Simultaneous detection and identification of Peste des petits ruminants Virus Lineages II and IV by MCA-Based real-time quantitative RT-PCR assay within single reaction.

BACKGROUND: Peste des petits ruminants (PPR) disease is a cross-species infectious disease that severely affects small ruminants and causes great losses to livestock industries in various countries. Distinguishing vaccine-immunized animals from naturally infected animals is an important prerequisite for the eradication of PPR. At present PPRV are classified into lineages I through IV, and only one vaccination strain, Nigeria/75/1, belongs to lineage II, but all of the epidemic strains in China at present are from lineage IV. RESULTS: To achieve this goal, we developed an SYBR Green I real-time qRT-PCR method for rapid detection and identification of PPRV lineages II and IV by analyzing different melting curve analyses. The negative amplification of other commonly circulating viruses such as orf virus, goat poxvirus, and foot-and-mouth disease virus demonstrated that primers targeting the L gene of PPRV were extremely specific. The sensitivity of the assay was assessed based on plasmid DNA and the detection limit achieved was 100 copies of PPRV lineages II and IV. CONCLUSION: Since the method has high sensitivity, specificity, and reproducibility, it will be effectively differentiated PPRV lineages II from PPRV lineages IV in PPRV infected animals.

A coordination strategy to achieve instant dissolution of a biomedical polymer in water via manual shaking.

Organic polymers with condensed long chains show slow dissolution kinetics in solvents, particularly in water, which has significantly hindered their potential applications where their instant dissolution without any assistance of a stirring machine, etc. is required. Herein, we put forward a strategy of rapid dissolution of chain-like polymers by coordinating with small molecular additives, using a thermogellable amphiphilic copolymer and CaCl2 for demonstration. We synthesized a block copolymer of poly(ethylene glycol) (PEG) and poly(lactide-co-glycolide) (PLGA) and prepared its powder after coordinating with calcium ions. Compared to the virgin copolymer, the composite was dissolved in water at a rate of over 104 fold, and simple manual shaking for half a minute could form its aqueous solution. Chelation using sodium citrate was further suggested to alleviate the possible biocompatibility problem caused by calcium ions. Finally, the potential application of the thermogels prepared by the rapid dissolution strategy for an instant use in hospitals was demonstrated in an ex vivo porcine model of a fluid cushion for endoscopic submucosal dissection. The mechanism was discussed, and the critical factor comes from the coordination between calcium ions and the PEG block in the copolymer. The strategy to introduce a solvable small molecular additive coordinated with the polymer chain at the molecular level is helpful for accelerating the dissolution of organic polymers in a solvent to a large extent.

Intelligent Paper-Free Sprayable Skin Mask Based on an In Situ Formed Janus Hydrogel of an Environmentally Friendly Polymer.

Traditional skin care masks usually use a piece of paper to hold the aqueous essences, which are not environmentally friendly and not easy to use. While a paper-free mask is desired, it is faced with a dilemma of moisture holding and rapid release of encapsulated bioactive substances. Herein, a paper-free sprayable skin mask is designed from an intelligent material-a thermogel which undergoes sol-gel-suspension transitions upon heating-to solve this dilemma. A synthesized block copolymer of poly(ethylene glycol) and poly(lactide-co-glycolide) with appropriate ratios can be dissolved in water, and thus easily mixed with a biological substance. The mixture is sprayable. After spraying, a Janus film is formed in situ with a physical gel on the outside and a suspension on the inside facing skin. Thus, both moisture holding and rapid release are achieved. Such a thermogel composed of biodegradable amphiphilic block copolymers loaded with nicotinamide as a skin mask is verified to reduce pigmentation on a 3D pigmented reconstructed epidermis model and further in a clinical study. This work might be stimulating for investigations and applications of biodegradable and intelligent soft matter in the fields of drug delivery and regenerative medicine.

A Facile Composite Strategy to Prepare a Biodegradable Polymer Based Radiopaque Raw Material for "Visualizable" Biomedical Implants.

