby suggesting that PCB-M had good stability. In vitro release analysis indicated that the rate of cumulative PCB release from PCB-M was greater than 90% in each medium compared with free PCB, which was less than 40%, thus pointing to a significantly improved solubility of PCB. In vivo pharmacokinetic results showed that oral biological availability of PCB-M increased 5.3 folds comparable to free PCB. The effects of PCB on osteoblasts and ALP activities were investigated; subsequently, zebrafish osteoporotic model was established with prednisolone to study the anti-osteoporotic effects of PCB and PCB-M. The results showed that PCB improved osteoporosis with PCB-M being more effective than free PCB. Finally, PCB-M can be used as a promising method to improve the solubility of PCB, while the bioavailability and anti-osteoporotic effect of PCB could be improved, thus laying a foundation for clinical use in the future.

A colloidal gold test strip assay for the detection of African swine fever virus based on two monoclonal antibodies against P30.

African swine fever (ASF), caused by African swine fever virus (ASFV), was first reported in Kenya in 1921, but an effective vaccine or antiviral drug is still not available for ASFV control. Rapid and effective diagnostics are key steps in managing ASF. We generated two monoclonal antibodies (MAbs) against the ASFV phosphoprotein P30 and designated these as 3H7A7 and 6H9A10. Epitope mapping revealed that MAb 3H7A7 and 6H9A10 recognized aa 144-154 and aa 12-18 of P30, respectively. A signal-amplified sandwich colloidal gold test strip for rapid detection of ASFV was developed based using these MAbs. Sensitivity and specificity analysis showed that the detection limit of the strip was 2.16 ng of P30. The strip only reacted with ASFV and did not react with other common porcine viruses. In detection tests using 153 clinical field samples including sera, plasma, anticoagulant-treated blood, and tissue, the strip had 95.42% concordance with real-time PCR. The new MAbs specific for P30 and the rapid colloidal gold test strip helped to reveal novel B cell epitopes in P30 and provide an efficient diagnostic test for on-site clinical detection of ASF.

JNK selective inhibitor, IQ-1S, protects the mice against lipopolysaccharides-induced sepsis.

Sepsis is a severe systemic inflammatory response induced by infection. Innate immunity recognizes pathogen components such as lipopolysaccharides (LPS), and mediates the polarization of immune cells and the release of cytokines. However, this process is also crucial for triggering sepsis and septic shock. To investigate the potential therapeutic function of 11H-indeno [1,2-b] quinoxalin-11-one oxime (IQ-1S) to sepsis, LPS plus d-galactosamine was used to establish a sepsis mouse model. Flow cytometry was performed to catalyze T cells and macrophages in mouse spleen. ELISA assay and qRT-PCR assay were performed to estimate the expression levels of cytokines and related genes including TNF-α, IL-6, IL-1ß, Nos2, Arg and Mrc. The protein levels of NF-κB, AP1, NF-Y, p-JNK2, JNK2, p-p38, p38, p-IκBα, IκBα, p-IKKß and IKKß were evaluated by Western blot assay. IQ-1S treatment significantly reduced mortality and lung inflammation in sepsis mice. IQ-1S treatment decreased the levels of inflammatory cytokines in sepsis mice. Polarization of M1 macrophages was suppressed by IQ-1S in vitro. IQ-1S significantly inhibited the activation of the JNK signaling pathway and reduced the phosphorylation level of JNK2 in sepsis mice. IQ-1S protected the mice against LPS-induced sepsis through inhibiting JNK signaling pathway.

Generation and immunogenicity assessment of ELPylated virus-like particles of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important pathogen affecting swine industry worldwide. The production of current PCV2 vaccines is time-consuming and expensive. Elastin-like polypeptides (ELP) undergo temperature-dependent inverse phase transition and ELPylated proteins can be purified simply by inverse transition cycling (ITC). METHODS: The Cap protein of PCV2b, together with the virus neutralizing (VN) epitopes of PCV2a, PCV2d and PCV2e, was expressed in E. coli as an ELPylated protein, and purified by ITC in the presence of mild detergents. For the control purpose, the Cap protein was also expressed as a His-tagged protein and purified by nickel affinity chromatography. The formation of ELPylated VLP (ELP-VLP) and His-tagged VLP (VLP) was revealed by transmission electron microscopy. Mice were immunized two times with the two forms of VLP and the antigen-specific IgG antibody, VN antibody, cytokine responses and immunoprotection against PCV2 challenge were compared. RESULTS: ELPylated Cap protein was expressed as a soluble protein and purified to 94.3% purity by ITC in the presence of 1% Triton X-100 and 0.5 M urea. His-tagged Cap fusion protein was expressed as insoluble inclusion bodies and purified to 90% purity under denatured conditions. The two purified fusion proteins assembled into VLP with similar morphology. Compared to immunization with VLP, immunization with ELP-VLP induced significantly (p < 0.01) stronger VN antibody response and slightly (p < 0.05) stronger Cap-specific IgG antibody response, cytokine production and immunoprotection against PCV2 challenge. CONCLUSION: A novel ELPylation platform for easy preparation of PCV2 VLP was established and the prepared ELP-VLP was more immunogenic than VLP. The ELPylation technology could be used for other VLP preparation and the prepared ELP-VLP could be developed as a novel PCV2 subunit vaccine.

