Quantitative phosphoproteomic analysis of chicken DF-1 cells infected with Eimeria tenella, using tandem mass tag (TMT) and parallel reaction monitoring (PRM) mass spectrometry.

Eimeria tenella is an obligate intracellular parasite which causes great harm to the poultry breeding industry. Protein phosphorylation plays a vital role in host cell-E. tenella interactions. However, no comprehensive phosphoproteomic analyses of host cells at various phases of E. tenella infection have been published. In this study, quantitative phosphoproteomic analysis of chicken embryo DF-1 fibroblasts that were uninfected (UI) or infected with E. tenella for 6 h (PI6, the early invasion phase) or 36 h (PI36, the trophozoite development phase) was conducted. A total of 10,122 phosphopeptides matched to 3,398 host cell phosphoproteins were identified and 13,437 phosphorylation sites were identified. Of these, 491, 1,253, and 275 differentially expressed phosphorylated proteins were identified in the PI6/UI, PI36/UI, and PI36/PI6 comparisons, respectively. KEGG pathway enrichment analysis showed that E. tenella modulated host cell processes through phosphorylation, including focal adhesion, regulation of the actin cytoskeleton, and FoxO signaling to support its early invasion phase, and modulating adherens junctions and the ErbB signaling pathway to favor its trophozoite development. These results enrich the data on the interaction between E. tenella and host cells and facilitate a better understanding of the molecular mechanisms underlying host-parasite relationships.

Title: Analyse phosphoprotéomique quantitative de cellules DF-1 de poulet infectées par Eimeria tenella, par spectrométrie de masse avec marqueur de masse en tandem (TMT) et surveillance des réactions parallèles (PRM). Abstract: Eimeria tenella est un parasite intracellulaire obligatoire qui cause de graves dommages à l'industrie de l'élevage de volailles. La phosphorylation des protéines joue un rôle essentiel dans les interactions entre la cellule hôte et E. tenella. Cependant, aucune analyse phosphoprotéomique complète des cellules hôtes à différentes phases de l'infection par E. tenella n'a été publiée. Dans cette étude, une analyse phosphoprotéomique quantitative de fibroblastes DF-1 d'embryon de poulet non infectés (NI) ou infectés par E. tenella pendant 6 h (PI6, la phase d'invasion précoce) ou 36 h (PI36, la phase de développement des trophozoïtes) a été réalisée. Un total de 10 122 phosphopeptides correspondant à 3 398 phosphoprotéines de cellules hôtes ont été identifiés et 13 437 sites de phosphorylation ont été identifiés. Parmi celles-ci, 491, 1 253 et 275 protéines différentiellement phosphorylées exprimées ont été identifiées respectivement dans les comparaisons PI6/NI, PI36/NI et PI36/PI6. L'analyse d'enrichissement de la voie KEGG a montré qu'E. tenella modulait les processus de la cellule hôte par phosphorylation, y compris l'adhésion focale, la régulation du cytosquelette d'actine et la signalisation FoxO, pour aider sa phase d'invasion précoce, et la modulation des jonctions adhérentes et de la voie de signalisation ErbB pour favoriser le développement de son trophozoïte. Ces résultats enrichissent les données sur l'interaction entre E. tenella et les cellules hôtes et facilitent une meilleure compréhension des mécanismes moléculaires sous-jacents aux relations hôtes­parasites.

Sensitive ex vivo human skin transdermal assay testing method with mass spectrometric analysis for cosmetics application.

BACKGROUND: Cosmetics manufacturers are focused on cosmetic delivery systems into the skin, but the level of diffusion of the systems in the skin tissues is not well understood. The current methods, such as Franz diffusion, assess analyte diffusion in the whole skin or artificial membranes, which has limitations for understanding skin delivery systems. AIMS: Our study aimed to create a transdermal delivery method which is based on dermal-epidermal separation of human skin, allowing us to assess each layer of skin separately for its efficacy. MATERIALS AND METHODS: During the experiment, resveratrol was used as the target analyte by applying it to the skin and then separating it into dermis and epidermis. Each layer is treated individually and subjected to a high-resolution mass spectrometry analysis to detect resveratrol levels. As a result, the efficiency of resveratrol diffusion in the dermal and epidermal layers of the skin can be evaluated. RESULTS: We found that resveratrol was detected in both the dermal and epidermal layers using our method. CONCLUSIONS: Hence, we developed a sensitive method for transdermal delivery testing that can be used to evaluate skin delivery systems for cosmetic or pharmaceutical purposes.