Enabling a biodegradable polymer radiopaque under X-ray is much desired for many medical devices. Physical blending of a present biodegradable polymer and a commercialized medical contrast agent is convenient yet lacks comprehensive fundamental research. Herein, we prepared a biodegradable polymer-based radiopaque raw material by blending poly(l-lactic acid) (PLLA or simply PLA) and iohexol (IHX), where PLA constituted the continuous phase and IHX particles served as the dispersed phase. The strong X-ray adsorption of IHX enabled the composite radiopaque; the hydrolysis of the polyester and the water solubility of the contrast agent enabled the composite biodegradable in an aqueous medium. The idea was confirmed by in vitro characterizations of the resultant composite, in vivo subcutaneous implantation in rats up to 6 months, and the clear visualization of a part of a biodegradable occluder in a Bama piglet under X-ray. We also found that the crystallization of PLA was significantly enhanced in the presence of the solid particles, which should be taken into consideration in the design of an appropriate biomaterial composite because crystallization degree influences the biodegradation rate and mechanical property of a material to a large extent. We further tried to introduce a small amount of poly(vinylpyrrolidone) into the blend of PLA and IHX. Compared to the bicomponent composite, the tricomponent one exhibited decreased modulus and increased elongation at break and tensile strength. This paves more ways for researchers to select appropriate raw materials according to the regenerated tissue and the application site.

Injectable Thermogel Generated by the "Block Blend" Strategy as a Biomaterial for Endoscopic Submucosal Dissection.

Endoscopic submucosal dissection is an established method for the removal of early cancers and large lesions from the gastrointestinal tract but is faced with the risk of perforation. To decrease this risk, a submucosal fluid cushion (SFC) is needed clinically by submucosal injection of saline and so on to lift and separate the lesion from the muscular layer. Some materials have been tried as the SFC so far with disadvantages. Here, we proposed a thermogel generated by the "block blend" strategy as an SFC. This system was composed of two amphiphilic block copolymers in water, so it was called a "block blend". We synthesized two non-thermogellable copolymers poly(d,l-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(d,l-lactide-co-glycolide) and blended them in water to achieve a sol-gel transition upon heating in both pure water and physiological saline. We explored the internal structure of the resultant thermogel with transmission electron microscopy, three-dimensional light scattering, 13C NMR, fluorescence resonance energy transfer, and rheological measurements, which indicated a percolated micelle network. The biosafety of the synthesized copolymer was preliminarily confirmed in vitro. The main necessary functions as an SFC, namely, injectability of a sol and the maintained mucosal elevation as a gel after injection, were verified ex vivo. This study has revealed the internal structure of the block blend thermogel and illustrated its potential application as a biomaterial. This work might be stimulating for investigations and applications of intelligent materials with both injectability and thermogellability of tunable phase-transition temperatures.

Biological sealing and integration of a fibrinogen-modified titanium alloy with soft and hard tissues in a rat model.

Percutaneous or transcutaneous devices are important and unique, and the corresponding biological sealing at the skin-implant interface is the key to their long-term success. Herein, we investigated the surface modification to enhance biological sealing, using a metal sheet and screw bonded by biomacromolecule fibrinogen mediated via pre-deposited synthetic macromolecule polydopamine (PDA) as a demonstration. We examined the effects of a Ti-6Al-4V titanium alloy modified with fibrinogen (Ti-Fg), PDA (Ti-PDA) or their combination (Ti-PDA-Fg) on the biological sealing and integration with skin and bone tissues. Human epidermal keratinocytes (HaCaT), human foreskin fibroblasts (HFF) and preosteoblasts (MC3T3-E1), which are closely related to percutaneous implants, exhibited better adhesion and spreading on all the three modified sheets compared with the unmodified alloy. After three-week subcutaneous implantation in Sprague-Dawley (SD) rats, the Ti-PDA-Fg sheets could significantly attenuate the soft tissue response and promote angiogenesis compared with other groups. Furthermore, in the model of percutaneous tibial implantation in SD rats, the Ti-PDA-Fg screws dramatically inhibited epithelial downgrowth and promoted new bone formation. Hence, the covalent immobilization of fibrinogen through the precoating of PDA is promising for enhanced biological sealing and osseointegration of metal implants with soft and hard tissues, which is critical for an orthopedic percutaneous medical device.

Physicochemical Properties and In Vitro Biocompatibility of Three Bacterial Nanocellulose Conduits for Blood Vessel Applications.

A novel design of bioreactor G-BNC, in combination with two previously reported designs of bioreactor were used to fabricate three small caliber bacterial nanocellulose (BNC) conduits (G-BNC, S-BNC and D-BNC). They were compared systematically with a clinically-used ePTFE graft. S-BNC possessed a laminated structure, the lowest BNC content, roughest luminal surface and weakest mechanical properties, and so might not be sufficiently strong for use as an artificial blood vessel alone. The D-BNC conduit possessed an unstratified structure with a fiber network that was more dense and the greatest BNC content, providing the strongest mechanical properties. G-BNC possessed a looser network with the smoothest luminal surface and greater hemocompatibility. Following comprehensive evaluation of mechanical properties and performance, we judge that D-BNC and G-BNC should possess greater potential in application as small caliber vascular grafts, however the patency of the three BNC conduits need be further verified in animal studies in vivo.

