A label-free activatable biosensor for in situ detection of exosomal microRNAs based on DNA-AgNCs and hairpin type nucleic acid probes.

Exosomal microRNA (miRNA) is a potential biomarker for cancer diagnosis, metastasis, and treatment. In situ detection of exosomal miRNA is an attractive option due to its simplicity and high accuracy. However, in situ exosomal miRNA detection has encountered challenges because of the low target abundance of targets and limited probe permeability. Herein, a label-free and activatable biosensor was developed for in situ exosomal miRNA assays by utilizing hairpin-shaped nucleic acid probes and DNA-hosted silver nanoclusters (DNA-AgNCs). The probe is directly internalized into the exosomes, and then hybridized with the target miRNA-21. Subsequently, the DNA-AgNCs are pulled closer to the G-rich sequence, ultimately leading to in situ red fluorescence activation. The biosensor not only can detect exosomal miRNA-21 but also distinguish cancer cells from normal cells. Under optimal reaction conditions, the detection limit (LOD) of exosomal miRNA-21 is 1.53 × 107 particles per mL. Furthermore, DNA-AgNCs are used as label-free signal elements for in situ detection of exosomal miRNAs for the first time, expanding the application of nanomaterials in this field. This strategy does not require tedious RNA extraction steps and expensive instruments, and may develop into a non-invasive diagnostic tool for ovarian cancer.

Investigation on the co-infections of Toxoplasma gondii with PRRSV, CSFV or PCV-2 in swine in part of China.

The objective of the present investigation was to estimate the prevalence of Toxoplasma gondii infection and co-infection with porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and porcine circovirus type 2 (PCV-2) in pigs in China. A total of 372 tissues or serum samples collected from pigs distributed in 9 provinces/municipalities of China during the period from February 2011 to November 2012 were assayed for T. gondii antigens and antibodies using enzyme linked immunosorbent assay (ELISA) technique, while the PCR was designed for the detection of the PRRSV, CSFV and PCV-2, respectively. The total positive rate of T. gondii, PRSSV, CSFV and PCV-2 was 9.14% (34/372), 50.00% (186/372), 37.10% (138/372) and 3.23% (12/372), respectively. Among the 34 T. gondii positive samples, 26 samples were simultaneously infected with T. gondii and viruses, while the remaining eight samples were infected with T. gondii alone. In addition, the co-infection rate of T. gondii with PRSSV, T. gondii with PRSSV and CSFV, T. gondii with PRSSV and PCV-2, T. gondii with CSFV and PCV-2, T. gondii with PRSSV, CSFV and PCV-2 was 1.61% (6/372), 4.03% (15/372), 0.27% (1/372), 0.27% (1/372) and 0.81% (3/372), respectively. The results of the present survey revealed that PRRSV and CSFV were the common pathogens co-existing with porcine toxoplasmosis in China, and both of them could increase the chances of T. gondii infection in pig. This is the first report of T. gondii co-infections with viruses in pigs. It is very important to understand the interactions of parasite and virus, and can be used as reference data for the control and prevention of co-infections of T. gondii and viruses in pigs.

High prevalence of torque teno sus virus in China and genetic diversity of the 5' non-coding region.

Members of the family Anelloviridae are emerging circular DNA viruses infecting many species of vertebrates including pigs. To date, members of two distinct genera, Iotatorquevirus, including torque teno sus virus 1a and torque teno sus virus 1b (TTSuV1a and TTSuV1b), and Kappatorquevirus, including torque teno sus virus k2a and torque teno sus virus k2b (TTSuVk2a and TTSuVk2b), have been identified in domestic pigs and wild boars. The goal of this study was to evaluate the prevalence and genetic diversity of these viruses based on 5' non-coding genes in Chinese swine herds experiencing clinical symptoms. One hundred eighty-five clinical samples from 11 different regions, collected during 2008-2009, were analyzed using a PCR method, and the results revealed a high TTSuV-positive rate of 78.9 % (146/185) in pigs. Moreover, we detected co-infection with multiple TTSuV strains in the same pig. Nucleotide sequencing results revealed greater genetic diversity within the genus Kappatorquevirus than within the genus Iotatorquevirus. In addition, TTSuVk2b, a novel virus discovered in New Zealand in 2012, was also identified in this study. In summary, the present work helps us obtain more knowledge about the epidemiology and genetic diversity of TTSuVs.