ΔNp63α mediates sulforaphane suppressed colorectal cancer stem cell properties through transcriptional regulation of Nanog/Oct4/Sox2.

Cancer stem cells (CSCs) play a key role in cancer initiation, development, metastasis, and recurrence. Previously, we found that sulforaphane (SFN), a natural compound obtained from cruciferous vegetables, inhibited colorectal CSCs via the downregulation of TAp63α. However, the role of ΔNp63α, another critical isoform of p63 which has been considered to contribute to cancer progression, in SFN-mediated colorectal CSCs inhibition remains unclear. Here, we showed that ΔNp63α expression was enhanced in sphere-forming colorectal cancer cells. Overexpression of ΔNp63α promoted the properties of CSCs, while downregulation of ΔNp63α suppressed those properties. Besides, ΔNp63α was found to activate the transcription of core CSCs genes including Nanog, Oct4, and Sox2. Furthermore, in vitro and in vivo experiments illustrated the regulatory effects of SFN on ΔNp63α and colorectal CSCs. These findings suggested for the first time that ΔNp63α activated the transcription of Nanog, Oct4, Sox2 and mediated the interventional effects of SFN on colorectal CSCs, thus providing a novel mechanism by which SFN inhibits colorectal CSCs.

Effects of isoprenylcysteine carboxylmethyltransferase silencing on the migration and invasion of tongue squamous cell carcinoma.

OBJECTIVES: The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT. METHODS: Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays. RESULTS: After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (P<0.05). The mRNA and total protein expression levels of RhoA in the two groups were not significantly different (P>0.05). The expression levels of RhoA membrane protein, ROCK1, MMP-2, and MMP-9 decreased (P<0.05). The migration and invasion abilities were inhibited (P<0.05). CONCLUSIONS: The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.

Effects of isoprenylcysteine carboxyl methyltransferase silencing on the proliferation and apoptosis of tongue squamous cell carcinoma.

OBJECTIVES: This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC). METHODS: Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry. RESULTS: The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (P<0.05). No significant difference in RhoA mRNA and protein expression was detected (P>0.05), but the expression of RhoA membrane protein decreased compared with the negative control group and blank control groups (P<0.05). Cyclin D1 expression decreased, whereas p21 expression significantly increased and the relative expression of ERK protein in the experimental group did not significantly different that in the control group (P>0.05). However, the phosphorylation level of ERK was significantly reduced (P<0.05). The cell cycles of TSCC CAL-27 and SCC-4 were altered in G1/S, cell proliferation activity was inhibited, and apoptosis was induced (P<0.05). CONCLUSIONS: Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.

TAp63α targeting of Lgr5 mediates colorectal cancer stem cell properties and sulforaphane inhibition.

Cancer stem cells (CSCs) have an established role in cancer progression and therapeutic resistance. The p63 proteins are important transcription factors which belong to the p53 family, but their function and mechanism in CSCs remain elusive. Here, we investigated the role of TAp63α in colorectal CSCs and the effects of sulforaphane on TAp63α. We found that TAp63α was upregulated in spheres with stem cell properties compared to the parental cells. Overexpression of TAp63α promoted self-renewal capacity and enhanced CSC markers expression in colorectal sphere-forming cells. Furthermore, we showed that TAp63α directly bound to the promoter region of Lgr5 to enhance its expression and activate its downstream ß-catenin pathway. Functional experiments revealed that sulforaphane suppressed the stemness of colorectal CSCs both in vitro and in vivo. Upregulation of TAp63α attenuated the inhibitory effect of sulforaphane on colorectal CSCs, indicating the role of TAp63α in sulforaphane suppression of the stemness in colorectal cancer. The present study elucidated for the first time that TAp63α promoted CSCs through targeting Lgr5/ß-catenin axis and participated in sulforaphane inhibition of the stem cell properties in colorectal cancer.

Sulforaphane inhibits epithelial-mesenchymal transition by activating extracellular signal-regulated kinase 5 in lung cancer cells.

