Single-cell profiling of African swine fever virus disease in the pig spleen reveals viral and host dynamics.

African swine fever, one of the major viral diseases of swine, poses an imminent threat to the global pig industry. The high-efficient replication of the causative agent African swine fever virus (ASFV) in various organs in pigs greatly contributes to the disease. However, how ASFV manipulates the cell population to drive high-efficient replication of the virus in vivo remains unclear. Here, we found that the spleen reveals the most severe pathological manifestation with the highest viral loads among various organs in pigs during ASFV infection. By using single-cell-RNA-sequencing technology and multiple methods, we determined that macrophages and monocytes are the major cell types infected by ASFV in the spleen, showing high viral-load heterogeneity. A rare subpopulation of immature monocytes represents the major population infected at late infection stage. ASFV causes massive death of macrophages, but shifts its infection into these monocytes which significantly arise after the infection. The apoptosis, interferon response, and antigen-presentation capacity are inhibited in these monocytes which benefits prolonged infection of ASFV in vivo. Until now, the role of immature monocytes as an important target by ASFV has been overlooked due to that they do not express classical monocyte marker CD14. The present study indicates that the shift of viral infection from macrophages to the immature monocytes is critical for maintaining prolonged ASFV infection in vivo. This study sheds light on ASFV tropism, replication, and infection dynamics, and elicited immune response, which may instruct future research on antiviral strategies.

Determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat by ultrahigh-performance liquid chromatography-tandem mass spectrometry.

RATIONALE: Succinic acid and lactic acid have been associated with diarrhea in weaned piglets. The level of succinic acid and lactic acid in serum, meat, and intestinal contents is important to elucidate the mechanism of diarrhea in weaned piglets. METHODS: A facile method was developed for the quantification of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS). The serum samples underwent protein precipitation with methanol. The meat and intestinal contents were freeze-dried and homogenized using a tissue grinding apparatus. Methanol-water mixture (80:20, v/v) was used for homogenizing the meat, while water was used for homogenizing the intestinal contents. An additional step of protein precipitation with acetonitrile was required for the intestinal contents. The resulting solution was diluted with water before being analyzed by UHPLC/MS/MS. Separation of succinic acid and lactic acid could be achieved within 3 min using a Kinetic XB-C18 column. RESULTS: The coefficients of variation for peak areas of succinic acid and lactic acid were less than 5.0%. The established method demonstrated good linearity as indicated by correlation coefficients exceeding 0.996. Additionally, satisfactory recoveries ranging from 88.58% to 108.8% were obtained. The detection limits (RS/N = 3) for succinic acid and lactic acid were determined to be 0.75 ng/mL and 0.02 µg/mL, respectively. CONCLUSION: This method exhibited high sensitivity, simplicity in operation, and small sample weight, making it suitable for quantitative determination of succinic acid and lactic acid in pigs' serum, intestinal contents, and meat. The method developed will provide valuable technical support in studying the metabolic mechanisms of succinic acid and lactic acid in pigs.

Enhanced patient-based real-time quality control using the graph-based anomaly detection.

OBJECTIVES: Patient-based real-time quality control (PBRTQC) is an alternative tool for laboratories that has gained increasing attention. Despite the progress made by using various algorithms, the problems of data volume imbalance between in-control and out-of-control results, as well as the issue of variation remain challenges. We propose a novel integrated framework using anomaly detection and graph neural network, combining clinical variables and statistical algorithms, to improve the error detection performance of patient-based quality control. METHODS: The testing results of three representative analytes (sodium, potassium, and calcium) and eight independent variables of patients (test date, time, gender, age, department, patient type, and reference interval limits) were collected. Graph-based anomaly detection network was modeled and used to generate control limits. Proportional and random errors were simulated for performance evaluation. Five mainstream PBRTQC statistical algorithms were chosen for comparison. RESULTS: The framework of a patient-based graph anomaly detection network for real-time quality control (PGADQC) was established and proven feasible for error detection. Compared with classic PBRTQC, the PGADQC showed a more balanced performance for both positive and negative biases. For different analytes, the average number of patient samples until error detection (ANPed) of PGADQC decreased variably, and reductions could reach up to approximately 95 % at a small bias of 0.02 taking calcium as an example. CONCLUSIONS: The PGADQC is an effective framework for patient-based quality control, integrating statistical and artificial intelligence algorithms. It improves error detection in a data-driven fashion and provides a new approach for PBRTQC from the data science perspective.

MALDI-TOF-MS for Rapid Screening and Typing of ß-Globin Variant and ß-Thalassemia through Direct Measurements of Intact Globin Chains.

