Transcriptomic analysis of the larva Taenia multiceps.

Taenia multiceps is an adult worm affiliated to Taeniidae family, Platyhelminthes phylum. The larvae of the parasite (Coenurus cerebralis) parasitic in the brain and spinal cord in domestic and wild ruminants or humans can led to a fatal central nervous system (CNS) disease. The aims of the present study were to define the transcriptome profiles of the larvae of T. multiceps by RNA-Seq approach, and to generate large functional gene datasets that could be used to predict the key molecular pathways linked to this cestode. Our results generated a total of 39,094,890 clean reads that were assembled from the sequence data in 90,833 contigs. Briefly, 70,253 unigenes with a mean length of 1492bp were formed. Based on a sequence similarity search against the databases (NR, Swissport, GO, COG, KEGG) using BLASTX with an E-value cutoff of 10-5, 40,465 of unigenes were identified as coding sequences (CDS) and 3261 were scanned by ESTScan. The present study carried out the transcriptome of the larval stage of T. multiceps, which provides a solid foundation for further studies in molecular biology and biochemistry as well as identification of candidate genes used in diagnosis and vaccine development.

Proteomic analysis of differentially expressed proteins in the three developmental stages of Trichinella spiralis.

Trichinella spiralis, an intracellular parasitic nematode, can cause severe foodborne zoonosis, trichinellosis. The life cycle of T. spiralis consists of adult (Ad), muscle larvae (ML) and newborn larvae (NBL). The protein profiles in different developmental stages of the parasite remain unknown. In the present study, proteins from lysates of Ad, ML and NBL were identified by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 4691 proteins were identified in all the developmental stages, of which 1067 proteins were differentially expressed. The number of up-regulated proteins in NBL was higher than that of the other two groups. The protein profiles from Ad, ML and NBL were compared in pairs. The identified proteins were involved in various functions of T. spiralis life cycle, including sexual maturity, metabolism, utilization of carbohydrates, lipids and nucleotides, and other crucial developmental processes that occur at distinct stages. Further investigation of the transcriptional levels of major sperm protein, serine protease, zinc finger protein, etc. from the different protein profiles using quantitative RT-PCR showed identical results to the iTRAQ analysis. The differentially expressed proteins that are involved in developmental regulation and host-parasite interactions should be further studied.

Cloning and characterization of thioredoxin peroxidases from Trichinella spiralis.

The intracellular parasitic nematode, Trichinella spiralis, can initiate a high level of oxidative stress, especially during rapid growth and generative propagation phases. Thioredoxin peroxidases (TPXs) protect helminths against oxidative stress, but none has been identified in T. spiralis. Here, 3 members of the TPX family were cloned from T. spiralis muscle larvae (ML). The lengths of TsTPX ORFs were 747bp, 588bp and 594bp, respectively, and the deduced proteins predicted to contain AhpC-TSA and 1-cys Prx_C domains. Interestingly, qRT-PCR data showed that TsTPX genes were expressed in all three developmental stages of T. spiralis. The TsTPX2 and TsTPX3 genes were up-regulated in day 3 adults (Ad3) compared with newborn larvae (NBL) and ML (P<0.05); expression levels of the TsTPX1 gene in ML were higher compared with Ad3 and NBL amounts (P<0.05). After prokaryotic expression, the reactivity of rTsTPX proteins was assessed by Western-blotting: only rTsTPX1 was specifically recognized by T. spiralis infection sera from pigs. Enzyme catalytic experiments showed that rTsTPX proteins could deoxidize H2O2 in the presence of DTT, with the catalytic ability increasing with protein concentration and time.

Sequence variation and bioinformatics analysis of Toxoplasma gondii GRA16 Gene.

Toxoplasmosis is caused by the intracellular protozoan Toxoplasma gondii. It is anopportunistic zoonosis in warm-blooded animals and humans, with a worldwide distribution. Toxoplasma gondii dense granule protein 16 (TgGRA16) can modulate some functions in host cells and is considered a significant virulent factor of the parasite. The present study reports sequence variation in TgGRA16 gene among T. gondii strains from different hosts and geographical locations, and the construction of phylogenetic relationships of these T. gondii strains based on sequences of TgGRA16, and analysis of B cell epitopes in TgGRA16. Our results showed that all TgGRA16 gene sequences were 1518 bp and the C+G contents ranged from 52.17% to 52.59%. Sequence variation in the TgGRA16 gene was 0-1.51%. Phylogenetic analysis revealed that TgGRA16 gene sequence could not be used to differentiate the different T. gondii genotypes. Six B cell epitopes were predicted in TgGRA16. These results indicated that TgGRA16 gene is not an ideal marker for studying genetic relationships of T. gondii isolates, but may represent a good vaccine candidate against toxoplasmosis.