Epithelial-mesenchymal transition (EMT) contributes to the initiation, invasion, metastasis and drug resistance of cancer. The function of extracellular signal-regulated kinase 5 (ERK5) in lung cancer progression remains elusive. In this study, we investigated the effect of sulforaphane (SFN) on lung cancer EMT and the role of ERK5 in its effect. Wound healing and Transwell assays were applied to examine the migratory and invasive capacity in vitro. Quantitative real-time polymerase chain reaction and immunoblotting analysis were performed to investigate the expression of mRNA and protein levels. Small-interfering RNA was used to silence ERK5. Xenograft model was used to confirm the effect of SFN in vivo. Enhanced EMT and decreased ERK5 activation were observed in lung cancer cells in comparison with normal human bronchial epithelial cells. SFN diminished the migratory and invasive capacity of lung cancer cells. Additionally, significantly increased expression of epithelial markers (E-cadherin and ZO-1), decreased expression of mesenchymal markers (N-cadherin and Snail1) and activation of ERK5 were observed after SFN treatment. The inhibitory effect of SFN on lung cancer cell EMT was attenuated by ERK5 silencing. SFN-induced EMT suppression and ERK5 activation were further confirmed in lung cancer xenograft mouse model. The present study illustrated for the first time that ERK5 activation mediates SFN suppression of lung cancer cell EMT. These findings could provide new insights into the function of ERK5 in EMT regulation and the potential therapeutic application of SFN in cancer intervention.

Genistein inhibits nasopharyngeal cancer stem cells through sonic hedgehog signaling.

Genistein, a soy derived isoflavanoid compound, exerts anticancer effects in various cancers. Nasopharyngeal cancer stem cells (NCSCs) are a small subpopulation of cancer cells which are responsible for initiation, progression, metastasis, and recurrence of nasopharyngeal cancer. The present study aimed to investigate the suppressive effects of genistein on NCSCs and its underlying mechanism. NCSCs were enriched from human nasopharyngeal cancer cell lines CNE2 and HONE1 through tumorsphere-forming assay. It was shown that genistein inhibited the tumorsphere formation capacity, decreased the number of EpCAM+ cells, downregulated the expression of NCSCs markers, suppressed cell proliferation, and induced apoptosis of NCSCs. Genistein suppressed the activity of Sonic hedgehog (SHH) signaling, which was important for the maintenance of NCSCs, while activation of SHH signaling by purmorphamine diminished the inhibitory effects of genistein on NCSCs. Our data suggested that genistein inhibited NCSCs through the suppression of SHH signaling. These findings support the use of genistein for targeting NCSCs.

Phenethyl isothiocyanate inhibits colorectal cancer stem cells by suppressing Wnt/ß-catenin pathway.

Cancer stem cells (CSCs) are considered to play essential roles in the process of origination, proliferation, migration, and invasion of cancer, and their properties are regulated by Wnt/ß-catenin pathway. Phenethyl isothiocyanate (PEITC) is a natural product obtained from cruciferous vegetables with anticancer activities. The present study aimed to investigate the inhibitory effect and the underlying mechanisms of PEITC on colorectal CSCs. In this study, we found that PEITC can significantly reduce the size and number of colorectal cancer cell spheroids in serum-free medium. With increasing PEITC concentrations (10-40 µM), the number of spheroids was reduced to about 10% of the control group, and the percentage of CD133+ cells was decreased by about 3-16 folds. PEITC also decreased the expression of CSC markers. Meanwhile, inhibition of proliferation as well as induction of apoptosis of colorectal CSCs was observed after PEITC treatment. Furthermore, through activating Wnt/ß-catenin pathway with LiCl, the inhibitory effects of PEITC on colorectal CSCs were diminished. Our data suggested that PEITC can be an effective inhibitor of colorectal CSCs by targeting Wnt/ß-catenin pathway.

Correction to: Wnt/ß-catenin signaling mediates the suppressive effects of diallyl trisulfide on colorectal cancer stem cells.

Unfortunately, the online published article has error in Figure 4. The correct Figure 4 is given here.

Wnt/ß-catenin signaling mediates the suppressive effects of diallyl trisulfide on colorectal cancer stem cells.

