Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection.

Toxoplasma gondii are obligate intracellular protoza, and due to their small genome and limited encoded proteins, they have to exploit host factors for entry, replication, and dissemination. Such host factors can be defined as host dependency factors (HDFs). Though HDFs are inessential for cell viability, they are critical for pathogen infection, and potential ideal targets for therapeutic intervention. However, information about these HDFs required by T. gondii infection is highly deficient. In this study, the genes of human foreskin fibroblast (HFF) cells were comprehensively edited using the lentiviral CRISPR-Cas9-sgRNA library, and then the lentivirus-treated cells were infected with T. gondii at multiplication of infection 1 (MOI = 1) for 10 days to identify HDFs essential for T. gondii infection. The survival cells were harvested and sent for sgRNA sequencing. The sgRNA sequence matched genes or miRNAs were potential HDFs. Some cells in the lentivirus-treated group could survive longer than those in the untreated control group after T. gondii infection. From a pool of 19,050 human genes and 1,864 human pri-miRNAs, 1,193 potential HDFs were identified, including 1,183 genes and 10 pri-miRNAs (corresponding with 17 mature miRNAs). Among them, seven genes and five mature miRNAs were validated with siRNAs, miRNA inhibitors, and mimics, respectively. Bioinformatics analysis revealed that, among the 1,183 genes, 53 potential HDFs were associated with regulation of host actin cytoskeleton and 23 potential HDFs coded immune negative regulators. This result indicated that actin dynamics were indispensable for T. gondii infection, and some host immune negative regulators may be involved in disarming host defenses. Our findings contribute to the current limited knowledge about host factors required by T. gondii infection and provide us with new targets for medication therapy and vaccine exploitation.

iTRAQ-based phosphoproteomic analysis reveals host cell's specific responses to Toxoplasma gondii at the phases of invasion and prior to egress.

Protein phosphorylation plays a key role in host cell-T. gondii interaction. However, the phosphoproteome data of host cell at various phases of T. gondii infection has not been thoroughly described. In this study, we assessed the host phosphoproteome data with isobaric tags for relative and absolute quantification (iTRAQ) method during the phases of T. gondii invasion (30 min post infection, PI) and prior to egress (28 h PI). Our iTRAQ analysis revealed a total of 665 phosphoproteins, among which the significantly regulated phosphoproteins in different between-group comparisons were further analyzed. Functional analysis of these significantly regulated phosphoproteins suggested that T. gondii modulated host cell processes through phosphorylation including cell cycle regulation, inducing apoptosis, blocking the synthesis of some inflammatory factors, mediating metabolism to support its proliferation at the infection phase prior to egress, and utilizing membrane and energy from host cell, reorganizing cytoskeleton to favor its invasion and PV formation at the phase of invasion. The phosphorylation level of Smad2, CTNNA1, and HSPB1 identified with western blot revealed a consistent trend of change with iTRAQ result. These newly identified and significantly regulated phosphoproteins from our phosphoproteome data may provide new clues to unravel the host cell's complex reaction against T. gondii infection and the interaction between the host cell and T. gondii.

The Differential Expression and Possible Function of Long Noncoding RNAs in Liver Cells Infected by Dengue Virus.

The function of long noncoding RNAs (lncRNAs) in liver injury resulted by dengue virus (DENV) infection have not yet been explored. The differential expression profiles of lncRNAs (as well as mRNAs) in the L-02 liver cells infected by DENV1, DENV2, or uninfected were compared and analyzed after a high throughput RNA seq. The significantly up-regulated and down-regulated lncRNAs (or mRNAs) resulted by DENV infection were identified with a cutoff value at log2 (ratio) ≥ 1.5 and log2 (ratio) ≤ -1.5 (ratio = the reads of the lncRNAs or mRNAs from the infection groups divided by the reads from the control group). Several differentially expressed lncRNAs were verified with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Target gene analysis, pre-miRNA prediction, and the lncRNA-mRNA co-expression network construction were performed to predict the function of the differentially expressed lncRNAs. The differentially expressed lncRNAs were associated with biosynthesis, DNA/RNA related processes, inhibition of estrogen signaling pathway, sterol biosynthetic process, protein dimerization activity, vesicular fraction in DENV1 infection group; and with protein secretion, methyltransferase process, host cell cytoskeleton reorganization and the small GTPase Ras superfamily, inhibition of cell proliferation, induction of apoptosis in DENV2 infection. LncRNAs might be novel diagnostic markers and targets for further researches on dengue infection and liver injury resulted by dengue virus.

Phosphoproteome of Toxoplasma gondii Infected Host Cells Reveals Specific Cellular Processes Predominating in Different Phases of Infection.

