[Gene cloning, heterologous expression and activity identification of latroeggtoxin-â ¥].

One of the distinct characters of Latrodectus tredecimguttatus is that its toxic components exist not only in the venomous glands, but also in the tissues outside the venomous glands and even in the eggs. Investigation on the toxins outside the venomous glands can deepen our understanding of spider toxins and discover new lead molecules with important application prospects. In order to explore the low-abundance proteinaceous toxins in the L. tredecimguttatus eggs, we used bioinformatic strategies to mine a gene sequence encoding a peptide toxin from the transcriptome of L. tredecimguttatus eggs, and then heterologously expressed the gene successfully with a 3'-RACE combined with nest PCR strategy. Biological activity analyses indicated that the expressed peptide toxin, named latroeggtoxin-Ⅵ (LETX-Ⅵ), could inhibit Na⁺ channel currents in ND7/23 cells and promote dopamine release from PC12 cells, without obvious toxicity against Periplaneta americana and bacteria as well as fungi including Staphylococcus aureus and Candida albicans, demonstrating that LETX-Ⅵ is a mammal-specific neurotoxin with a potential application prospect in development of the tool reagents for neurobiological study and the drugs for treating related diseases.

Gene cloning, heterologous expression and activity identification of latroeggtoxin-Ⅵ / 生物工程学报

One of the distinct characters of Latrodectus tredecimguttatus is that its toxic components exist not only in the venomous glands, but also in the tissues outside the venomous glands and even in the eggs. Investigation on the toxins outside the venomous glands can deepen our understanding of spider toxins and discover new lead molecules with important application prospects. In order to explore the low-abundance proteinaceous toxins in the L. tredecimguttatus eggs, we used bioinformatic strategies to mine a gene sequence encoding a peptide toxin from the transcriptome of L. tredecimguttatus eggs, and then heterologously expressed the gene successfully with a 3'-RACE combined with nest PCR strategy. Biological activity analyses indicated that the expressed peptide toxin, named latroeggtoxin-Ⅵ (LETX-Ⅵ), could inhibit Na⁺ channel currents in ND7/23 cells and promote dopamine release from PC12 cells, without obvious toxicity against Periplaneta americana and bacteria as well as fungi including Staphylococcus aureus and Candida albicans, demonstrating that LETX-Ⅵ is a mammal-specific neurotoxin with a potential application prospect in development of the tool reagents for neurobiological study and the drugs for treating related diseases.

First report on the prevalence of Fasciola hepatica in the endangered Père David's deer (Elaphurus davidianus) in China.

BACKGROUND: Fasciola hepatica is an important zoonotic parasite that causes fasciolosis in a broad range of animals. No information is available about the prevalence of F. hepatica in Père David's deer (Elaphurus davidianus), an endangered species in the world. Therefore, the purpose of the study was to evaluate the prevalence of fasciolosis in Père David's deer in the Dafeng Elk National Natural Reserve, Jiangsu province, China. RESULTS: In this study, 142 fecal samples from Père David's deer were analyzed for F. hepatica by microscopy and nest-PCR. Only one sample was positive for F. hepatica according to microscopy examination, while 18 of 142 (12.68, 95%CI: 2.841-22.45%) samples were positive for F. hepatica according to nest-PCR results. CONCLUSIONS: This is the first report of prevalence of F. hepatica in Père David's deer. The prevalence data indicated that F. hepatica was also present in this endangered animal, which may cause a potential threat to this precious species.

Development of a nest-PCR for detection of Fasciola hepatica DNA in the intermediate snail host, Radix cucunorica, and the prevalence in northwestern China.

Fasciolosis, a foodborne zoonotic disease, caused by Fasciola species which is considered an important problem for human health and livestock husbandry development. Snails are intermediate hosts of F. hepatica, the epidemiological surveillance of snails can evaluate the transmission risk of this disease in human and livestock. In this study, we developed a nest-polymerase chain reaction (nest-PCR) to detect the DNA of F. hepatica in Radix cucunorica, a prevalent intermediate host of this parasite in northwestern China. The nest-PCR was used to amplify a 208 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica with two pairs of primers. The method was able to detect up to 0.16 fg genomic DNA in a 25 µL PCR reaction system even effected with high concentrations of snail DNA, and no cross reaction was observed from the genomic DNA of Paramphistomum cervi, Clonorchis sinensis, Orientobilharzia turkestanicum, Metorchis orientalis, Dicrocoelium chinensis. To evaluate the transmission risk of this disease, 409 snail samples collected from different areas of Gansu province were used to detect and analyze the transmission risk of F. hepatica in this area. Of 409 snail samples, the overall prevalence is 43.76%. The prevalence was 92.75% in Gannan Tibetan Autonomous Prefecture, while no snail was positive for F. hepatica in Linxia Hui Autonomous Prefecture. The nest-PCR was firstly used to detect the infection of F. hepatica in snail. It is a novel, useful and convenient method with high sensitivity and specificity. This study is the first report about the epidemiological surveillance of snail infection by F. hepatica in northwestern China, which will help to evaluate the transmission risk of F. hepatica in northwestern China.

