Periodically poled self-frequency-doubling green laser fabricated from Nd:Mg:LiNbO3 single crystal.

Although a breakthrough in the fabrication of green laser diodes has occurred, the high costs associated with the difficulty of manufacture still present a great obstacle for its practical application. Another approach for producing a green laser, by combining a laser device and a nonlinear crystal, entails the fabrication of complex structures and exhibits unstable performance due to interface contact defects, thus limiting its application. In this work, we report the fabrication by domain engineering of high quality periodically poled LiNbO3, co-doped with Nd³âº and Mg²âº, which combines a laser medium and a high efficiency second harmonic conversion crystal into a single system that is designed to overcome the above problems. An 80 mW self-frequency doubling green laser was constructed for the first time from a periodically poled Nd:Mg:LiNbO3 crystal of 16 mm in length. This crystal can be used for developing compact, stable, highly efficient mini-solid-state-lasers, which promise to have many applications in portable laser-based spectroscopy, photo-communications, terahertz wave generation, and laser displays.

Full-length genomic characterizations of two canine parvoviruses prevalent in Northwest China.

Canine parvovirus (CPV) can cause acute hemorrhagic diarrhea and fatal myocarditis in young dogs. Currently, most studies have focused on the evolution of the VP2 gene, whereas the full-length genome of CPV has been rarely reported. In this study, the whole genomes of CPV-LZ1 and CPV-LZ2 strains prevalent in Northwest China were determined and analyzed in comparison with those of the reference CPVs. The genome sequences of both LZ strains consisted of 5053 nucleotides. CPV-LZ1 and CPV-LZ2 strains were designated as new CPV-2a and CPV-2b, respectively. Sequence alignment analysis results revealed that these two new strains underwent specific unique variations during the process of local adaption. The left non-translated regions of these strains formed a Y-shaped hairpin structure, whereas the right non-translated regions lacked the reiteration of DNA sequence. A phylogenetic tree constructed from 33 whole coding regions of CPVs showed a strong spatial clustering, and these two strains belonged to the Chinese strain cluster lineage. This study provides a method to obtain the full-length genome of CPV. The isolation and characterization of these viruses adds incrementally to the knowledge of the full-length genome of CPV. The results from this study also provide insight into the molecular epidemiology and genetic diversity of the CPV field isolates from Northwest China and can be useful in preventing and controlling CPV infection in this region.

Self-assembly of virus-like particles of canine parvovirus capsid protein expressed from Escherichia coli and application as virus-like particle vaccine.

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus (CPV). Current commercial vaccines are mainly attenuated and inactivated; as such, problems concerning safety may occur. To resolve this problem, researchers developed virus-like particles (VLPs) as biological nanoparticles resembling natural virions and showing high bio-safety. This property allows the use of VLPs for vaccine development and mechanism studies of viral infections. Tissue-specific drug delivery also employs VLPs as biological nanomaterials. Therefore, VLPs derived from CPV have a great potential in medicine and diagnostics. In this study, small ubiquitin-like modifier (SUMO) fusion motif was utilized to express a whole, naturalVP2 protein of CPV in Escherichia coli. After the cleavage of the fusion motif, the CPV VP2 protein has self-assembled into VLPs. The VLPs had a size and shape that resembled the authentic virus capsid. However, the self-assembly efficiency of VLPs can be affected by different pH levels and ionic strengths. The mice vaccinated subcutaneously with CPV VLPs and CPV-specific immune responses were compared with those immunized with the natural virus. This result showed that VLPs can effectively induce anti-CPV specific antibody and lymphocyte proliferation as a whole virus. This result further suggested that the antigen epitope of CPV was correctly present on VLPs, thereby showing the potential application of a VLP-based CPV vaccine.

Development and characterization of a recombinant infectious bronchitis virus expressing the ectodomain region of S1 gene of H120 strain.

Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is a highly contagious chicken disease, and can lead to serious economic losses in poultry enterprises. The continual introduction of new IBV serotypes requires alternative strategies for the production of timely and safe vaccines against the emergence of variants. Modification of the IBV genome using reverse genetics is one way to generate recombinant IBVs as the candidates of new IBV vaccines. In this study, the recombinant IBV is developed by replacing the ectodomain region of the S1 gene of the IBV Beaudette strain with the corresponding fragment from H120 strain, designated as rBeau-H120(S1e). In Vero cells, the virus proliferates as its parental virus and can cause syncytium formation. The peak titer would reach 10(5.9) 50% (median) tissue culture infective dose/mL at 24 h post-infection. After inoculation of chickens with the recombinant virus, it demonstrated that rBeau-H120(S1e) remained nonpathogenic and was restricted in its replication in vivo. Protection studies showed that vaccination with rBeau-H120 (S1e) at 7-day after hatch provided 80% rate of immune protection against challenge with 10(3) 50% embryos infection dose of the virulent IBV M41 strain. These results indicate that rBeau-H120 (S1e) has the potential to be an alternative vaccine against IBV based on excellent propagation property and immunogenicity. This finding might help in providing further information that replacement of the ectodomain fragment of the IBV Beaudette S1 gene with that from a present field strain is promising for IBV vaccine development.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

Fabrication of novel hierarchical structured Fe3O4 @LnPO4 (Ln=Eu, Tb, Er) multifunctional microspheres for capturing and labeling phosphopeptides.

Novel core-shell structured Fe3O4@LnPO4 (Ln=Eu, Tb, Er) multifunctional microspheres with a magnetic Fe3O4 core and a LnPO4 shell covered with spikes are synthesized for the first time through the combination of a homogeneous precipitation approach and an ion-exchange process. Their potential for selective capture, rapid separation, and easy mass spectrometry (MS) labeling of the phosphopeptides from complex proteolytic digests are evaluated. These affinity microspheres can improve the specificity for capture of the phosphopeptides, realize fast magnetic separation, enhance the MS detection signals, and directly identify phosphopeptides through 80 Da mass loss in the mass spectra. The synthesis strategy could become a general and effective technique for similar core-shell hierarchical structures.

Monodisperse REPO4 (RE = Yb, Gd, Y) hollow microspheres covered with nanothorns as affinity probes for selectively capturing and labeling phosphopeptides.

Rare-earth phosphate microspheres with unique structures were developed as affinity probes for the selective capture and tagging of phosphopeptides. Prickly REPO(4) (RE = Yb, Gd, Y) monodisperse microspheres, that have hollow structures, low densities, high specific surface areas, and large adsorptive capacities were prepared by an ion-exchange method. The elemental compositions and crystal structures of these affinity probes were confirmed by energy-dispersive spectroscopy (EDS), powder X-ray diffraction (XRD), and Fourier-transform infrared (FTIR) spectroscopy. The morphologies of these compounds were investigated using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and nitrogen-adsorption isotherms. The potential ability of these microspheres for selectively capturing and labeling target biological molecules was evaluated by using protein-digestion analysis and a real sample as well as by comparison with the widely used TiO(2) affinity microspheres. These results show that these porous rare-earth phosphate microspheres are highly promising probes for the rapid purification and recognition of phosphopeptides.

Immunization of mice by hollow mesoporous silica nanoparticles as carriers of porcine circovirus type 2 ORF2 protein.

BACKGROUND: Porcine circovirus type 2 (PCV2) is a primary etiological agent of post-weaning multi-systemic wasting syndrome (PMWS), which is a disease of increasing importance to the pig industry worldwide. Hollow mesoporous silica nanoparticles (HMSNs) have gained increasing interest for use in vaccines. METHODS: To study the potential of HMSNs for use as a protein delivery system or vaccine carriers. HMSNs were synthesized by a sol-gel/emulsion(oil-in-water/ethanol) method, purified PCV2 GST-ORF2-E protein was loaded into HMSNs, and the resulting HMSN/protein mixture was injected into mice. The uptake and release profiles of protein by HMSNs in vitro were investigated. PCV2 GST-ORF2-E specific antibodies and secretion of IFN-γ were detected by enzyme-linked immunosorbent assays, spleen lymphocyte proliferation was measured by the MTS method, and the percentage of CD4+ and CD8+ were determined by flow cytometry. RESULTS: HMSNs were found to yield better binding capacities and delivery profiles of proteins; the specific immune response induced by PCV2 GST-ORF2-E was maintained for a relatively long period of time after immunization with the HMSN/protein complex. CONCLUSION: The findings suggest that HMSNs are good protein carriers and have high potential for use in future applications in therapeutic drug delivery.

Lanthanum silicate coated magnetic microspheres as a promising affinity material for phosphopeptide enrichment and identification.