The invasion of Toxoplasma gondii tachyzoites into the host cell results in extensive host cell signaling activation/deactivation that is usually regulated by the phosphorylation/dephosphorylation. To elucidate how T. gondii regulates host cell signal transduction, the comparative phosphoproteome of stable isotope labeling with amino acids in cell culture-labeled human foreskin fibroblast cells was analyzed. The cells were grouped (Light [L], Medium [M], and Heavy [H] groups) based on the labeling isotope weight and were infected with T. gondii for different lengths of time (L: 0 hour; M: 2 hours; and H: 6 hours). A total of 892 phosphoproteins were identified with 1,872 phosphopeptides and 1,619 phosphorylation sites. The M versus L comparison revealed 694 significantly regulated phosphopeptides (436 upregulated and 258 downregulated). The H versus L comparison revealed 592 significantly regulated phosphopeptides (146 upregulated and 446 downregulated). The H versus M comparison revealed 794 significantly regulated phosphopeptides (149 upregulated and 645 downregulated). At 2 and 6 hours post-T. gondii infection, the most predominant host cell reactions were cell cycle regulation and cytoskeletal reorganization, which might be required for the efficient invasion and multiplication of T. gondii. Similar biological process profiles but different molecular function categories of host cells infected with T. gondii for 2 and 6 hours, which suggested that the host cell processes were not affected significantly by T. gondii infection but emphasized some differences in specific cellular processes at this two time points. Western blotting verification of some significantly regulated phosphoprotein phosphorylation sites was consistent with the mass spectra data. This study provided new insights into and further understanding of pathogen-host interactions from the host cell perspective.

Risk of drug resistance in Plasmodium falciparum malaria therapy-a systematic review and meta-analysis.

Plasmodium falciparum is responsible for the vast majority of the morbidity and mortality associated with malaria infection globally. Although a number of studies have reported the emergence of drug resistance in different therapies for P. falciparum infection, the degree of the drug resistance in different antimalarials is still unclear. This research investigated the risk of drug resistance in the therapies with different medications based on meta-analyses. Relevant original randomized control trials (RCTs) were searched in all available electronic databases. Pooled relative risks (RRs) with 95% confidence intervals (95% CIs) were used to evaluate the risk of drug resistance resulting from different treatments. Seventy-eight studies were included in the meta-analysis to compare drug resistance in the treatment of P. falciparum infections and yielded the following results: chloroquine (CQ) > sulfadoxine-pyrimethamine (SP) (RR = 3.67, p < 0.001 ), mefloquine (MQ) < SP (RR = 0.26, p < 0.001), artesunate + sulfadoxine-pyrimethamine (AS + SP) > artemether + lumefantrine (AL) (RR = 2.94, p < 0.001), dihydroartemisinin + piperaquine (DHA + PQ) < AL (RR = 0.7, p < 0.05), and non-artemisinin-based combination therapies (NACTs) > artemisinin-based combination therapies (ACTs) (RR = 1.93, p < 0.001); no significant difference was found in amodiaquine (AQ) vs. SP, AS + AQ vs. AS + SP, AS + AQ vs. AL, or AS + MQ vs. AL. These results presented a global view for the current status of antimalarial drug resistance and provided a guidance for choice of antimalarials for efficient treatment and prolonging the life span of the current effective antimalarial drugs.

Evaluation of aminotransferase abnormality in dengue patients: A meta analysis.

Dengue virus is a type of flavivirus transmitted by Aedes mosquitoes. The symptoms of infection by this virus range from asymptomatic or mild symptomatic dengue fever (DF) to dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). Significant abnormality in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) has been shown in a large number of dengue infection cases and to be indicator for liver injury provided that there are no other combined infections or liver injury. This study aims to assess the abnormal levels of liver aminotransferase in dengue patients. The related literature was searched in multiple databases, including PubMed, Embase, Google Scholar and Cochrane Library. The literature was selected through strict inclusion and exclusion criteria, and the quantitative synthesis of the liver aminotransferase abnormality was performed with R software. The fixed or random effects model was employed based on the results of the statistical test for homogeneity. In total, 15 studies were included. The proportion of AST abnormality with 95% confidence interval (95% CI) was 0.80 (95% CI: 0.56-0.92) in DHF patients and 0.75 (95% CI: 0.63-0.84) in DF patients; the proportion of ALT abnormality was 0.54 (95% CI: 0.34-0.73) in DHF patients and 0.52 (95% CI: 0.41-0.63) in DF patients. Serum ALT and AST levels may be indicators for evaluating liver injury in dengue infection and for diagnosis and treatment effect.

[Secretion and Distribution of Rhoptry Protein 16 during Toxoplasma gondii Invasion into Host Cells].

