[Transcriptome sequencing and bioinformatics analysis of Tyrophagus putres-centiae].

OBJECTIVE: To obtain the transcriptome data of Tyrophagus putrescentiae, so as to provide insights into the subsequent functional studies. METHODS: The mixture of male and female T. putrescentiae was sequenced using the Illumina HiSeqTM 2000 high-throughput sequencing platform. Unigenes were obtained after assembling the sequencing data using the Trinity software and compared with the protein sequences in the RefSeq non-redundant protein sequence (NR) database, nucleotide sequence (NT) database, Swiss-Prot database, Kyoto encyclopedia of genes and genomes (KEGG) database and clusters of orthologous groups (COG) database, and the function of the Unigenes was annotated. In addition, the coding DNA sequences (CDS) were predicted through alignment of the Unigenes in NR and Swiss-Prot protein databases. The SSR loci were identified by analysis of the Unigenes in T. putrescentiae with the MISA software, and the SNPs were detected using the SOAPsnp technique. RESULTS: A total of 4.67 GB high-quality data were obtained from raw sequencing data. A total of 51 271 Unigenes were obtained after assembling the sequencing data, with a total length of 41 848 995 nucleotide (nt) and a mean length of 816 nt. A total of 29 053 annotated Unigenes were obtained following comparisons with the public protein databases, and 27 443 CDS were predicted. In addition, there were 23 092 SSR loci and 148 027 SNPs identified. CONCLUSIONS: The database of T. putrescentiae transcriptome is created by sequencing, and a large number of T. putrescentiae transcripts are obtained, which provides a basis for the subsequent functional studies of allergy-related genes.

Wnt/beta-Catenin Signaling Contributes to Vincristine-Induced Neuropathic Pain.

Chemotherapy-induced neuropathic pain (CNP) is the major dose-limiting factor in cancer chemotherapy. However, the mechanisms underlying CNP remain elusive. In the present study, CNP was induced by repeated intraperitoneal injection of vincristine (VCR) into male C57BL/6J mice. VCR administration caused significant activation of Wnt/beta-catenin signaling, which led to the activation of astrocytes, microglia, the release of inflammatory cytokines tumour necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1) and the activation of subsequent mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) signaling pathway in CNP mice. Blocking Wnt/beta-catenin signaling by intrathecal administration of the inhibitors of Wnt response (IWR) effectively attenuated VCR-induced neuropathic pain. Furthermore, IWR inhibited the activation of astrocytes, microglia, TNF-alpha, MCP-1 and MAPK/ERK signaling in the spinal cord, which was triggered by VCR-induced Wnt/beta-catenin signaling upregulation. These results suggest that Wnt/beta-catenin signaling plays a critical role in VCR-induced neuropathic pain and provides evidence for potential interfering with Wnt/beta-catenin signaling to ameliorate VCR-induced neuropathic pain.

Tyrophagus putrescentiae group 4 allergen allergenicity and epitope prediction.

INTRODUCTION AND OBJECTIVES: Allergen-specific immunotherapy (ASIT) is the only allergic disease-modifying therapy available for children and adults, and recombinant allergens are an interesting approach to improve allergy diagnosis and ASIT. Tyrophagus putrescentiae is a common storage mite that produces potent allergens. The aim of this study was to express and characterize recombinant group 4 allergen protein of T. putrescentiae (Tyr p 4), and to further investigate allergenicity and potential epitopes of Tyr p 4. MATERIALS AND METHODS: The cDNA encoding Tyr p 4 was generated by RT-PCR and subcloned into pET-28a(+) plasmid. The plasmid was then transformed into E. coli cells for expression. After purification by nickel affinity chromatography and identification by SDS-PAGE, recombinant Tyr p 4 protein was used for a skin prick test and an ELISA to determine the allergic response. RESULTS: Study participants' allergic response rate to Tyr p 4 protein was 13.3% (16/120). Eight B-cell epitopes and three T-cell epitopes of Tyr p 4 were predicted. CONCLUSIONS: Similar to group 4 allergens of other species of mite, allergenicity of Tyr p 4 is weak. The expression, characterization and epitope prediction of recombinant Tyr p 4 protein provide a foundation for further study of this allergen in the diagnosis and ASIT of storage mite allergy.

[Cloning and sequence analysis of leptin receptor overlapping transcript-like 1 gene from Dermatophagoides farinae].