BACKGROUND: Traditional phenotype-based screening for ß-globin variant and ß-thalassemia using hematological parameters is time-consuming with low-resolution detection. Development of a MALDI-TOF-MS assay using alternative markers is needed. METHODS: We constructed a MALDI-TOF-MS-based approach for identifying various ß-globin disorders and classifying thalassemia major (TM) and thalassemia intermedia (TI) patients using 901 training samples with known HBB/HBA genotypes. We then validated the accuracy of population screening and clinical classification in 2 separate cohorts consisting of 16 172 participants and 201 ß-thalassemia patients. Traditional methods were used as controls. Genetic tests were considered the gold standard for testing positive specimens. RESULTS: We established a prediction model for identifying different forms of ß-globin disorders in a single MALDI-TOF-MS test based on δ- to ß-globin, γ- to α-globin, γ- to ß-globin ratios, and/or the abnormal globin-chain patterns. Our validation study yielded comparable results of clinical specificity (99.89% vs 99.71%), and accuracy (99.78% vs 99.16%) between the new assay and traditional methods but higher clinical sensitivity for the new method (97.52% vs 88.01%). The new assay identified 22 additional abnormal hemoglobins in 69 individuals including 9 novel ones, and accurately screened for 9 carriers of deletional hereditary persistence of fetal hemoglobin or δß-thalassemia. TM and TI were well classified in 178 samples out of 201 ß-thalassemia patients. CONCLUSIONS: MALDI-TOF-MS is a highly accurate, predictive tool that could be suitable for large-scale screening and clinical classification of ß-globin disorders.

Distribution of abnormal IgG glycosylation patterns from rheumatoid arthritis and osteoarthritis patients by MALDI-TOF-MSn.

Glycosylation is a post-translational modification essential for maintaining the structure and function of proteins. Abnormal N-glycan patterns have been found in various diseases compared to healthy controls. A decrease in terminal galactosylated N-glycans of serum IgG in rheumatoid arthritis (RA) and osteoarthritis (OA) may be involved in their immunopathogenesis. However, how glycan patterns differ between RA and OA remains unclear. Here, we identified 15 glycan forms of serum IgG from RA and OA using MALDI-TOF MS. We found that IgG galactosylation represented a suitable candidate for differentiating RA from healthy controls (AUC > 0.9). Then, we performed binary logistic regression to screen out three bisecting N-acetylglucosamine (GlcNAc) glycoforms for distinguishing between OA and RA. Combined ROC analysis of the selected glycans yielded an AUC of 0.81 between OA and RA and an AUC of 0.79 between OA and RF/ACPA negative RA. Similar results were found in the validation set. In conclusion, our analysis demonstrates that RA and OA are distinguished on the basis of their different IgG glycan patterns, which thus serve as suitable candidates as biomarkers for reliably identifying clinical conditions such as RA and OA.

Characterization of IgG glycosylation in rheumatoid arthritis patients by MALDI-TOF-MSn and capillary electrophoresis.

An analytical method based on the combination of multistage mass spectrometry (MSn) and capillary electrophoresis (CE) was developed for the analysis of immunoglobulin G (IgG) glycosylation in rheumatoid arthritis (RA) patients. It has been recently suggested that IgG glycosylation defect may be involved in RA immunopathogenesis. Complete characterization of glycans, including both qualitative and quantitative analysis, requires a combination of different techniques, and accurate, robust, sensitive, and high-throughput methodologies are important for analysis of clinical samples. In the present study, N-glycosylation of IgG in RA patients and in healthy people was characterized through identification of the released glycans using multistage matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MSn), and quantitation by CE. Assignment of the IgG N-glycan structures was made through branching pattern analysis by MSn with high-throughput. Further accurate quantitation indicated that galactosylation and sialylation of IgG N-glycans in RA cases were significantly lower than in healthy subjects. The results indicate that CE coupled with MSn can identify abnormal glycosylation of IgG in RA patients compared with healthy people, and that the present work is useful for RA mechanism studies and RA diagnosis. Graphical Abstract Qualitative and quantitative analysis of IgG glycosylation in rheumatoid arthritis patients by MALDI-TOF-MSn and capillary electrophoresis.

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.

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.

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.

Room-temperature sulfur doped NiMoO4 with enhanced conductivity and catalytic activity for efficient hydrogen evolution reaction in alkaline media.

Transition metal oxides have been acknowledged for their exceptional water splitting capabilities in alkaline electrolytes, however, their catalytic activity is limited by low conductivity. The introduction of sulfur (S) into nickel molybdate (NiMoO4) at room temperature leads to the formation of sulfur-doped NiMoO4 (S-NiMoO4), thereby significantly enhancing the conductivity and facilitating electron transfer in NiMoO4. Furthermore, the introduction of S effectively modulates the electron density state of NiMoO4 and facilitates the formation of highly active catalytic sites characterized by a significantly reduced hydrogen absorption Gibbs free energy (ΔGH*) value of -0.09 eV. The electrocatalyst S-NiMoO4 exhibits remarkable catalytic performance in promoting the hydrogen evolution reaction (HER), displaying a significantly reduced overpotential of 84 mV at a current density of 10 mA cm-2 and maintaining excellent durability at 68 mA cm-2 for 10 h (h). Furthermore, by utilizing the anodic sulfide oxidation reaction (SOR) instead of the sluggish oxygen evolution reaction (OER), the assembled electrolyzer employing S-NiMoO4 as both the cathode and anode need merely 0.8 V to achieve 105 mA cm-2, while simultaneously producing hydrogen gas (H2) and S monomer. This work paves the way for improving electron transfer and activating active sites of metal oxides, thereby enhancing their HER activity.

Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

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.

Early mesodermal development in the patellogastropod Lottia goshimai.

Mesodermal development is essential to explore the interlineage variations in the development of spiralians. Compared with model mollusks such as Tritia and Crepidula, knowledge about the mesodermal development of other molluscan lineages is limited. Here, we investigated early mesodermal development in the patellogastropod Lottia goshimai, which shows equal cleavage and has a trochophore larva. The endomesoderm derived from the 4d blastomere, that is, the mesodermal bandlets, was situated dorsally and showed a characteristic morphology. Investigations of the potential mesodermal patterning genes revealed that twist1 and snail1 were expressed in a proportion of these endomesodermal tissues, while all of the five genes we investigated (twist1, twist2, snail1, snail2, and mox) were expressed in ventrally located ectomesodermal tissues. Relatively dynamic snail2 expression suggests additional roles in various internalization processes. By tracing snail2 expression in early gastrulae, the 3a211 and 3b211 blastomeres were suggested to be the precursors of the ectomesoderm, which elongated to become internalized before division. These results help to understand the variations in the mesodermal development of different spiralians and explore the different mechanisms by which ectomesodermal cells are internalized, which has important evolutionary implications.

An improved compact-form antisaturation model-free adaptive control algorithm for a class of nonlinear systems with time delays.

To solve the time-delay problem and actuator saturation problem of nonlinear plants in industrial processes, an improved compact-form antisaturation model-free adaptive control (ICF-AS-MFAC) method is proposed in this work. The ICF-AS-MFAC scheme is based on the concept of the pseudo partial derivative (PPD) and adopts equivalent dynamic linearization technology. Then, a tracking differentiator is used to predict the future output of a time-delay system to effectively control the system. Additionally, the concept of the saturation parameter is proposed, and the ICF-AS-MFAC controller is designed to ensure that the control system will not exhibit actuator saturation. The proposed algorithm is more flexible, has faster output responses for time-delay systems, and solves the problem of actuator saturation. The convergence and stability of the proposed method are rigorously proven mathematically. The effectiveness of the proposed method is verified by numerical simulations, and the applicability of the proposed method is verified by a series of experimental results based on double tanks.

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.

Optimization of electrotransfection conditions of mammalian cells with different biological features.

We introduced eukaryotic expression plasmid pEGFP-N1 encoding green fluorescent protein (GFP) genes into cells with different biological features through electroporation. The effects of conditions, including voltage, capacitor flow, pulse cycle, DNA dosage and buffer, on transfection efficiency were investigated based on fluorescent microscopy and posttransfection survival rate of cells by staining with trypan blue. Better electrotransfection outcomes were achieved in the following epithelial cells: Vero cells at 300 V/850 µF, PK15 cells at 300 V/500 µF, MDCK cells at 200 V/600 µF, F81 cells at 200 V/500 µF, cancer cells MB49 at 300 V/400 µF, Hela cells at 200 V/450 µF, HF-29 cells at 300 V/800 µF and B16F1 cells at 200 V/650 µF. Among fibroblast cells, better electrotransfection was achieved in BHK21 cells at 300 V/600 µF and ST cells at 200 V/750 µF. RPMI-1640 medium without antibiotics and serum demonstrated higher electrotransfection efficiency and cell survival rate than other cell culture media as electroporation buffer. Our findings further prove that electroporation transfection is an effective method for genetic transfection. Cells with different biological features require varying transfection conditions to obtain higher transfection efficiency of target genes.

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.

Upconversion nanoparticles modified with aminosilanes as carriers of DNA vaccine for foot-and-mouth disease.

The potential of the upconversion nanoparticles NaYF(4):Yb/Er@silica(UCPs)/plasmid DNA (pcDNA3.1/VP1-GFP) complex in inducing immune responses was evaluated using the UCPs as carriers of the foot-and-mouth disease virus (FMDV AsiaI/Jiangsu2005) DNA vaccine. The UCPs protection against DNaseI degradation was measured using an in vitro inhibition assay. The expression of the plasmid in vivo was determined via confocal microscopy. Its biocompatibility was evaluated through cytotoxicity assay. Based on the results, the aminosilane-modified UCPs can electrostatically bind, condense, and protect plasmid DNA. Cell viability assays demonstrated that the cytotoxicity of the UCPs/plasmid DNA complex is lower than that of the cationic lipid/plasmid DNA complex, and that the transfection efficiency of UCPs is the same as that of the cationic lipid. Furthermore, the UCP/plasmid DNA complex was intramuscularly administered to guinea pigs. Humoral and cellular immune responses were detected using indirect enzyme-linked immunosorbent assay (ELISA), micro-neutralization assay, and T-lymphocyte proliferation assay. Anti-FMDV specific antibodies, neutralizing antibodies, and T-lymphocyte proliferation responses were induced after vaccination. In the challenge test, all the guinea pigs vaccinated with the UCPs/plasmid DNA complex were fully protected from the FMDV challenge. The current study encourages the use of UCPs as an effective nanosystem for gene delivery to cells for in vitro and in vivo vaccination, and other therapeutic applications.