PURPOSE: Cancer stem cells (CSCs) are responsible for colorectal cancer (CRC) initiation, growth, and metastasis. Garlic-derived organosulfur compound diallyl trisulfide (DATS) possesses cancer suppressive properties. Wnt/ß-catenin signaling is a key target for CSCs inhibition. However, the interventional effect of DATS on colorectal CSCs has not been clarified. We aimed to illustrate the regulation of Wnt/ß-catenin in DATS-induced colorectal CSCs inhibition. METHODS: Serum-free medium culture was used to enrich colorectal CSCs. SW480 and DLD-1 sphere-forming cells were treated with different concentrations of DATS for 5 days; LiCl and ß-catenin plasmids were used to stimulate the activity of Wnt/ß-catenin pathway. The size and number of colonspheres were detected by tumorsphere formation assay; the expression of colorectal CSCs-related genes was detected by Western blotting and qRT-PCR; the capacities of colorectal CSCs proliferation and apoptosis were detected by Cell Counting Kit-8, Hoechst 33258 cell staining and flow cytometry, respectively. RESULTS: The levels of colorectal CSCs markers were elevated in the tumorspheres cells. DATS efficiently suppressed the activity of colorectal CSCs, as evidenced by reducing the size and number of colonspheres, decreasing the expression of colorectal CSCs markers, promoting apoptosis and inhibiting the proliferation of colorectal CSCs. Moreover, DATS suppressed the activity of Wnt/ß-catenin pathway, while upregulation of Wnt/ß-catenin diminished the inhibitory effect of DATS on colorectal CSCs. CONCLUSIONS: Wnt/ß-catenin pathway mediates DATS-induced colorectal CSCs suppression. These findings support the use of DATS for targeting colorectal CSCs.

(-)-Epigallocatechin-3-Gallate Inhibits Colorectal Cancer Stem Cells by Suppressing Wnt/ß-Catenin Pathway.

The beneficial effects of tea consumption on cancer prevention have been generally reported, while (-)-Epigallocatechin-3-gallate (EGCG) is the major active component from green tea. Cancer stem cells (CSCs) play a crucial role in the process of cancer development. Targeting CSCs may be an effective way for cancer intervention. However, the effects of EGCG on colorectal CSCs and the underlying mechanisms remain unclear. Spheroid formation assay was used to enrich colorectal CSCs from colorectal cancer cell lines. Immunoblotting analysis and quantitative real-time polymerase chain reaction were used to measure the alterations of critical molecules expression. Immunofluorescence staining analysis was also used to determine the expression of CD133. We revealed that EGCG inhibited the spheroid formation capability of colorectal cancer cells as well as the expression of colorectal CSC markers, along with suppression of cell proliferation and induction of apoptosis. Moreover, we illustrated that EGCG downregulated the activation of Wnt/ß-catenin pathway, while upregulation of Wnt/ß-catenin diminished the inhibitory effects of EGCG on colorectal CSCs. Taken together, this study suggested that EGCG could be an effective natural compound targeting colorectal CSCs through suppression of Wnt/ß-catenin pathway, and thus may be a promising agent for colorectal cancer intervention.

Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/ß-catenin and Sonic Hedgehog Pathways.

Cancer stem cells (CSCs) are highly implicated in the progression of human cancers. Thus, targeting CSCs may be a promising strategy for cancer therapy. Wnt/ß-catenin and Sonic Hedgehog pathways play an important regulatory role in maintaining CSC characteristics. Natural compounds, such as curcumin, possess chemopreventive properties. However, the interventional effect of curcumin on lung CSCs has not been clarified. In the present study, tumorsphere formation assay was used to enrich lung CSCs from A549 and H1299 cells. We showed that the levels of lung CSC markers (CD133, CD44, ALDHA1, Nanog and Oct4) and the number of CD133-positive cells were significantly elevated in the sphere-forming cells. We further illustrated that curcumin efficiently abolished lung CSC traits, as evidenced by reduced tumorsphere formation, reduced number of CD133-positive cells, decreased expression levels of lung CSC markers, as well as proliferation inhibition and apoptosis induction. Moreover, we demonstrated that curcumin suppressed the activation of both Wnt/ß-catenin and Sonic Hedgehog pathways. Taken together, our data suggested that curcumin exhibited its interventional effect on lung CSCs via inhibition of Wnt/ß-catenin and Sonic Hedgehog pathways. These novel findings could provide new insights into the potential therapeutic application of curcumin in lung CSC elimination and cancer intervention. Copyright © 2017 John Wiley & Sons, Ltd.

High expression of XPA confers poor prognosis for nasopharyngeal carcinoma patients treated with platinum-based chemoradiotherapy.