Novel Fe(3)O(4)@La(x)Si(y)O(5) affinity microspheres consisting of a superparamagnetic Fe(3)O(4) core and an amorphous lanthanum silicate shell have been synthesized. The core-shell-structured Fe(3)O(4)@La(x)Si(y)O(5) microspheres, with a mean size of ca. 480 nm, had rough lanthanum silicate surfaces and displayed relatively strong magnetism (47.2 emu g(-1)). This novel affinity material can be used for selective capture, rapid magnetic separation, and part dephosphorylation (which plays an important role in identifying phosphopeptides in MS) of the phosphopeptides in a peptide mixture. Its ability to selectively trap and magnetically isolate as well as label the phosphopeptides was evaluated using a standard phosphorylated protein (ß-casein) and a real sample (human serum). Phosphopeptides and their corresponding label ions were detected for concentrations of ß-casein as low as 1 × 10(-9) M and in mixtures of ß-casein and BSA with molar ratios as low as 1:50. In addition, this affinity material, with its labeling properties, is superior to commercial TiO(2) beads in terms of interference from non-phosphopeptide molecules. These results reveal that the lanthanum silicate coated magnetic microspheres represent a promising affinity material for the rapid purification and recognition of phosphopeptides.

Synthesis and characterization of [Eu(DBM)3phen]Cl3 @ SiO2-NH2 composite nanoparticles.

Fluorescent rare earth complex Eu(DBM)3(phen)]Cl3@SiO2-NH2 nanoparticles were synthesized by combination of solvent precipitation method and Stöber method. The morphologies, structure, surface and optical properties of the samples were characterized by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), fourier transform infrared (FTIR) spectroscopy, thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), and fluorescence spectrophotometer (FS). The observation from FE-SEM images indicate that the obtained samples are spherical and uniform nanoparticles with a tunable average sizes from 140 nm to 300 nm. TEM results verify a core-shell structure of the nanoparticles. The FTIR spectrum confirms the characteristic vibration absorption peaks of the complex [Eu(DBM)3(phen)]Cl3@SiO2-NH2. TGA result indicates that the complex is stable below 200 degrees C. The photoluminescence analysis shows that the complex has Eu3+ characteristic red luminescence and broader excitation peak from 200 nm to 450 nm that can meet the demands of fluorescent confocal imaging. The amino groups are directly introduced to the [Eu(DBM)3(phen)]Cl3@SiO2-NH2 nanoparticles surface by using APS (3-aminopropyl triethoxysilane). This makes the surface modification and bioconjugation of the nanoparticles easier. The nano-sized spheres could be provided a basis for further expansion of its application in biomedical imaging, biological detection and fluorescent nanoprobes.

Synthesis of the Fe3O4@SiO2@SiO2-Tb(PABA)3 luminomagnetic microspheres.

In this paper, we describe the synthesis and characterization of a luminomagnetic microspheres with core-shell structures (denoted as Fe3O4@ SiO2 @SiO2-Tb(PABA)3). The luminomagnetic microspheres were characterized by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometer (VSM), and photoluminescence spectrophotometer (PL). The SEM observation shows that the microsphere consists of the magnetic core with about 400 nm in average diameter and silica shell doped with terbium complex with an average thickness of about 90 nm. It has a saturation magnetization of 15.8 emu/g and a negligible coercivity at room temperature and exhibits strong green emission peak from 5D4 --> 7F5 transition of Tb3+ ions. The luminomagnetic microspheres with good magnetic response and fluorescence probe property as well as water-dispersibility would have potential medical applications, such as time-resolved fluoroimmunoassay (TR-FIA), fluorescent imaging, and magnetic resonance imaging (MRI).

Synthesis and characterization of nano-PVP/EuL3 luminescent complex.

Nano-sized rare earth europium [Eu(III)]-benzoic acid [HL] luminescent complex powders have been synthesized in polyvinylpyrrolidone matrix by precipitation method. The chemical constitution of the complex has been demonstrated as PVP/EuL3 by a combination of elemental analysis, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and fourier-transform infrared spectroscopy (FT-IR). X-ray diffraction analysis (XRD) and scanning electron microscopy (SEM) have shown that the complex is a new nanocrystal whose structure is totally different from the ligand. Thermogravimetric/Differential thermal analysis (TG/DTA) have indicated that the luminescent complex is thermally stable below 500 degrees C. Photoluminescence spectra (PL) have shown that the nanocomplex can emit Eu3+ characteristic red fluorescence under ultraviolet excitation.