Cloning, identification, and bioinformatics analysis of a putative aquaporin TsAQP from Trichinella spiralis.

Vaccination as a preventative strategy against Trichinella spiralis infection is an ongoing effort, although no ideal vaccine candidates have been identified until now. Identification of more effective antigens that have a role in essential life stages of the parasite and that may be effective vaccine candidates is therefore of importance. In the present study, we identified a novel aquaporin gene (TsAQP) from T. spiralis, and the potential antigenicity of TsAQP was evaluated by epitope prediction. A total of 11 post-translational modification sites were predicted in the protein and fell into 4 categories: N-glycosylation; casein kinase II phosphorylation; protein kinase C phosphorylation; and N-myristoylation sites. TsAQP is a membrane intrinsic protein with high hydrophobicity; the main hydrophobic domains comprised up to 38.5% of the protein and were distributed at amino acid positions 21-43, 54-71, 83-91, 107-121, 163-174, 187-200, and 242-261. The protein consisted mainly of helices (39.58%) and loops (50%). The advanced structure of TsAQP was predicted using homology modeling, which showed that the protein was formed from 6 membrane-spanning domains connected by 5 loops. Based on these analyses, 6 potential B-cell epitopes and 4 potential T-cell epitopes were further predicted. These results suggest that TsAQP could be a promising antigen candidate for vaccination against T. spiralis.

Molecular characterization of enolase gene from Taenia multiceps.

Taenia multiceps is a cestode parasite with its larval stage, known as Coenurus cerebralis, mainly encysts in the central nervous system of sheep and other livestocks. Enolase is a key glycolytic enzyme and represents multifunction in most organisms. In the present study, a 1617bp full-length cDNA encoding enolase was cloned from T. multiceps and designated as TmENO. A putative encoded protein of 433 amino acid residues that exhibited high similarity to helminth parasites. The recombinant TmENO protein (rTmENO) showed the catalytic and plasminogen-binding characteristics after the TmENO was subcloned and expressed in the pET30a(+) vector. The TmENO gene was transcribed during the adult and larval stages and was also identified in both cyst fluid and as a component of the adult worms and the metacestode by western blot analysis. Taken together, our results will facilitate further structural characterization for TmENO and new potential control strategies for T. multiceps.

Low-level sequence variation in Toxoplasma gondii calcium-dependent protein kinases among different genotypes.

The causative agent of toxoplasmosis, Toxoplasma gondii, can infect virtually all nucleated cell types of warm-blooded animals. In this study, we examined the sequence variation in calcium-dependent protein kinase 2 (CDPK2) genes among 13 T. gondii strains from different hosts and geographical locations. The results showed that the lengths of the complete CDPK2 DNA and cDNA sequences were 3671-3673 and 2136 bp, respectively, and the sequence variation was 0-0.9% among different T. gondii strains. Phylogenetic analysis based on the CDPK2 gene sequences revealed that T. gondii strains of the same genotypes were clustered in different clades. Further analysis of all the other T. gondii CDPK genes in genotype I (GT1), II (ME49), or III (VEG) strains indicated the T. gondii CDPK gene family is quite conserved, with sequence variation ranging from 0 to 1.40%. We concluded that CDPK2 as well as all the other CDPK genes in T. gondii cannot be used as proper markers for studying the variants of different T. gondii genotypes from different hosts and geographical locations, but their sequence conservation may be a useful feature promoting them as anti-T. gondii vaccine candidates in further studies.

Seroprevalence and genotype of Chlamydia in pet parrots in China.

Parrots are one of the most popular pet birds in China, and can harbour Chlamydia which has significance for human and animal health. We investigated, by indirect haemagglutination assay, the seroprevalence of Chlamydia infection in four species of parrots, namely budgerigars (Melopsittacus undulatus), lovebirds (Agapornis sp.), cockatiels (Nymphicus hollandicus) and Alexandrine parakeets (Psittacula eupatria) that were collected from Weifang and Beijing cities, North China and explored the association between potential risk factors and chlamydial seropositivity. We further determined the genotype of Chlamydia in 21 fresh faecal samples based on the ompA sequence by reconstruction of phylogenetic relationships. Of the 311 parrots examined, 35·37% (95% confidence interval 30·06-40·68) were seropositive, and species, gender, age, season and geographical location were identified as risk factors. Two PCR-positive samples represented Chlamydia psittaci genotype A. The occurrence of C. psittaci genotype A in the droppings of two pet parrots in China suggests potential environmental contamination with Chlamydiaceae and may raise a public health concern.