Objective: To examine the secretion and localization of Toxoplasma gondii Rhoptry protein 16 （ROP16） during invasion of different strains of T. gondii into host cells. Methods: The Tgrop16 gene was amplified by PCR on the cDNA of T. gondii RH strain, subcloned into the plasmid pET-32a（+）, and expressed in Escherichia coli BL21（DE3） under the induction of isopropyl ß-D-1-thiogalactopyranoside. New Zealand rabbit was immuned with the expressed recombinant protein TgROP16 to produce polyclonal anti-TgROP16 antibody. The specificity and sensitivity of the polyclonal antibody were examined by Western blotting and indirect ELISA, respectively. The transcriptional and protein levels of Tgrop16 in T. gondii RH strain and Pru strain were determined by real-time PCR and Western blotting, respectively. The secretion and distribution of TgROP16 in human foreskin fibroblasts (HFFs) during the invasion by T. gondii RH strain and Pru strain were examined by indirect immunofluorescence assay (IFA). Results: Western blotting showed a specific band at M(r) of ～100 000, indicating that the specific rabbit-derived anti-TgROP16 polyclonal antibody was capable of recognizing TgROP16. Indirect ELISA revealed a titer of 1:25 600 for the antibody. The relative expression level of Tgrop16 in Pru strain［（7.786±0.206）］ was 7 times than that in RH strain［（1.000±0.110）］（P<0.05） as detected by real-time PCR, and TgROP16 protein level was higher in RH strain than in Pru strain. IFA showed that TgROP16 was localized on the apical complex of the unrecruited tachyzoite of T. gondii before invasion and was secreted out of the recruited tachyzoite after invasion. Conclusion: The anti-TgROP16 polyclonal antibody has high specificity and sensitivity. The TgROP16 protein level is higher in the RH strain than in the Pru strain. For both strains, TgROP16 is localized on the apical complex of the unrecruited tachyzoite before invasion and secreted out of the recruited tachyzoite during invasion.

Multiple Sources of Infection and Potential Endemic Characteristics of the Large Outbreak of Dengue in Guangdong in 2014.

A large outbreak of dengue, with the most documented cases, occurred in Guangdong China in 2014. Epidemiological studies and phylogenetic analysis of the isolated dengue virus (DENV) showed this outbreak was attributed to multiple sources and caused by at least two genotypes of DENV-1 (Genotypes I and III) and two genotypes of DENV-2 (Cosmopolitan and Asian I Genotypes). A retrospective review and phylogenetic analysis of DENV isolated in Guangdong showed that DENV-1 Genotype I strains were reported continuously during 2004-2014, Genotype III strains were reported during 2009-2014 ; DENV-2 Cosmopolitan and Asian I Genotype strains were reported continuously during 2012-2014. At least 45,171 cases were reported in this outbreak, with 65.9% of the patients in the 21-55-year-old group. A trend toward a decrease in the daily newly emerged cases lagged by approximately 20 days compared with the mosquito density curve. Several epidemiological characteristics of this outbreak and the stably sustained serotypes and genotypes of DENV isolated in Guangdong suggest that Guangdong has been facing a threat of transforming from a dengue epidemic area to an endemic area. The high temperature, drenching rain, rapid urbanization, and pandemic of dengue in Southeast Asia may have contributed to this large outbreak of dengue.

A Systematic Review and Meta-Analysis of the Efficacy of Anti-Toxoplasma gondii Medicines in Humans.

No effective drug and definitive "gold standard" treatment for Toxoplasma gondii (T. gondii) infection has been available so far, though some medicines have been commonly used in the treatment of T. gondii infection, such as spiramycin, azithromycin, traditional Chinese medicine (TCM), pyrimethamine- sulfadiazine (P-S), trimethoprim-sulfamethoxazole (TMP-SMX), and pyrimethamine-clindamycin (P-C). A systematic review and meta-analysis were performed to compare the efficacies of these conventional medicines in the treatment. Cohort studies for the treatment of acute T. gondii infection were searched from PubMed, Google Scholar, ect. All the cases number for different group extracted from each included literature were input to meta-analysis 3.13 software to calculate the pooled negative conversion rate (NCR), cure rate (CR) or vertical transmission rate based on their sample size and weight. The pooled NCR with 95% confidence intervals (CI) was used to evaluate the overall rate of a diagnosis positive result conversion to a negative result after treatment, which of spiramycin, azithromycin and TCM were 83.4% (95%CI, 72.1%-90.8%), 82.5% (95%CI, 75.9%-87.6%), and 85.5% (95%CI, 71.3%-93.3%) respectively, with no statistical difference between them. The pooled CR with 95% CI was used to evaluate the overall rate of complete disappearance of clinical symptoms for toxoplasmic encephalitis after therapy, which of P-S, TMP-SMX, and P-C were 49.8% (95%CI, 38. 8% -60.8%), 59.9% (95%CI, 48.9%-70.0%), and 47.6% (95%CI, 24.8%-71.4%) respectively, with no statistical difference between them. Primary T. gondii infection in pregnancy was treated mainly with spiramycin alone or combined with other drugs, and the pooled rate of vertical transmission was about 9.9% (95%CI, 5.9%-16.2%) after therapy. Toxoplasmic encephalitis in AIDS patients was usually treated with sulfonamides combined with other drugs and the pooled CR was 49.4% (95%CI, 37.9%-60.9%).