High prevalence of bovine viral diarrhea virus 1 in Chinese swine herds.

Nested RT-PCR was used to investigate bovine viral diarrhea virus in 511 specimens collected from Chinese pigs exhibiting clinical symptoms between 2007 and 2010. Of these, 137 samples were BVDV-positive and the BVDV prevalence rate was 23.1% (9/39) in 2007, 27.7% (44/159) in 2008, 33.6% (34/101) in 2009, and 23.6% (50/212) in 2010. Twenty of 137 BVDV-positive samples were used for further genetic analysis of the 5'-UTR. Phylogenetic analysis revealed that they were BVDV-1 and subtyped into BVDV-1a, BVDV-1b, BVDV-1m, BVDV-1o and an unknown subgenotype. This study showed that BVDVs were highly prevalent in Chinese pig herds and appropriate measures should be taken to control BVDV prevalence in pig herds.

[Study on liver injury of Oncomelania hupensis caused by Eomecon chinanthe sanguinarine].

OBJECTIVE: To analyze the effects of Eomecon chinanthe sanguinarine (SAN) on glucogen, enzyme activity and lipid peroxidation of Oncomelania hupensis liver so as to explore the mechanism of SAN against Oncomelania hupensis. METHODS: SAN was extracted and purified from the dry powder of Eomecon chionantha. Oncomelania hupensis were immersed in 5 mg/L sanguinarine (50 Oncomelania hupensis per 500 ml solution) or clean water at 25 degrees C for 36 h, the livers were isolated from live snails. Total glucogen content, malondialdehyde (MDA) level, activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), peroxidase (POD) were determined respectively and the data were analyzed by independent t test. RESULTS: The glucogen content of snail livers in the SAN group and the control group were (12.151 +/- 0.204) and (18.113 +/- 0.163) mg/g respectively, the difference between the two groups was significant (P < 0.05); the MDA levels of the two groups were (5.298 +/- 0.441) and (4.351 +/- 0.197) nmol/mgprot respectively, and the difference was not significant (P > 0.05); the activities of ALT, AST, ACP, AKP, SOD in the SAN group were (2.760 +/- 0.076) U/mgprot, (68.723 +/- 2.295) U/mgprot, (407.949 +/-19.868) U/gprot, (191.287 +/- 0.771) U/ gprot and (48.452 +/- 0.193) U/mgprot respectively, the activities of these enzymes in the control group were (1.104 +/- 0.000) U/mgprot, (49.448 +/- 1.626) U/mgprot, (344.475 +/- 30.186) U/gprot, (121.905 +/- 3.127) U/gprot and (38.814 +/- 2.765) U/mgprot respectively, the activities of ALT, AST, ACP, AKP and SOD were significantly increased after immersed in 5 mg/L SAN for 36 h, the differences were significant (All P values < 0.05); yet the difference of POD between the SAN group [(22.170 +/- 0.018) U/mgprot] and the control group [(21.747 +/- 0.264) U/mgprot] was not significant (P > 0.05). CONCLUSION: SAN can destroy liver functions of Oncomelania hupensis through decreasing glucogen content and changing activities of some important enzymes in snail liver.

Co-existence of multiple strains of porcine circovirus type 2 in the same pig from China.

Pigs are often co-infected by different viral strains from the same virus. Up to now, there are few reports about co-existence of different porcine circovirus type 2 (PCV2) strains in China. The aim of this study was to evaluate it in Chinese swine herds. 118 PCV2 positive DNAs isolated from diseased pigs identified by classic PCR were re-detected using a modified differential PCR assay. The results indicated that co-existence rates of PCV2 were 32.2% (38/118) in diseased pigs and 0% (0/41) in asymptomatic pigs. Four PCV2 complete genomes were cloned from two co-infected samples and their nucleotide (nt) identities were 95%-97.3%. The phylogenetic analysis showed that four PCV2 strains were divided into different genotypes, PCV2a, PCV2b, PCV2d and PCV2e, respectively. In addition, co-existence were not detected in 41 serum samples from healthy pigs but PCV2 single infection (31.7%, 13/41) existed. These data revealed that the co-existence of different strains of PCV2 might contribute to the development of more severe clinical symptoms for pigs. This is the first report confirming the co-existence of different PCV2 strains in Chinese swine herds. Meanwhile, this study could help us to understand new infection and prevalence forms of PCV2 clinically.