Sequence variation and bioinformatics analysis of Toxoplasma gondii GRA16 Gene

Toxoplasmosis is caused by the intracellular protozoan Toxoplasma gondii. It is anopportunistic zoonosis in warm-blooded animals and humans, with a worldwide distribution. Toxoplasma gondii dense granule protein 16 (TgGRA16) can modulate some functions in host cells and is considered a significant virulent factor of the parasite. The present study reports sequence variation in TgGRA16 gene among T. gondii strains from different hosts and geographical locations, and the construction of phylogenetic relationships of these T. gondii strains based on sequences of TgGRA16, and analysis of B cell epitopes in TgGRA16. Our results showed that all TgGRA16 gene sequences were 1518 bp and the C+G contents ranged from 52.17% to 52.59%. Sequence variation in the TgGRA16 gene was 0-1.51%. Phylogenetic analysis revealed that TgGRA16 gene sequence could not be used to differentiate the different T. gondii genotypes. Six B cell epitopes were predicted in TgGRA16. These results indicated that TgGRA16 gene is not an ideal marker for studying genetic relationships of T. gondii isolates, but may represent a good vaccine candidate against toxoplasmosis.

Identification and bioinformatic analysis of a putative calcium-dependent protein kinase (CDPK6) from Toxoplasma gondii.

Toxoplasma gondii is recognized as an opportunistic human pathogen with a worldwide distribution. Development of effective vaccines is considered the only ideal way to control T. gondii infection. However, only one live vaccine is commercially available for use in sheep and goats. Therefore, the identification of more effective antigenic proteins is very important. In this study, we identified a novel putative calcium-dependent protein kinase of T. gondii, TgCDPK6, and further analyzed its potential antigenicity using a bioinformatic approach. The physical and chemical characteristics, transmembrane domain, epitopes, advanced structure, and functional sites of TgCDPK6 were predicted by multiple bioinformatic approaches. Twenty-six post-translational modification sites were identified in the protein. The secondary structure showed that 58.35% amino acids of TgCDPK6 are exposed to the solvent interface, and the high hydrophilic domains were distributed in amino acid positions 21-59, 68-81, 156-205, 245-271, 280-294, 297-324, 334-356, 367-393, 474-498, and 543-553. The advanced structure of TgCDPK6 was developed by a homology modeling method and was validated by PROCHECK, which showed that most amino acid residues were in the most favored regions. Using these analyses, 10 potential epitopes were predicted. The results indicated that TgCDPK6 could be a vaccine candidate antigen against T. gondii.

Characterization of the Toxoplasma gondii hsp60 gene sequences from different hosts and geographical locations.

The intracellular protozoan Toxoplasma gondii is one of the most successful parasites, with the ability to invade all warm-blooded animals, including humans. T. gondii heat shock protein 60 (TgHSP60) plays an important role in intracellular survival and in the differentiation of the parasite, and is also recognized as being associated with its virulence. In the present study, we examined sequence variation in the hsp60 coding region among five T. gondii isolates from different hosts and geographical regions, which were compared with the corresponding sequences of strains ME49, 76K, and GT1 available in the ToxoDB databases. The length of the T. gondii hsp60 sequence was 1728 bp for all strains, and the A+T content ranged from 41.96 to 42.13%. The sequence alignment of the 8 T. gondii strains identified 20 variable positions (0-1.44%) and showed 1.16% overall sequence variation, suggesting a relatively considerable sequence diversity. Phylogenetic analysis of hsp60 sequences using Bayesian inference and maximum parsimony differentiated the two major clonal lineage types into their respective clusters, and thus separated atypical strains from classical genotypes. The results of the present study suggested that the coding region of the hsp60 gene may represent a novel genetic marker for intraspecies phylogenetic analyses of T. gondii.

Toxoplasma gondii rhoptry protein 38 gene: sequence variation among isolates from different hosts and geographical locations.