Distribution and characteristics on species of Mycobacterium leprae in China / 临床检验杂志

Objective To realize the distribution and characteristics of Mycobacterium leprae (M.leprae) species and its single nucleotide polymorphisms (SNPs) spreading currently in China.Methods A total of 171 cutaneous lesion specimen of leprosy patients from 22 provinces were collected.The 16S rRNA conservative region of Mycobacterium leprae was amplified by nest PCR and the positive products were sequenced directly and aligned by BLAST.The SNPs of M.leprae were genotyped by restricted fragment length polymorphism for the PCR products.Results The 171 specimen were all Mycobacterium leprae since the amplified fragments of DNA samples were 99％ similar to the Br4923 of M.leprae from Brazil.No new species (M.lepromatosis) was found.Among the 85 samples genotyped for SNPs,SNP3,SNP1 and SNP2 accounted for 78.8％ (67/85),20％ (17/85) and 1.2％ (1/85) respectively.There was no sample with SNP4 genotype to be detected.Among the 171 sequencing specimen,130 showed mutation C-T at 251 bp of 16S rRNA.There was no difference for mutant rate of 16S rRNA gene and SNP genotype among the samples with different clinical pathological types.Certain associations between 16S rRNA C251T mutation and SNP genotype were found.Most of the samples with C251T mutations of 16S rRNA sequence were SNP3,only a few were SNP1 but not SNP2.There was significant difference of SNP genotype distribution among the patients from different regions.The distribution rate of SNP3 genotype in the samples from inland region (97.1％,34/35)was significantly higher than that from coastal region (66％,33/50) (x2 =11.96,P ＜ 0.01) . There was significant difference of the gene mutation rate of 16S rRNA sequence among the patients from different regions.The mutation rate of 16S rRNA in the samples from inland region (94.8％,92/97) was significantly higher than that from coastal region (51.4％,38/74) (x2 =43.56,P ＜0.01).Conclusion C251T mutation in 16S rRNA gene sequence of M.leprae may associate with SNP type suggesting that the characteristics of geographical distribution presented in different genotypes of M.leprae.No new species of M.leprae was found in this study.

Investigation of babesia spp. Infections in blood donors in Guangxi, China / 第二军医大学学报

Objective To know about the Babesia infection situation in the blood donors in Guangxi, China, so as to provide a basical epidemical date, and to provide scientific proofs for safety blood transfusion. Methods A total of 1 900 peripheral blood samples were obtained from tubes of used blood bagstaking from Guangxi, China in 2013. Babesia spp. infections in the donors were tested using Babesia 18S rRNA and β tubulin Nest-PCR, morphological observation, and indirect immunofluorescence assay (IFA). Results We found that the positive rate of Babesia infection was 2. 53% (48/1 900) in Guangxi, with all cases caused by Babesia microti. The PCR sensitivity of Babesia 18S rRNA primer was much higher than that of β tubulin. In 38 blood the erythrocytes were found to have circular and dense nuclear, with ring-form thin cytoplasm under microscope. The specific fluorescence was not observed when Nest-PCR positive specimens with IFA at ≥ 1: 64 antibody concentration, and was taken as negative. Conclusion There is a certain proportion of Babesia microti infection in Guangxi. For those patients with immunocompromised condition who needs blood transfusion, Babesia microti should been tested in blood donors.

A Cross-sectional study on tick-borne Rickettsiae infections among murine-like animals / 预防医学

Objective Toinvestigatethestatusoftick-borneRickettsiaeinfectionsamongmurine-likeanimalsin differentareasofZhejiangprovince.Methods Liverandspleensamplesofmurine-likeanimalscapturedthroughnight trapping method were collected from Anji,Jinhua and Tiantai County according to their geographic locations and historical detection of Rickettsiae .Nest-PCR tests were used to determine the presence of the 16S rRNA genes of Anaplasma and Ehrlichia ,and the heat shock protein genes (groEL)of Rickettsiae (including typhus and spotted fever group)and Orientiainthesesamples.Results Atotalof851murine-likeanimalsbelongingto14specieswerecaptured.The predominant species were Rattus confucianus (30.32%),Apodemus agrarius (18.80%) and Thallomys paedulcus (1 1.75%)and they were significantly different among three areas (P<0.05 ).48 Rickettsia positive were found in 562 tested samples with the positive rate of 8.54%,among which the percentage of Anaplasma,typhus group Rickettsia, Orientia,Ehrlichia and spotted fever group Rickettsia were 3.38%,1.78%,1.78%,1.07% and 0.53% respectively. The positive rates of Anaplasma in Jindong (4.76%)and Anji (4.27%)were significantly higher than that in Tiantai (P<0.05 )while the spotted fever group Rickettsia were found only in Tiantai County.Moreover,Rattus confucianus-the predominant species of Zhejiang Province-had the highest infection rate of tick-borne Rickettsiae up to 14.97%.Co-infections with several Rickettsiae were existed among the same species.Conclusion Rickettsiae infections exist widely among different areas of Zhejiang province and the positive rates are significantly different among species.