The RNA profile of porcine parvovirus 4, a boca-like virus, is unique among the parvoviruses.

PPV4 transcribes its genome from a single promoter, and the RNAs are generated via alternate splicing coupled with alternate polyadenylation, a strategy similar to that of the bocaviruses; however, several differences were detected. The PPV4 ORF1 codes for four NS proteins, while the bocavirus ORF1 codes for 1-3 NS proteins. Whereas the VP1/VP2 capsid proteins of bocavirus are encoded by a single RNA, VP1 and VP2 of PPV4 are encoded by two separate RNAs. While ORF3 of PPV4 encodes two NP proteins, ORF3 of bocavirus codes for only one NP polypeptide. Taken together, PPV4 is unique among the parvoviruses.

Detection of a novel porcine parvovirus, PPV4, in Chinese swine herds.

To determine whether the novel porcine parvovirus type 4 (PPV4) recently reported in America is prevalent in China, a set of specific primers was designed and used for molecular survey of PPV4 among the clinical samples collected from various provinces of China between 2006 and 2010. The results showed that PPV4 is present in Chinese swine herds at a rate of 2.09% (12/573) among the clinical samples examined and 0.76% (1/132) among the samples taken from healthy animals. We also noted that PPV4 was not detected in samples taken prior to 2009. Analysis of the coding sequences showed that the Chinese and American PPV4 genome sequences are closely related with greater than 99% nucleotide sequence identity. Similar to a previous study, viral genomes in head-to-tail configuration of various lengths of the non-coding region were detected. Our findings confirmed that PPV4 is a unique recently discovered virus in pigs. Phylogenetically, PPV4 is most closely related to bovine parvovirus 2 (BPV2, which is not a Bocavirus and is not assigned to any Parvovirinae genus) and shares limited ORF1 (33.6%) and ORF2 (24.5%) amino acid identity. With respect to genome structure and organization, PPV4 encodes an ORF3 in the middle of the viral genome that resembles the Bocavirus genus. However, the PPV4 ORF3 encoded protein shares minimal amino acid identity with the ORF3 encoded proteins of the Bocavirus genus.

[Identification of differentially expressed proteins in the liver of Oncomelania snails induced by Eomecon chinanthe sanguinarine].

OBJECTIVE: To identify the differentially expressed proteins in the liver of Oncomelania snails induced by Eomecon chinanthe sanguinarine. METHODS: Sanguinarine was extracted and purified from the dry powder of Eomecon chinanthe. Oncomelania snails were immersed in 5 mg/L sanguinarine (50 Oncomelania snails per 500 ml) or pure water for 36 h (25°C) and the livers were isolated from live snails. Total liver proteins were extracted and separated by two-dimensional gel electrophoresis. Electrophoretogram was analyzed by Image Master 2D 5.0 software. The differentially expressed proteins between sanguinarine group and pure water group were selected and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing of tryptic peptides. RESULTS: In terms of protein spots, 433 ± 14 and 385 ± 12 were observed in sanguinarine group and in water group respectively. The eleven identified differentially expressed proteins included tropomyosin, hypothetical protein XP_533132, actin 87E, keratin 6A, beta-tubulin, mitochondrial inner membrane protein isoform 4, keratin 2, allatostatin precursor, ENSANGP00000020184, actin-3 and ENSANGP00000013943. Among them, hypothetical protein XP_533132 and ENSANGP00000013943 were down-regulated and the other nine proteins were up-regulated in sanguinarine group. CONCLUSION: Sanguinarine could alter the expression of proteins in livers of Oncomelania snails.

2-, 3-, and 4-(1-Oxo-1H-2,3-dihydroisoindol-2-yl)benzoic acids and their corresponding organotin carboxylates: synthesis, characterization, fluorescent, and biological activities.

Three novel organotin complexes with general formula Sn(OH)(bz)(2)L (bz = benzyl, HL = 2-, 3-, or 4-(1-oxo-1H-2,3-dihydroisoindol-2-yl)benzoic acid) and one of their ligands were prepared and characterized. In vitro antifungal and antibacterial activities of these complexes and ligands were investigated with the representative strains of Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Their fluorescence properties have also been discussed.