Toxoplasma gondii is an obligate intracellular parasite that is able to infect almost all mammalian species, and may lead to toxoplasmosis of the host. In the present study, we examined sequence variation in rhoptry protein 38 (ROP38) genes among T. gondii isolates collected from different hosts and geographical regions. The complete ROP38 gene from 13 T. gondii isolates was amplified and sequenced. The results of sequence alignments showed that the lengths of the entire ROP38 gene ranged from 2646 to 2650 bp, with a sequence variation of 0.2-1.1%, among the 13 T. gondii isolates. This result indicated low sequence variation in the ROP38 gene. Phylogenetic analysis of ROP38 sequences using Bayesian inference showed that the clustering of the 13 T. gondii isolates was not consistent with their respective genotypes. This result indicates that the ROP38 gene is not a suitable genetic marker for population genetic studies of different T. gondii genotypes from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis, and hence worthy of further research.

The applicability of Ki-67 marker for renal epithelioid angiomyolipoma: experience of ten cases from a single center.

In order to present our experience with 10 cases of renal epithelioid angiomyolipoma (EAML) and validate the applicability of Ki-67 (proliferation marker) for EAML, we reviewed medical records of 10 consecutive cases diagnosed EAML from January 2005 to February 2012 at our department. Clinical data were collected and analyzed and pathology slides were reviewed. The immunohistochemical reactions for Ki-67 were performed and tumors showed positive expression were estimated. Active follow-up was performed to investigate the association between Ki-67 expression and the prognosis. The mean age and tumor size of the patients was 43.6 years (range 32-56) and 8.2 cm (range 2-15 cm), respectively. Seven were females while three were males. Radical nephrectomy was performed in 6 patients, partial nephrectomy in 3, and renal artery ligation in 1. The immunohistochemical reactions for HMB-45 (Human Melanoma Black), SMA (Smooth Muscle Actin) were positive but for S-100 were negative. The number of patients showing positive/negative Ki-67 expression was 5/5. The survival rate of the positive group was 20% (1/5) while 100% (5/5) of the negative group during the median follow-up time of 26.75 months (range 1-53). Recurrence, metastasis and death due to disease occurred in 1 (10%), 3 (30%) and 4 (40%) patients, respectively. Higher expression (positive) of Ki-67 indicates the presence of EAML and poor prognosis of patients. Surgical excision including radical and partial nephrectomy is a considerable approach to the treatment for its malignant potential.

Fluoride and chlorhexidine release from filled resins.

Resin-based materials that release either fluoride or chlorhexidine have been formulated for inhibiting caries activity. It is not known if the two agents, when incorporated into one material, would interact and affect their release potential. We hypothesized that the ratio of fluoride to chlorhexidine incorporated into a resin, and the pH of the storage medium, will affect their releases from the material. The material investigated contained 23 wt% of filler, and the ratios of calcium fluoride to chlorhexidine diacetate were 8/2, 5/5, and 2/8. The release was conducted in pH 4, 5, and 6 acetate buffers. The results showed that release of either agent increased as the pH of the medium decreased. The presence of fluoride salt substantially reduced the chlorhexidine release, while the presence of a specific quantity of chlorhexidine significantly increased fluoride release. This interaction can be utilized to optimize the release of either agent for therapeutic purposes.

Controlled release of chlorhexidine from UDMA-TEGDMA resin.

Chlorhexidine salts are available in various formulations for dental applications. This study tested the hypothesis that the release of chlorhexidine from a urethane dimethacrylate and triethylene glycol dimethacrylate resin system can be effectively controlled by the chlorhexidine diacetate content and pH. The filler concentrations were 9.1, 23.1, or 33.3 wt%, and the filled resins were exposed to pH 4 and pH 6 acetate buffers. The results showed that Fickian diffusion was the dominant release mechanism. The rates of release were significantly higher in pH 4 buffer, which was attributed to the increase of chlorhexidine diacetate solubility at lower pH. The higher level of filler loading reduced the degree of polymerization, leading to a greater loss of organic components and higher chlorhexidine release rates.

Effect of CaF2 content on rate of fluoride release from filled resins.

Information on the time-dependent release of fluoride from filled resins containing fluoride particles as a function of particle content and solution pH is limited. This study characterized the fluoride ion release from filled resins containing CaF2 particles as a function of filler content and pH. Urethane dimethacrylate and triethylene glycol dimethacrylate resins were used to make filled-resin disks containing 9.09, 23.08, or 33.33 mass% CaF2 filler. Fluoride ion release for the 9.09 mass% concentration was independent of pH. Increasing the filler content from 9.09 to 33.33 mass% increased the fluoride release rate in pH 4.0 buffer solution, because of greater surface degradation. Fluoride ion release from disks stored in pH 6.0 buffer solutions occurred mainly by diffusion from disk surfaces, while fluoride release from disks in pH 4.0 buffers was controlled by diffusion from disk surfaces and degeneration of the resin matrix, which exposed more CaF2 particle surface area.