An ShK-domain serine protease of Eriocheir sinensis regulates the PO activity to resist Spiroplasma eriocheiris infection.

A novel serine protease contains two ShK-domain was found from the Chinese mitten crab Eriocheir sinensis (EsShK-SP). The full-length EsShK-SP cDNA is 1927 bp and contains a 1260-bp open reading frame encoding a protein of 420 amino acids, including a signal peptide, two ShK domain, and Tryp-SPC domain. Quantitative real-time PCR showed that EsShK-SP was expressed mainly in the hemocytes, gills, intestine, and nerve, but weakly in heart, muscle, and hepatopancreas. After infected with Spiroplasma eriocheiris, the expression of EsShK-SP was significantly up-regulated from 1 d to 9 d. The Tryp-SPC domain was ligated with pGEX-4T-1 vector and prokaryotic expressed to obtain recombinant protein rSPC. When rSPC and S. eriocheiris stimulated the hemocytes of E. sinensis, the PO activity was significantly up-regulated. The subcellular localization revealed that recombinant EsShK-SP was mainly located in the cytoplasm of Drosophila S2 cells. Both absolute real-time PCR and confocal laser scanning microscope results showed that over-expression of EsShK-SP in S2 cells could decrease the copy number of S. eriocheiris. Meanwhile, the over-expression of EsShK-SP also increased the PO activity and cell viability of S2 cells. After EsShK-SP RNA interference using dsRNA, the expression levels of proPO and activity of PO decreased significantly from 48 h to 96 h. The knockdown of EsShK-SP by RNAi resulted in the copy number of S. eriocheiris in the EsShK-SP silenced group was significantly increased compared to the control groups during S. eriocheiris infection. Meanwhile, the survival rate of crabs decreased in the EsShK-SP-dsRNA group. The above results indicated that EsShK-SP plays an important immune role during E. sinensis against S. eriocheiris through regulation of the proPO system.

Half-life extension of porcine interferon-α by fusion to the IgG-binding domain of streptococcal G protein.

Recombinant interferon-α (rIFN-α) has been widely used for treating viral infections. However, the clinical efficacy of unmodified rIFN-α is limited due to small molecular size and rapid clearance from circulation. In this study we developed a novel strategy for half-life extension of porcine IFN-α (PoIFN-α) by fusion to the immunoglobulin (Ig)-binding C2 domain of streptococcal protein G (SPG). The coding sequences for PoIFN-α6 and SPG C2 domain, with a tobacco etch virus (TEV) protease recognition sequence introduced at the 5-end, were cloned into an elastin-like polypeptide (ELP) fusion expression vector and expressed as an ELP-PoIFNα-C2 fusion protein. After optimization of the conditions for soluble protein expression and purification, the fusion protein was purified to more than 90% purity by two rounds of inverse transition cycling (ITC) in the presence of 0.5% Triton X-100. After cleavage with self-aggregating peptide ELK-16-tagged tobacco etch virus protease, the protease was removed by quick centrifugation and PoIFNα-C2 protein was recovered by an additional round of ITC with 98% purity. Western blotting analysis showed that PoIFNα-C2 protein had the specific affinity for pig IgG binding. The antiviral assay showed that PoIFNα-C2 protein had potent antiviral activities against vesicular stomatitis virus and porcine pseudorabies virus. After single intravenous or subcutaneous injection into rats, PoIFNα-C2 protein showed 16- or 4-fold increase in serum half-life with significantly improved bioavailability.

Recombinant Adenoviruses Displaying Matrix 2 Ectodomain Epitopes on Their Fiber Proteins as Universal Influenza Vaccines.