Development and validation of an ELISA using a protein encoded by ORF2 antigenic domain of porcine circovirus type 2.

BACKGROUND: The capsid protein (ORF2) is a major structural protein of porcine circovirus type 2 (PCV2). A simple and reliable diagnostic method based on ORF2 protein immunoreactivity would serve as a valuable diagnostic method for detecting serum antibodies to PCV2 and monitoring PCV infection. Here, we reported an indirect enzyme-linked immunosorbent assay (I-ELISA) by using an antigenic domain (113-147AA) of ORF2-encoded antigen, expressed in E. coli, for diagnosis of PCV infection. RESULTS: The ELISA was performed on 288 serum samples collected from different porcine herds and compared with an indirect immunofluorescent assay (IFA). In total, 262 of 288 samples were positive as indicated by both I-ELISA and IFA. The specificity and sensitivity of I-ELISA were 87.7% and 93.57%. CONCLUSIONS: This ELISA is suitable for detection and discrimination of PCV2 infection in both SPF and farm antisera.

Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the open reading frames 1a of highly pathogenic porcine reproductive and respiratory syndrome virus genome was developed. The 10 reference strains, 1 clinical isolation strain and 122 positive samples were tested. Positive reactions were confirmed for all strains and specimens by reverse transcription loop-mediated isothermal amplification and nested reverse transcription polymerase chain reaction (RT-PCR). The results showed this detection technique is more reliable and convenient for rapid and sensitive diagnosis of highly pathogenic porcine reproductive and respiratory syndrome virus infection.

Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification.

A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). The amplification could be finished in 60 min under isothermal condition at 64 degrees C by employing a set of four primers targeting the cap gene of PCV2. The LAMP assay showed higher sensitivity than the conventional PCR, with a detection limit of five copies per tube of purified PCV2 genomic DNA. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 1 (PCV1), porcine parvovirus (PPV), porcine pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection rate of PCV2 LAMP for 86 clinical samples was 96.5% and appeared greater than that of the PCR method. The LAMP assay reported can provide a rapid yet simple test of PCV2 suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Development of reverse transcription loop-mediated isothermal amplification for rapid detection of H9 avian influenza virus.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a unique gene amplification method that can be completed within 45 min at 63 degrees C. In this study, RT-LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the H9 subtype of avian influenza virus (AIV). The experiment results from the reference strains demonstrated that the established RT-LAMP sensitivity was 10-fold higher than that of RT-PCR, with the detection limit of 10 copies per reaction, and no cross-reactivity was observed from the samples of other related viruses including H5N1, H3N2 subtype of AIV and Newcastle disease virus. Furthermore, a total of 112 clinical samples were tested by RT-LAMP, RT-PCR, and virus isolation, respectively. All of the 85 positive specimens identified by virus isolation were also positive by RT-LAMP, while 7 of these samples were missed by RT-PCR. These results suggest that the present RT-LAMP system may provide a new avenue for the recognition of H9 subtype virus, and may be employed to screen for potential carriers in wild and domestic birds.

Rapid discrimination of H5 and H9 subtypes of avian influenza viruses and Newcastle disease virus by multiplex RT-PCR.

Avian influenza and Newcastle disease are the highly contagious and most economically important diseases in poultry industry throughout the world. A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed for the rapid and specific discrimination of H5 and H9 subtypes of avian influenza viruses (AIV) and Newcastle disease virus (NDV). Three sets of specific primers were applied in the assay based on the sequences of the hemagglutinin gene of H5-AIV, H9-AIV and fusion protein gene of NDV. 59 clinical samples including the throat washes, oral swabs, and cloacal scrapings were detected by mRT-PCR and single RT-PCR (sRT-PCR), respectively. The results indicated that the sensitivity and specificity of mRT-PCR were in accordance with sRT-PCR. The mRT-PCR developed in this study may therefore provide a new avenue to rapid detection of these important pathogens in one reaction.

Viroporin Activity of the Foot-and-Mouth Disease Virus Non-Structural 2B Protein.