In this study, we tried to explore if xeroderma pigmentosum complementation group-A (XPA) expression is likely a prognostic prediction factor for locally advanced nasopharyngeal carcinoma (NPC) patients treated with platinum-based chemoradiotherapy, which was considered to bring chemotherapy-related severe toxicity compared with radiotherapy alone. Firstly, MTT assay revealed that downregulating XPA expression in NPC HONE1 and CNE1 cells decreased IC50 of cisplatin and sensitized cells to cisplatin. XPA expression was detected by immunohistochemistry in cancer tissues from locally advanced NPC patients treated with platinum-based chemoradiotherapy. The relationships between XPA expression and clinicopathologic features, overall survival and progression-free survival of patients were evaluated. The results showed that XPA expression was not associated with clinicopathologic parameters, but was likely an independent prognostic factor for patient survival. High XPA level predicts a poor prognosis, and the prediction values were higher in subgroups of younger, higher EBV antibody titer, or treated with concurrent chemoradiotherapy. Combining XPA levels and T/N classifications, we successfully classified these patients into low, medium and high risk groups for platinum-based chemoradiotherapy. These findings suggest that XPA levels may be a potential predictor of prognosis in locally advanced NPC patients treated with platinum-based chemoradiotherapy, and helpful for selecting patients likely to need and benefit from this treatment in future.

Nutrition support can bring survival benefit to high nutrition risk gastric cancer patients who received chemotherapy.

PURPOSE: The aim of our study is firstly to evaluate the prevalence and prognostic value of nutrition risk in gastric cancer patients and secondly to explore whether the nutrition support can prolong the survival of advanced gastric cancer patients. METHODS: It contained two study periods. In the first period, we prospectively evaluated the nutritional risk of gastric adenocarcinoma patients from 2009 to 2011 using the method of European Nutritional Risk Screening (NRS) 2002. The Kaplan-Meier method and log-rank test were used to evaluate the prognostic value of high nutrition risk. The second period was between 2012 and 2013. We prospectively gave the nutrition support to stage IV gastric cancer patients whose NRS is ≥3. RESULTS: There were 830 patients in the first period, 50.7% patients with a NRS ≥ 3. Patients with NRS ≥ 3 presented a significantly higher percentage of stage IV diseases, elevated values of C-reactive protein, and hypoproteinemia. The median survival was significantly higher in NRS < 3 patients (31.9 vs. 25.7 months, P < 0.001). Multivariate analysis confirmed that NRS status was an independent prognostic factor. There were 347 patients in the second period. Young, male, and good response to chemotherapy were more likely to have the NRS shift to <3 after nutrition support. The median survival was 14.3 and 9.6 months for patients with and without NRS shift, respectively, P = 0.001. CONCLUSIONS: NRS ≥ 3 was an independent adverse prognostic factor in gastric cancer patients. For stage IV patients whose NRS ≥ 3, the nutrition support might be helpful to improve the prognosis.

Adenovirus-mediated delivery of interferon-γ gene inhibits the growth of nasopharyngeal carcinoma.

BACKGROUND: Interferon-γ (IFN-γ) is regarded as a potent antitumor agent, but its clinical application is limited by its short half-life and significant side effects. In this paper, we tried to develop IFN-γ gene therapy by a replication defective adenovirus encoding the human IFN-γ (Ad-IFNγ), and evaluate the antitumoral effects of Ad-IFNγ on nasopharyngeal carcinoma (NPC) cell lines in vitro and in xenografts model. METHODS: The mRNA levels of human IFN-γ in Ad-IFNγ-infected NPC cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), and IFN-γ protein concentrations were measured by enzyme-linked immunosorbent assay (ELISA) in the culture supernatants of NPC cells and tumor tissues and bloods of nude mice treated with Ad-IFNγ. The effects of Ad-IFNγ on NPC cell proliferation was determined using MTT assay, cell cycle distribution was determined by flow cytometry analysis for DNA content, and cells apoptosis were analyzed by Annexin V-FITC/7-AAD binding assay and hoechst 33342/PI double staining. The anti-tumor effects and toxicity of Ad-IFNγ were evaluated in BALB/c nude mice carrying NPC xenografts. RESULTS: The results demonstrated that Ad-IFNγ efficiently expressed human IFN-γ protein in NPC cell lines in vitro and in vivo. Ad-IFNγ infection resulted in antiproliferative effects on NPC cells by inducing G1 phase arrest and cell apoptosis. Intratumoral administration of Ad-IFNγ significantly inhibited the growth of CNE-2 and C666-1 cell xenografts in nude mice, while no significant toxicity was observed. CONCLUSIONS: These findings indicate IFN-γ gene therapy mediated by replication defective adenoviral vector is likely a promising approach in the treatment of nasopharyngeal carcinoma.