Influenza is a zoonotic disease that poses severe threats to public health and the global economy. Reemerging influenza pandemics highlight the demand for universal influenza vaccines. We developed a novel virus platform using extracellular domain IV of the matrix 2 protein (M2e), AdC68-F3M2e, by introducing three conserved M2e epitopes into the HI loop of the chimpanzee adenovirus (AdV) fiber protein. The M2e epitopes were expressed sufficiently on the AdV virion surface without affecting fiber trimerization. Additionally, one recombinant adenovirus, AdC68-F3M2e(H1-H5-H7), induced robust M2e-specific antibody responses in BALB/c mice after two sequential vaccinations and conferred efficient protection against homologous and heterologous influenza virus (IV) challenges. We found that the use of AdV with tandem M2e epitopes in fiber is a potential strategy for influenza prevention.IMPORTANCE Influenza epidemics and pandemics severely threaten public health. Universal influenza vaccines have increasingly attracted interest in recent years. Here, we describe a new strategy that incorporates triple M2e epitopes into the fiber protein of chimpanzee adenovirus 68. We optimized the process of inserting foreign genes into the AdC68 structural protein by one-step isothermal assembly and demonstrated that this 225-bp HI loop insertion could be well tolerated. Furthermore, two doses of adjuvant-free fiber-modified AdC68 could confer sufficient protection against homologous and heterologous influenza virus infections in mice. Our results show that AdC68-F3M2e could be pursued as a novel universal influenza vaccine.

A novel method for concentration of porcine reproductive and respiratory syndrome virus from the environmental samples using self-aggregating peptide-tagged CD151-binding capture.

Environmental surveillance of porcine reproductive and respiratory syndrome virus (PRRSV) represents a key issue in control of the disease. CD151 has recently been recognized as one of several receptors for PRRSV. We describe here a novel method for concentration of PRRSV from the environmental samples by CD151-binding capture. After fusion to self-aggregating peptide ELK16, the large extracellular loop (LEL) of porcine CD151 and its two segments (namely N63 and C63) were expressed in E. coli as protein aggregates. The three fusion proteins were purified to high purities by regular centrifugation and washing with Triton X-100. Viral binding assay showed that the C63-ELK16 protein, but not ELK16-N63 protein, had the specific binding affinity for PRRSV. The C63-ELK16 protein could bind to, and eluted from, PRRSV in pH-, temperature-, and time-dependent manners with a final virus recovery of 44.7%. By using PRRSV-spiked and experimentally infected pig fecal slurry samples, the C63-ELK16 binding capture-combined quantitative RT-PCR was shown to have higher detection sensitivity than the conventional RT-PCR. Although the viral RNA could be detected in the experimentally infected pig samples with or without C63-ELK16 binding capture, infectious PRRSV was not isolated without C63-ELK16 binding capture. Therefore, the CD151-binding capture method established offers sufficient recovery and quickness and will facilitate environmental PRRSV surveillance.

A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease.

Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-γ (His-BoIFN-γ) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 × 10(4) units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-γ (rBoIFN-γ) was purified to 98.3% purity with 63% recovery. The rBoIFN-γ had an antiviral activity of 1.6 × 10(3) units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification.

Single-step concentration and purification of adenoviruses by coxsackievirus-adenovirus receptor-binding capture and elastin-like polypeptide-mediated precipitation.

A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the "spiked" PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples.

The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd.

Staphylococci are the leading pathogens of bovine mastitis which is difficult to control. However, the published data on the prevalence of staphylococcal species, virulence and antibiotic resistance genes in bovine mastitis from China are limited. In this study, 104 out of 209 subclinical mastitis milk samples from a single Chinese dairy herd were cultured-positive for staphylococci (49.8%), which were further identified as coagulase-positive staphylococci (CPS) or coagulase-negative staphylococci (CNS). According to the partial tuf and/or 16S rRNA gene sequence, the 28 CPS isolates were confirmed to be Staphylococcus aureus (26.9%), and 76 CNS isolates were assigned to 13 different species (73.1%) with Staphylococcus arlettae, Staphylococcus sciuri, Staphylococcus xylosus and Staphylococcus chromogenes as the dominant species. In the 28 S. aureus isolates, the most prevalent general virulence genes were coa, Ig and eno (100%), followed by hla (96.4%), hlb (92.9%), fib (92.9%), clfA (89.3%), clfB (85.7%) and nuc (85.7%). Both exotoxin and biofilm-associated genes were significantly less prevalent than the previously reported. Although 19 different virulence gene patterns were found, only one was dominant (32.1%). The prevalence of blaZ (82.1%) or mecA gene (35.7%) was much higher than the previously reported. In the 76 CNS isolates, the virulence genes were significantly less prevalent than that in the S. aureus isolates. Among the 4 main CNS species, S. chromogenes (n = 12) was the only species with high percentage (75%) of blaZ gene, while S. sciuri (n = 12) was the only species with the high percentage (66.7%) of mecA gene. The most of antibiotic resistance genes were present as multi-resistance genes, and the antibiotic resistances were attributed by different resistance genes between resistant S. aureus and CNS isolates. These data suggest that the prevalence of staphylococcal species, virulence and antibiotic resistance in the mastitis milk from the Chinese dairy herd are different from the previously reported, and that the herd- or farm-based diagnosis of staphylococcal bovine mastitis is required.

Inhibition of porcine reproductive and respiratory syndrome virus infection by recombinant adenovirus- and/or exosome-delivered the artificial microRNAs targeting sialoadhesin and CD163 receptors.