[Screening and identification of differentially expressed proteins between adult female and male worms of Schistosoma japonicum].

OBJECTIVE: To screen and identify differentially expressed proteins between adult female and male worms of Schistosoma japonicum(S.japonicum). METHODS: Two rabbits infected with the cercaria were perfused with saline in carotid, and approximately two hundred adult female and two hundred male worms of S.japonicum were collected. Approximately 300 microg soluble and hydrophobic proteins of adult female and male worms of S.japonicum were extracted and then the proteins were separated by two-dimensional gel electrophoresis respectively. The analysis using ImageMaster Platinum 2D 5.0 resulted in differentially expressed proteins between adult female and male worms, which were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry sequencing of tryptic peptides. RESULTS: There were (255 +/- 10) and (224 +/- 12) spots detected for soluble proteins and (200 +/- 11) and (132 +/- 8) spots for hydrophobic proteins from adult female and male worms respectively. Six differential proteins were identified, five up-regulated proteins in female worms were thioredoxin, putative ferritin-1 heavy chain, chain B in solution structure of the human ubiquitin-conjugating-enzyme-like protein Mms2-Ubiquitin Complex, heat shock protein 10, cytoplasmic fatty acid binding protein variant H; while only one up-regulated proteins in male worms was identified as 48 kDa histamine receptor subunit peptide 4. CONCLUSION: Several differentially expressed proteins between female and male worms of S. japonicum were recognized through screening and identifying differential proteins between female and male worms of S.japonicum.

[Identification of porcine reproductive and respiratory syndrome virus regulation sequence in 3'-untranslated region].

Based on our established infectious clone of PRRSV, designated as pCBC2, a series of mutagenesis of 3'-untranslated region (3'-UTR) at primary structure and secondary structure level were constructed. Then the full length mutant clones were transfected into MARC-145 cells, from which the influences of the discrete 3'-UTR mutation on PRRSV replication and transcription were analyzed. The properties of the rescued mutant viruses were then further characterized by Northern Blot and plaque morphology analysis. Our results demonstrated that PRRSV could tolerate more than 41 nucleotides deletion and 23nt insertion in the 3'-UTR, however, minor changes in the conserved stem loop region destroyed virus infectivity. To sum up, the stem-loop structure was essential for virus viability, but 5' end of the 3'-UTR tolerates certain level of nucleotide deletion or insertion. This is the first report to define the essential sequence and secondary structure for PRRSV genome replication and it is useful for future research about the regulation element.

[Expression of truncated Toxoplasma gondii GRA8 in the prokaryotic expression plasmids].

OBJECTIVE: To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids. METHODS: The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting. RESULTS: The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were constructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain. CONCLUSION: The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.

[Cloning and characterization of new genes of Schistosoma japonicum].

OBJECTIVE: To clone and characterize new genes of Schistosoma japonicum, Sj, and to provide efficient vaccine candidates. METHODS: Sj adult cDNA library was screened with rabbit sera raised against male worm soluble antigen. The inserted cDNA fragments from the positively selected clones were amplified with PCR and further sequenced, as well as characterized through internet NCBI GenBank software. RESULTS: Eleven positive clones were obtained and two were verified by GenBank as new, including a novel gene designated as Sj-P8 (GenBank accession No. AF517843) and a new partial cDNA of Sj myosin (GenBank accession No. AY770506). The two new genes encoded a transmembrane protein of 75 amino acids and a myosin protein fragment of 212 amino acids respectively. CONCLUSION: The newly obtained genes may provide useful information for the research on Sj vaccine.

[Expression and purification of Toxoplasma gondii GRA4 gene in prokaryotic system].

OBJECTIVE: To construct a prokaryotic expression system containing the dense granule protein 4 (GRA4) of Toxoplasma gondii, purify the expressed protein and detect its immunogenicity. METHODS: The specific fragment of GRA4 gene was amplified by PCR. After subcloning the prokaryotic expression recombinant pET-GRA4, the expressed product was purified with His Bind affinity chromatography and analyzed by Western blot. BALB/c mice were immunized with the GRA4 recombinant protein, and the antibody IgG titer was detected by ELISA. RESULTS: The pET-GRA4 prokaryotic expression system was obtained. The MW of the expressed protein was Mr 40,000 and formed in inclusion body. After purification, the recombinant protein could be specifically recognized by the T. gondii infected rabbit serum. Mice immunized with the purified recombinant protein elicited high titer of IgG antibody. CONCLUSION: The pET-GRA4 recombinant protein was successfully expressed and purified, which shows the immunogenicity.