Great efficacy of sulfachloropyrazine-sodium against acute murine toxoplasmosis.

OBJECTIVE: To identify more effective and less toxic drugs to treat animal toxoplasmosis. METHODS: Efficacy of seven kinds of sulfonamides against Toxoplasma gondii (T. gondii) in an acute murine model was evaluated. The mice used throughout the study were randomly assigned to many groups (10 mice each), which either remained uninfected or were infected intraperitoneally with tachyzoites of T. gondii (strains RH and CN). All groups were then treated with different sulfonamides and the optimal treatment protocol was determined candidates. Sulfadiazine-sodium (SD) was used for comparison. RESULTS: The optimal therapy involved gavaging mice twice per day with 250 mg/kg bw of sulfachloropyrazine-sodium (SPZ) for five days. Using this protocol, the average survival time and the time-point of 50% fatalities were prolonged significantly compared with SD treatment. Treatment with SPZ protected 40% of mice from death, and the heart and kidney tissue of these animals was parasite-free, as determined by nested-PCR. SPZ showed excellent therapeutic effects in the treatment of T. gondii in an acute murine model and is therefore a promising drug candidate for the treatment and prevention of T. gondii in animals. CONCLUSIONS: It can be concluded that the effective drug sulfachloropyrazine may be the new therapeutic options against animal toxoplasmosis.

Evaluation of long-term toxicity of Ad/hIFN-, an Adenoviral vector encoding the human interferon-gamma gene, in nonhuman primates.

Interferon-gamma(IFN-gamma) plays an important role in the immunomodulation and growth inhibition of many tumor cells, but its clinical application is limited by its systemic toxicity. Ad/hIFN-gamma, a nonreplicating adenoviral vector encoding human IFN-gamma, has been reported to inhibit tumor growth in vitro and in a xenograft model. In this study, the long-term toxicity of Ad/hIFN-gamma was assessed in cynomolgus macaques (Macaca fascicularis). Thirty animals were enrolled into 5 groups, and administered intramuscularly, respectively, Ad/hIFN-gamma (3.3 x 10(10), 3.3 x 10(11), or 3.3 x 10(12) VP/kg), Ad/LacZ (vector control, 3.3 x 10(11) VP/kg), or excipient 3 times per week for 8 weeks, followed by a 4-week recovery period. At 12 weeks all experimental animals appeared generally healthy, and there were no statistically significant differences in body weight, urinalysis, hemogram, blood biochemistry, and electrocardiogram results between the treatment and control groups. No significant toxic effects were noted on macroscopic and microscopic examinations of organs and tissues. Preliminary investigation of the immunotoxicity of Ad/IFN-gamma indicated that anti-adenoviral and anti-hIFN-gamma antibodies were generated. These data demonstrate that long-term, high-dose intramuscular administration of Ad/IFN-gamma was not notably toxic and might be safe for clinical therapeutic use.

[Construction of subtractive cDNA libraries of the sporogony stage of Eimeria tenella by suppression subtractive hybridization].

In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella, the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver, respectively, the cDNAs from sporozoites of E. tenella was used tester, Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver, the cDNAs from sporulated ooceysts was used tester, one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%, 96% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries, respectively, thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts, eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries, nine ESTs shared significant identity with previously described, the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.

[Comparison of the proteome of the sporutated oocysts of Eimeria tenella diclazuril sensitive strain with diclazuril resistant strain].

Two-dimensional electrophoresis (2-DE) was employed to compare the proteome of Diclazuril-resistance Eimeria tenella with that of sensitive strains for identifying unique proteins of these stains. 5 protein spots were found to express differentially. Four spots which remarkably were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in NCBInr database search, two protein spots in gel were identified as Eimeria tenella sporulated oocyst TA4 antigen protein, Heat shock 70kD protein, two protein spots were functional proteins of Eukaryote. These proteins are potentially basic work for finding molecular mechanism about drug-resistance of Eimeria tenella and new marker in the detection of resistance of Eimeria tenella.