Structural mechanics of 3D-printed poly(lactic acid) scaffolds with tetragonal, hexagonal and wheel-like designs.

While various porous scaffolds have been developed, the focused study about which structure leads to better mechanics is rare. In this study, we designed porous scaffolds with tetragonal, hexagonal and wheel-like structures under a given porosity, and fabricated corresponding poly(lactic acid) (PLA) scaffolds with three-dimensional printing. High-resolution micro-computed tomography was carried out to calculate their experimental porosity and confirm their high interconnectivity. The theoretical and experimental compressive properties in the longitudinal direction were characterized by finite element analysis method and electromechanical universal testing system, respectively. Thereinto, the scaffold with the tetragonal structure exhibited higher mechanical strength both theoretically and experimentally. Creep and stress relaxation behaviors of the scaffolds revealed that the tetragonal scaffold had less significant viscoelasticity. Immersion dynamic mechanical analysis was performed to test their cycle-loading fatigue behaviors in the simulated body fluid at 37 °C; the tetragonal scaffold exhibited the latest fatigue beginning point at 4400 cycles, which indicated a better anti-fatigue performance; the hexagonal and wheel-like ones exhibited the middle and earliest fatigue beginning points at 3200 and 2500 cycles, respectively. What is more, cytocompatibility and histocompatibility of the scaffolds with all of the structures were confirmed by cell counting kit-8 assay in vitro and three-month subcutaneous implantation in rats in vivo. Hence, the key property difference of the three examined structures comes from their mechanics; the tetragonal structure exhibited better mechanics in the longitudinal direction examined in this study, which could be taken into consideration in design of a porous scaffold for tissue engineering and regeneration.

Performance improvements of the BNC tubes from unique double-silicone-tube bioreactors by introducing chitosan and heparin for application as small-diameter artificial blood vessels.

In order to improve property of bacterial nano-cellulose (BNC) to achieve the requirements of clinical application as small caliber vascular grafts, chitosan (CH) was deposited into the fibril network of the BNC tubes fabricated in unique Double-Silicone-Tube bioreactors. Heparin (Hep) was then chemically grafted into the BNC-based tubes using EDC/NHS crosslinking to improve performance of anticoagulation and endothelialization. Physicochemical and mechanical property, blood compatibility, and cytocompatibility were compared before and after compositing. The results indicated that strength at break was increased but burst pressure decreased slightly after compositing. Performance of the BNC tubes was improved remarkably after introducing chitosan and heparin. The EDC/NHS crosslinking catalyzed both amide bonds and ester bonds formation in the BNC/CH-Hep composites. Three-dimensional surface structure and roughness were firstly obtained and discussed in relation to the hemocompatibility of BNC-based tubes. This work demonstrates the heparinized BNC-based tubes have great potential in application as small-diameter vascular prosthesis.

[Epidemiological study of human caliciviruses among children with acute diarrhea in Lulong county, 1999 - 2001].

OBJECTIVE: To investigate the epidemiological characteristus of human caliciviruses (HuCVs) among children under 5 years of age with acute diarrhea and to estimate the disease burden in Lulong county. METHODS: HuCVs were detected by enzyme linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Some PCR amplicons were cloned and sequenced. Phylogenetic tree was constructed for strain characterization. The rate of HuCVs-attributed hospitalization was estimated according to the positive rate of HuCVs detection in fecal specimens collected from hospitalized diarrhea patients. RESULTS: Between July 1999 and June 2001, 708 fecal specimens were collected, of which 393 rotavirus-negative and 5 rotavirus-positive specimens were detected for HuCVs. Thirty-one point six percentage of fecal specimens from patients with diarrhea was HuCVs positive. Among inpatients, HuCVs positive rate was 17.5%. HuCVs detection was mainly distributed in 3 - 17 mouth-old children, in winter. All 11 strains belonged to NLV GII in which 6 strains GII-3, 2 strains GII-4 and 3 strains GII-7, and they shared 55.1% - 100% nucleotide identity. NLV GII-4 and GII-7 were identified in 2000, while NLV GII-3 and GII-7 in 2001. The preliminary estimate of HuCVs-attributed hospitalization rate was 3.6 per thousand. CONCLUSION: Human caliciviruses with different genotypes circulated among children in Lulong county with GII NLVs were the prevalent strains. The disease burden of HuCVs was second to rotavirus.