OBJECTIVE: To obtain the leptin receptor overlapping transcript-like 1 encoding gene (LepROTL1 gene) from Dermatophagoides farina, investigate the molecular characteristics of the gene and construct a prokaryotic expression vector to express this gene. METHODS: The LepROTL1 gene-encoding sequence fragments were captured based on the transcriptome sequencing results, and the full-length gene fragments were amplified from total RNA of D. farinae using a RT-PCR assay, and used to construct the expression plasmid pET28a(+)-LepROTL1, followed by sequencing. The plasmid was transformed into E. coli BL21 (DE3) T1R for the induction of IPTG expression. The expression product was characterized by SDS-PAGE and Western blotting. Bioinformatics analyses were performed to analyze the sequence and the molecular characteristics of its encoded protein. RESULTS: The amplification products of the RT-PCR assay showed a clear band on agarose gel electrophoresis, and sequencing analysis of the pET28a(+)-LepROTL1 plasmid showed 417 bp in length of the coding gene from the start codon ATG to the termination codon TAA. Following the plasmid transformation into E. coli and induction with IPTG, a specific band was seen on SDS-PAGE, indicating successful expression. Bioinformatics analysis showed that the LepROTL1 gene-encoded protein was composed of 134 amino acids, and had a relative molecular weight of 14 378.13 Da, a hydrophilicity index of 1.149, and certain hydrophobicity. The secondary structure was composed of alpha-helix (19 aa, 14.18%), extended strand (48 aa, 35.82%) and random coil (67 aa, 50.00%). The deduced amino acid sequence was used to obtain homologous genes by BLAST, and the phylogenetic tree showed that D. farinae was clustered with D. pteronyssinus. CONCLUSIONS: The full-length sequences and expression plasmid of the LepROTL1 gene are obtained, and the molecular features of the gene are demonstrated using bioinformatics analyses, which provide insights into further studies on the gene.

[Expression level and clinical significance of LncRNA HOXA11-AS in esophageal squamous cell carcinoma patients].

Objective: To explore the expression of long noncoding RNA (lncRNA) HOXA11-AS in esophageal squamous cell carcinoma tissues and the relationship of HOXA11-AS level with clinical outcomes. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the expression level of HOXA11-AS in cell lines HET-1A, EC9706, EC109, and in tumor tissue and paired adjacent tissue samples from 73 ESCC patients who received surgical resection.The correlations of the expression level of HOXA11-AS with clinicopathological features and prognosis were also analyzed. Results: The relative expression levels of HOXA11-AS in tumor tissue and paired adjacent tissue were 0.832±0.387 and 2.486±1.087, respectively, with significant difference (P<0.001). The expression of HOXA11-AS was upregulated in 63.0%(46/73)ESCC tissues. The relative expression levels of HOXA11-AS in HET-1A, EC-9706 and EC-109 cells were 1.000, 23.553±3.221 and 17.217±1.968, respectively. The expression level of HOXA11-AS was upregulated in ESCC cell lines (P<0.001). High expression level of HOXA11-AS was correlated with histological grade and lymph node metastasis of ESCC patients (P<0.05). However, it was not associated with the age, gender, depth of infiltration and TNM staging (P>0.05). The median overall survival (OS) and median disease-free survival (DFS) of patients with low HOXA11-AS expression were 43 months and 42 months, respectively, significantly longer than 37 months and 28 months of patients with HOXA11-AS high expression (P<0.05). Cox model multivariate analysis showed that the expression of HOXA11-AS and lymph node metastasis were independent factors of poor prognosis of ESCC patients. Conclusions: The expression of HOXA11-AS is upregulated in esophageal cancer cell lines and tissues. High expression of HOXA11-AS is associated with poor prognosis of ESCC patients.Therefore, LncRNA HOXA11-AS may serve as a predictive marker of postoperative ESCC patients.

[Management of early bronchopleural fistula after pneumonectomy].

OBJECTIVE: To investigate the management of early bronchopleural fistula(BPF) within one month after pneumonectomy. METHODS: A total of 30 cases developed BPF within one month after pneumonectomy in recent 30 years were reviewed from First Hospital of Jilin University. All patients were treated by reoperation, closed drainage with or without open thoracic window, or endobronchial glue. RESULTS: Seventeen patients were returned to the operating room for reclosure of BPF, one patients died of BPF and 16 reoperations were successful.Twelve cases were treated by closed drainage with or without followed open thoracic window. In 6 patients who were managed by closed drainage alone, 2 cases died of BPF and 4 bronchopleural fistulas closed spontaneously. In 6 patients treated with closed drainage and later with open thoracic window, one case died of BPF. One patient successfully underwent endoscopic glue to close BPF with injection three times. CONCLUSION: Within one month postoperatively, BPF without established empyema with pus should be reoperated if the patient had enough cardiopulmonary reserve and could there fore tolerate reoperation. Contamination of pleural space is not the contraindication to reoperation. Reclosure via original thoracotomy and repair the fistula with pedicled intercostal muscle flap are convenient and efficient. Drainage with or without followed open thoracic window is appropriate for the patients with limited cardiopulmonary reserve, impaired general condition, secondary to empyema with pus, early recurred cancer, or some small fistulas.

Ecological stabilization of thickened wastewater sludge from CAST process.