Viroporins are a family of low-molecular-weight hydrophobic transmembrane proteins that are encoded by various animal viruses. Viroporins form transmembrane pores in host cells via oligomerization, thereby destroying cellular homeostasis and inducing cytopathy for virus replication and virion release. Among the Picornaviridae family of viruses, the 2B protein encoded by enteroviruses is well understood, whereas the viroporin activity of the 2B protein encoded by the foot-and-mouth disease virus (FMDV) has not yet been described. An analysis of the FMDV 2B protein domains by computer-aided programs conducted in this study revealed that this protein may contain two transmembrane regions. Further biochemical, biophysical and functional studies revealed that the protein possesses a number of features typical of a viroporin when it is overexpressed in bacterial and mammalian cells as well as in FMDV-infected cells. The protein was found to be mainly localized in the endoplasmic reticulum (ER), with both the N- and C-terminal domains stretched into the cytosol. It exhibited cytotoxicity in Escherichia coli, which attenuated 2B protein expression. The release of virions from cells infected with FMDV was inhibited by amantadine, a viroporin inhibitor. The 2B protein monomers interacted with each other to form both intracellular and extracellular oligomers. The Ca(2+) concentration in the cells increased, and the integrity of the cytoplasmic membrane was disrupted in cells that expressed the 2B protein. Moreover, the 2B protein induced intense autophagy in host cells. All of the results of this study demonstrate that the FMDV 2B protein has properties that are also found in other viroporins and may be involved in the infection mechanism of FMDV.

Novel Ti4+-chelated magnetic nanostructured affinity microspheres containing N-methylene phosphonic chitosan for highly selective enrichment and rapid separation of phosphopeptides.

Magnetic composite particles immobilized with metal affinity ions show high potential in the phosphoproteome mass-spectrometric (MS) analysis. However, the preparation of this kind of material still suffers from a complicated synthesis procedure and high cost. In this work, the magnetic nanostructured composite microspheres (MPCS) incorporated into N-methylene phosphonic chitosan were first fabricated via a facile one-pot synthesis strategy and titanium ions (Ti4+) were subsequently immobilized onto the MPCS to form MPCS-Ti4+ affinity particles. The uniform spherical MPCS-Ti4+ affinity particles with abundant chelated titanium ions have a particle size distribution between 126 nm and 280 nm, and they display superparamagnetism with a saturation magnetization (Ms) value of 44.75 emu g-1. The amount of the immobilized Ti4+ ions was estimated to be 11.6 wt% by EDS. The MPCS-Ti4+ exhibited excellent dispersibility in aqueous solution, high affinity selectivity for phosphopeptides and quite fast magnetic separation within 10 s as well as good reusability. Thus, the prepared magnetic MPCS-Ti4+ nanostructured affinity materials will possess great potential in phosphoproteome research.

Novel core-shell cerium(IV)-immobilized magnetic polymeric microspheres for selective enrichment and rapid separation of phosphopeptides.

In this work, novel magnetic polymeric core-shell structured microspheres with immobilized Ce(IV), Fe3O4@SiO2@PVPA-Ce(IV), were designed rationally and synthesized successfully via a facile route for the first time. Magnetic Fe3O4@SiO2 microspheres were first prepared by directly coating a thin layer of silica onto Fe3O4 magnetic particles using a sol-gel method, a poly(vinylphosphonic acid) (PVPA) shell was then coated on the Fe3O4@SiO2 microspheres to form Fe3O4@SiO2@PVPA microspheres through a radical polymerization reaction, and finally Ce(IV) ions were robustly immobilized onto the Fe3O4@SiO2@PVPA microspheres through strong chelation between Ce(IV) ions and phosphate moieties in the PVPA. The applicability of the Fe3O4@SiO2@PVPA-Ce(IV) microspheres for selective enrichment and rapid separation of phosphopeptides from proteolytic digests of standard and real protein samples was investigated. The results demonstrated that the core-shell structured Fe3O4@SiO2@PVPA-Ce(IV) microspheres with abundant Ce(IV) affinity sites and excellent magnetic responsiveness can effectively purify phosphopeptides from complex biosamples for MS detection taking advantage of the rapid magnetic separation and the selective affinity between Ce(IV) ions and phosphate moieties of the phosphopeptides. Furthermore, they can be effectively recycled and show good reusability, and have better performance than commercial TiO2 beads and homemade Fe3O4@PMAA-Ce(IV) microspheres. Thus the Fe3O4@SiO2@PVPA-Ce(IV) microspheres can benefit greatly the mass spectrometric qualitative analysis of phosphopeptides in phosphoproteome research.