[Studies on fractionation and protective immunity of the membrane antigen from hepato-portal juveniles of Schistosoma japonicum].

OBJECTIVE: To explore the immunological characteristics of the membrane antigen from hepato-portal juveniles of Schistosoma japonicum and its protective immunity against S. japonicum (Sj) in mice. METHODS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and enzyme-linked immune electro-transfer blot(EITB) methods were used to recognize the membrane antigens from hepato-portal schistosomula (SjHmAg) by infected rabbit sera (IRS) and normal rabbit sera (NRS). Kunming mice were immunized subcutaneously three times(0, 2, 4 weeks) with SjHmAg. Each mouse was challenged with 40 +/- 1 cercariae. Six weeks later the mice were killed, worms and liver eggs were counted. RESULTS: 7 major protein bands appeared on SDS-PAGE. IRS mainly reacted specifically with SjHmAg of 23, 33 and 63 kDa. Compared with the control groups, the reduction rate of worms and eggs per gram liver in the experimental group were 16.2% and 54.4%, respectively. CONCLUSION: Different protein components from SjHmAg are obtained using SDS-PAGE, and the antigen can induce a protective immunity against Sj in mice.

[Study on diagnosis of schistosomiasis by ELISA using periodate-treated soluble egg antigen].

OBJECTIVE: To improve SEA-ELISA, an immunodiagnostic assay for schistosomiasis. METHODS: Soluble egg antigen (SEA) of Schistosoma japonicum was treated with sodium periodate (SP) in order to oxidate its glycosylated epitopes. ELISA using the treated SEA was then performed to detect specific antibodies to SEA in the sera of schistosomiasis patients. RESULTS: Serum samples were tested by ELISA using SEA treated with sodium periodate (SP-SEA-ELISA), including 64 sera from cases with chronic schistosomiasis japonica, 119 sera from normal individuals in non-endemic area, 34 sera from patients with clonorchiasis, 33 sera of paragonimiasis cases and 36 sera from patients with cysticercosis. The results showed that its sensitivity (98.4%) was similar to that of the routine SEA-ELISA (100.0%) (P > 0.05) and the specificity is higher than that of the SEA-ELISA (P < 0.05). SP-SEA-ELISA showed a higher negative rate (89.0%) for sera of schistosomiasis patients 12 months post-treatment than that of the SEA-ELISA (42.1%). CONCLUSION: Use of SP-SEA can increase the specificity of ELISA, reduce cross-reactivity with serum samples from cases infected with other parasites and improve its value in evaluating therapeutic efficacy.

Molecular cloning, expression and vaccination of a novel gene Sj-MA of Schistosoma japonicum.

In order to obtain new gene and to develop the new vaccine candidate of immunoprotection against Schistosoma japonicum (Sj), Sj adult worm cDNA library was screened with anti-sera to soluble male adult worm antigen and resulted in the discovery of a novel gene designated as Sj-MA. Sequence analysis showed that Sj-MA as a complete cDNA contains one open reading frame. It was deduced to contain 249 amino acid residues and encode a 28.8 kD soluble protein with plenty of phosphorylation sites, supposing its action in signal transduction. Furthermore, Sj-MA cDNA was cloned into a prokaryotic expression vector pGEX-5X to construct the recombinant plasmid which was transformed and highly expressed in E. coli as a 54.8 kD glutathione-S-transferase (GST) fusion protein. The fusion protein rSj-MA/GST could be recognized with both anti-male adult worm sera and anti-GST sera in Western blotting. Mice vaccinated with the fusion protein revealed significant worm reduction rate of 34.29% (P<0.001), compared with the control groups. Taken together, the novel gene Sj-MA can be expressed in E. coli as a fusion protein that can elicit immunity against Schistosoma japonicum, suggesting its potential as a new vaccine candidate.