Wastewater sludge ecological stabilization (WWSES) pilot scale experiments were conducted for thickening treatment and disposal of sludge which came from Cyclic Activated Sludge Technology (CAST) process. The study was performed over the periods from June to November 2005 and from May to November 2006, on a bed of 80 m2. The sludge loadings were stopped for the winter from December 2005 and resumed in May 2006. The results shows that dried sludge layer has higher permeation coefficients of 0.15-1.3 m/h. It is suggested that the percolate did not filtrate downwards evenly, part of percolate filtrates downwards along stems, roots and cracks existing in dried sludge which have lower flow resistance. The relationship of dried sludge thickness and operation time is in accord with quadratic equation under fluctuating sludge loadings. Linear regression equation can indicate dried sludge thickness variation under fixed sludge loading. In comparison with natural ones, coarse protein content of Phragmites australis roots in the system is twice as high, coarse fiber content of roots, coarse fat content of stems and leaf are obviously higher; and coarse protein content of Typha augustifolia in the system are obviously higher, while coarse fat and coarse fiber contents have no significant difference.

An effective indirect fluorescent antibody test for diagnosis of intestinal acariasis.

Adult mites' bodies of Dermatophagoides farinae were used as antigen in an indirect fluorescent antibody test (IFAT) to detect mite-specific IgG in sera of 48 patients with intestinal acariasis based on stool examination. Antibody titers with positive reaction ranged from 1:4 to 1:512 in 48 patients with intestinal acariasis. If antibody titers > or = 1:16 is regarded as being positive, the positive rate of patients detected with IFAT was 92%.

Biological application of multi-component nanowires in hybrid devices powered by F1-ATPase motors.

In this paper, construction of hybrid device by integrating nanowires with F(1)-ATPase motors is described. The nickel nanowires and multi-segment nanowires, including gold and nickel, were fabricated by electrochemical deposition in nanoporous templates. The nickel nanowires functionalized by biotinylated peptide can be assembled directly onto F(1)-ATPase motors to act as the propellers. If the multi-component nanowires, including gold and nickel, were selectively functionalized by the thiol group modified ssDNA and the synthetic peptide, respectively, the biotinylated F(1)-ATPase motors can be attached to the biotinylated peptide on nickel segment of the nanowires. Then, the multi-component nanowires can also be used as the propellers, and one may observe the rotations of the multi-component nanowires driven by F(1)-ATPase motors. Therefore, introduction of multiple segments along the length of a nanowire can lead to a variety of multiple chemical functionalities, which can be selectively bound to cells and special biomolecules. This method provides an insight for the construction of other hybrid devices with its controlling arrangement of different biomolecule on designed nanometer scale structures.

Evaluation of heavy metal inhibition of activated sludge by TTC and INT-electron transport system activity tests.

TTC and INT-electron transport system activity tests were compared for assessing heavy metal inhibition of activated sludge. The median inhibitory concentrations (IC50s) of Cu2+, Zn2+, Cd2+, Hg2+, Ni2+, Pb2+ and Ag+ measured via TTC test were lower than those measured via INT test, which indicates that the INT-electron transport system activity test was less sensitive to heavy metal toxicity than the TTC test. Tested heavy metals brought about similar decrease in TTC-electron transport system activity and COD removal rate, but less decrease in INT-electron transport system activity than COD removal rate, which suggests that the TTC-electron transport system activity is a better parameter for reflecting heavy metal inhibition of activated sludge than INT-electron transport system activity. The ranking of tested heavy metals in order of decreasing toxicity based on TTC test was Hg2+, Cd2+, Cu2+, Ag+, Zn2+, Ni2+ and Pb2+, and the ranking based on INT test was Hg2+, Ag+, Cu2+, Cd2+, Zn2+, Ni2+ and Pb2+.

Acaroid mites (Acari: Astigmata) in Chinese traditional medicines.

Almost 2000 samples of the dried plants and animals used in Chinese traditional medicine were collected from a storehouse in Huainan city, Anhui province, China, where they had been kept for at least 6 months. When examined, every sample was found to contain acaroid mites. The mites included representatives of 44 species belonging to 22 genera and seven families. It seems that samples of Chinese traditional medicine frequently become severely infested with mites while in storage. More importance should be attached to protecting the medicines against mite infestation and to protecting against acariasis those who ingest or simply handle the medicines.

Choice of lasers for laser angioplasty.

Thrombi in test-tube, intraarterial thrombi and pig aorta wall were in vitro irradiated with continuous CO2 laser, short pulsal and ultrashort pulsal YAG laser (with pulse-width of 10 ns and 40 ps, respectively) and their double frequency laser and excimer laser with a wave-length of 308 nm and pulse-width of 20 ns. Their effect of vaporizing and ablating (photodecomposing) thrombi and their thermal injuring effect on adjacent tissues were compared and assessed in order to select optimal laser with little thermal injuring and more rapid vaporizing or ablating thrombi effect for laser angioplasty. The experimental results showed that excimer laser, ultrashort pulsal YAG laser and its double frequency laser, and double frequency laser of short pulsal YAG laser, with laser beam and blood vessel kept in a coaxial position, can effectively vaporize or ablate thrombi without thermal injury to vessel wall. So they may be used for laser angioplasty. Of them, especially, excimer laser and double frequency laser of ultrashort pulsal YAG laser have short wave-length and high peak power, and more effectively ablate thrombi than others, so they proved to be optimal lasers for laser angioplasty.