BACKGROUND: The current vaccines failed to provide substantial protection against porcine reproductive and respiratory syndrome (PRRS) and the new vaccine development faces great challenges. Sialoadhesin (Sn) and CD163 are the two key receptors for PRRS virus (PRRSV) infection of porcine alveolar macrophages (PAMs), but the artificial microRNA (amiRNA) strategy targeting two viral receptors has not been described. METHODS: The candidate miRNAs targeting Sn or CD163 receptor were predicted using a web-based miRNA design tool and validated by transfection of cells with each amiRNA expression vector plus the reporter vector. The amiRNA-expressing recombinant adenoviruses (rAds) were generated using AdEasy Adenoviral Vector System. The rAd transduction efficiencies for pig cells were measured by flow cytometry and fluorescent microscopy. The expression and exosome-mediated secretion of amiRNAs were detected by RT-PCR. The knock-down of Sn or CD163 receptor by rAd- and/or exosome-delivered amiRNA was detected by quantitative RT-PCR and flow cytometry. The additive anti-PRRSV effect between the two amiRNAs was detected by quantitative RT-PCR and viral titration. RESULTS: All 18 amiRNAs validated were effective against Sn or CD163 receptor mRNA expression. Two rAds expressing Sn- or CD163-targeted amiRNA were generated for further study. The maximal rAd transduction efficiency was 62% for PAMs at MOI 800 or 100% for PK-15 cells at MOI 100. The sequence-specific amiRNAs were expressed efficiently in and secreted from the rAd-transduced cells via exosomes. The expression of Sn and CD163 receptors was inhibited significantly by rAd transduction and/or amiRNA-containing exosome treatment at mRNA and protein levels. Both PRRSV ORF7 copy number and viral titer were reduced significantly by transduction of PAMs with the two rAds and/or by treatment with the two amiRNA-containing exosomes. The additive anti-PRRSV effect between the two amiRNAs was relatively long-lasting (96 h) and effective against three different viral strains. CONCLUSION: These results suggested that Sn- and CD163-targeted amiRNAs had an additive anti-PRRSV effect against different viral strains. Our findings provide new evidence supporting the hypothesis that exosomes can also serve as an efficient small RNA transfer vehicle for pig cells.

Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC.

Purification of recombinant proteins is a major task and challenge in biotechnology and medicine. In this paper we report a novel single-step recombinant protein purification system which was based on elastin-like peptide (ELP)-mediated reversible phase transition and FrpC self-processing module (SPM)-mediated cleavage. After construction of a SPM-ELP fusion expression vector, we cloned the coding sequence for green fluorescent protein (GFP), the Fc portion of porcine IgG (pFc) or human ß defensin 3 (HBD3) into the vector, transformed the construct into Escherichia coli, and induced the fusion protein expression with IPTG. The target-SPM-ELP fusion proteins GFP-SPM-ELP, Fc-SPM-ELP and HBD3-SPM-ELP were expressed in a soluble form and efficiently purified from the clarified cell extracts by two rounds of inverse transition cycling (ITC). Under the optimized conditions, the SPM-mediated cleavage efficiencies for the three fusion proteins ranged from 92% to 93%. After an additional round of ITC, the target proteins GFP, pFc and HBD3 were recovered with purities ranging from 90% to 100% and yields ranging from 1.1 to 36mg/L in shake flasks. The endotoxin levels in all of the three target proteins were <0.03EU/mg. The three target proteins were functionally active with the expected molecular weights. These experimental results confirmed the high specificity and efficiency of SPM-mediated cleavage, and suggested the applicability of SPM-ELP fusion system for purification of recombinant proteins.

Preparation, characterization, pharmacokinetics and ulcerative colitis treatment of hyperoside-loaded mixed micelles.

At present, ulcerative colitis (UC) has become a global disease due to its high incidence. Hyperoside (HYP) is a naturally occurring flavonoid compound with many pharmacological effects. This study aimed to develop HYP-loaded mixed micelles (HYP-M) to improve oral bioavailability of HYP and to evaluate its therapeutic effect on UC. The prepared HYP-M exhibited stable physical and chemical properties, smaller particle size (PS) (21.48 ± 1.37 nm), good polydispersity index (PDI = 0.178 ± 0.013), negative Zeta potential (ZP) (- 20.00 ± 0.48 mV) and high entrapment rate (EE) (89.59 ± 2.03%). In vitro release and in vivo pharmacokinetic results showed that HYP-M significantly increased the releasing rate of HYP, wherein its oral bioavailability was 4.15 times higher than that of free HYP. In addition, HYP-M was more effective in the treatment of UC than free HYP. In conclusion, HYP-M could serve as a novel approach to improve bioavailability and increase anti-UC activity of HYP.