Phylogenetic analysis of the S7 gene does not segregate Chinese strains of bluetongue virus into a single topotype.

Bluetongue virus (BTV) infection of ruminants is endemic throughout tropical and subtropical regions of the world. The S7 gene segments of prototype Chinese strains of BTV serotypes 1, 2, 3, 4, 12, 15, and 16 were sequenced and compared to the same genes of prototype strains of BTV from the US, Australia, and South Africa. The S7 genes and predicted VP7 proteins of the Chinese viruses were relatively conserved, with the notable exception of serotype 15. Furthermore, phylogenetic analysis of the S7 genes did not predict geographic origin of the various strains of BTV.

Active ion secretion and permeability of rabbit maxillary sinus epithelium, impact of staphylococcal enterotoxin B.

The aim of this study was to investigate the impact of staphylococcal enterotoxin B (SEB), a model superantigen, on the physiologic functions of rabbit maxillary sinus epithelium. Rabbit sinus mucosae were separated under a surgical microscope and mounted in Ussing chambers to record short-circuit current, conductance, and permeability to horseradish peroxidase. The results showed that SEB evoked increases in sinus epithelial cell baseline short-circuit current, conductance, and permeability to horseradish peroxidase. When tumor necrosis factor-alpha (TNF-alpha) was added to the Ussing chambers, we got results similar to those obtained by SEB stimulation in vitro; the effects of SEB on sinus epithelial cells could be blocked by pretreatment with anti-TNF-alpha antibody. These results demonstrate that SEB is able to alter the function of sinus epithelial cells and to affect the capability of the epithelial defensive barrier, which may be mediated by TNF-alpha.

Chemical durability of Dicor and fluorocanasite-based glass-ceramics.

Fluorocanasite (Al2O3-CaO-F-K2O-Na2O-SiO2) glass-ceramics exhibit fracture toughness values of up to 5.0 MPa x m1/2. However, their chemical durability is not adequate for dental applications. The objective of this study was to test the hypothesis that an increased concentration of Al2O3 can increase the chemical durability of fluorocanasite-based glass-ceramics. Glass frits containing 2 wt% (CAN2), 5 wt% (CAN5), and 10 wt% Al2O3 (CAN10) were melted individually, poured into a graphite mold, and cut into 16-mm-diam. x 2-mm-thick disks. Each disk was crystallized at 850 degrees C for 6 hrs. The disks were immersed in a solution of de-ionized-distilled water, 4% acetic acid, or a pH 1 buffer solution, and sealed in 90-mL Teflon containers. Corrosion testing was performed by means of vibrational motion at 60 cycles per min in a shaker-bath at 80 degrees C for 15 days. Solution analyses were performed by means of a pH meter, an atomic absorption spectrophotometer, and an inductively coupled plasma spectrometer. Samples exposed to 4% acetic acid solution exhibited a mean weight loss rate (WLR) for the control group (Dicor) of 0.04+/-0.01 mg/cm2 day, which was significantly lower (p < or = 0.0001) than the mean WLR of the CAN2 (1.08+/-0.02 mg/cm2 x day), CAN5 (1.31+/-0.02 mg/cm2 x day), and CAN10(1.51+/-0.05 mg/cm2 x day) groups. The reduced durability of fluorocanasite-based glass-ceramics with increasing Al2O3 concentration is most likely associated with a more uniform distribution of smaller crystals during heat treatment of the glass.

[Effects of tripchlorolide (T4) of Tripterygium Wilfordii Hook on the production of immunoglobulins by peripheral blood mononuclear cells and by synovial cells of rheumatoid arthritis patients in vitro].

Tripchlorolide(T4) is an active ingredient recently isolated from Tripterygium Wilfordii Hook. The in vitro effects of T4 on the peripheral blood mononuclear cells(PBMC) of healthy persons(n = 10), the digested single synovium cells(DSSC) (n = 3) and PBMC(n = 6) from RA patients on production of immunoglobulins (Ig) were studied using Elisa method. The results showed that T4 at concentrations from 5 ng.ml-1 to 35 ng.ml-1 significantly reduced the production of Ig by PWM-stimulated PBMC of healthy persons in a dose dependent manner. At concentration of 25 ng.ml-1, T4 also reduced the Ig secretion of RA-PBMC and DSSC. It is hoped that T4 would be an encouraging new drug of herbal nature for the treatment of RA.