Cloning and sequence analysis of SLC25A13 transcripts in human amniocytes.

OBJECTIVE: To amplify the entire ORF of SLC25A13 cDNA which encodes citrin, a liver-type mitochondrial aspartate-glutamate carrier, and to investigate sequence feature of the transcripts for this gene in cultured human amniocytes. This study will provide laboratory evidences for prenatal diagnosis of NICCD at mRNA level. METHODS: Total RNA was extracted from cultured amniocytes, and cDNA was synthesized, and then nest PCR was performed to amplify the entire ORF sequences of SLC25A13. The PCR products were purified, cloned, sequenced, and aligned with the genomic DNA of SLC25A13 to analyze the alternative splicing pattern. RESULTS: The entire ORF of SLC25A13 gene was successfully amplified. Three splice variants of SLC25A13, i.e., SLCA (normal mRNA), SLCB (CAG insertion between exon 9-10) and SLCC (exon 5-11 skipping), were identified in the subjects. However, no abnormal mRNA from the allele with mutation 851del4 was detected in the amniocytes cultured from a carrier fetus of this mutation. CONCLUSIONS: This study demonstrated that the entire ORF of SLC25A13 cDNA can be successfully amplified from cultured human amniocytes, and revealed exon 5-11 skipping as a novel SLC25A13 transcript. Normal mRNA occupied majority of the transcripts in normal control and heterozygous amniocytes which contained normal allele and 851del4 mutation, indicating that the two fetuses wouldn't suffer from NICCD. These SLC25A13 transcription features provided laboratory evidences for prenatal diagnosis of NICCD.

Sequence analysis for Hexon genes of types 3,7 and 11 of adenoviruses identified in Beijing / 中华流行病学杂志

Objective The objective of this study was to develop a rapid,sensitive and specific method for identifying and typing for adenovirus from clinical specimens and to learn about the viruses identified in Beijing on the molecular bases.Methods Primers were designed using hexon gene of adenovirus as target.One primer pair was designed as universal primers for amplifying a 1278 bp gene fragment located at the hcxon gene of adenovirus types 3,7,11 and 21.Four primer pairs located within the region of this 1278 bp fragment were designed specifically for amplifying adenovirusea types 3,7,II and 21.Which were used for a multiplex nest-PCR in a single tube.The products from this multiplex nest-PCR were 502 bp(for type 3),311 bp(for type 7),880 bp(for type 11)and 237 bp(for type 21)，respectively.The type of the adenovirus being tested could be determined after agarose eleetrophoresis analysis of the PCR products．Sequencing was performed for part of the Hexon genes at the 5'end from types 3．7 and 11 strains isolated from clinical specimens and the sequences were compared with corresponding genes published in GenBank．Results PCR products with predicted sizes were visualized in the agarose gel for prototype strains of adenovirus types 3,7,11 and 21,but not for other respiratory viruses,indicating that the technique is specific for typing without cross reaction with other viruses.Out of 61 clinicaI specimens which had been proved to be adenovirus positive by tissue culture and/or immunofluerescence assay,37 were found as adenovirus type 3(37161,60.73％),17 8S adenovirus type 7 (17/61,27.9％),3 Was adenovirus type 11(3161,4.9％).1 Was positive for both type 3 and 7(1／61,1.6％),suggesting that the patient Was co-infected with type 3 and 7 adenoviruses．No adenovirns type 21 was detected.Out of the 61 positive specimens,three showed positive on both tissue culture and immunofluerescence but could not be identified under the methods we used,suggesting that these 3 strains (4.9％)were with the types other than types 3,7,11 and 21．Data from sequence analysis indicated that adeno~ruses types of 3.7 and 11 in this study shared high homology with corresponding types of the strains published in GenBank.Three of the type 3 adenovirus in this study shared highest homology with the adenovirus type 3 identified in Guangzhou,China in 2005.Three of the type 7 adenovirus shared highest homology with the adenovirus type 7 identified in Japan in 1998 and 3 of type 11 adenovirus shared highest homology with the adenovirus type 11 identified in Japan in 2004.Comparing with types 3 and 7.The type 11 in this study showed highest diversity with the corresponding type in GenBank,indicated by the dispersing of the varied amino acids within the region of HVRl and HVR, of the Hexon genes.Conclusion This multiplex nest-PCR method had the advantages of rapid,sensitive and specific and could be used for identilying types of adenoviruses in clinical specimens.Although adenovirus types 3.7 and 1l from Beijing strains shared high homology with the corresponding genes in GenBank,some variances were noticed,especially in type 11 strains.