[Observation of the Feline Intestinal Epithelial Cell Infected with Toxoplasma gondii].

Small intestine samples of neonatal cat were aseptically collected from the jejunum-ileum region and digested with collagenase XI/dispase I. Immunohistochemistry results showed that feline intestinal epithelial cells were successfully isolated and could be cultured. Cytokeratin was positive in the cytoplasm of feline intestinal epithelial cells. The cells were infected with the bradyzoites of Toxoplasma gondii Prugniaud strain, and the rupture of the cells was observed on the 72nd day post-infection. The sexual stage of T. gondii did not occur, however.

[Preparation and purification of polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and its application in immunofluorescence localization].

OBJECTIVE: To prepare and purify polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and apply it to immunofluorescence localization. METHODS: The constructed recombinant plasmid pET32a-ROP2 was transformed into E. coli BL21 (DE3) and the protein was expressed under the condition of 0.5 mmol/L IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body, respectively. The washed inclusion bodies were dissolved in urea. New Zealand White rabbits were immunized with 200 microg purified recombinant ROP2 mixing with the same volume of Freund's adjuvant for 3 times at interval of 14 days and 19 days, respectively. Rabbit serum was collected at 10 days after the last immunization. Polyclonal antibody in rabbit serum was purified with HiTrap Protein G HP affinity purification column. Indirect ELISA and Western blotting were used to detect antibody titer and specificity of polyclonal antibody against the recombinant ROP2. The polyclonal antibody was used to the localization of ROP2 on the parasitophorous vacuole membrane in human foreskin fibroblasts infected by Toxoplasma tachyzoites by the immunofluorescence method. RESULTS: The recombinant ROP2 protein was obtained and specific rabbit-derived polyclonal antibody was prepared. Indirect ELISA confirmed that the rabbit-derived polyclonal antibody titer reached 1:102400, and the recombinant ROP2 protein was recognized by specific polyclonal antibody. Immunofluorescence localization test showed that the ROP2 protein was located on the parasitophorous vacuole membrane. CONCLUSION: The rabbit-derived polyclonal antibody against ROP2 is prepared, and used in immunofluorescence localization of ROP2 on parasitophorous vacuole membrane.

A meta analysis on risks of adverse pregnancy outcomes in Toxoplasma gondii infection.

OBJECTIVE: Quantified risks of congenital Toxoplasma gondii infection and abnormal pregnancy outcomes following primary maternal infection were evaluated with meta- analysis based on published studies. METHODS: The related literatures were searched in multiple literature databases regardless of languages. Odds ratio (OR) and 95% confidence interval (CI) were used to evaluate the risks of vertical transmission of Toxoplasma gondii and abnormal pregnancy outcomes following primary maternal infection with meta-analysis. RESULTS: 53 of the 2632 searched literatures were included in our analysis. The incidence of abnormal pregnancy outcomes in T. gondii infected pregnant women (infected group) was significantly higher than that in the uninfected pregnant women (control group) (OR = 5.10; 95% CI, 3.85-6.75). Toxoplasma gondii infection rate in the abnormal-pregnancy-outcome group was significantly higher than in the normal-pregnancy group (OR = 3.71; 95% CI, 3.31-4.15). The pooled rate of vertical transmission was 20% (95% CI, 15%-26%) in maternal infection of T. gondii. The incidences of vertical transmission in women who were infected in the first, second or third trimester of pregnancy were 5% (95%CI, 2%-16%), 13% (95%CI, 7%-23%), and 32% (95%CI, 24%-41%), respectively. The rates of vertical transmission in women who were treated with spiramycin-only, PSF (pyrimethamine + sulfadiazine + folinic acid) or PS (pyrimethamine + sulfadiazine) combined with spiramycin, or other untypical treatments were 13% (95%CI, 7%-22%), 13%(95%CI, 7%-25%), and 24%(95%CI, 18%-32%), respectively. CONCLUSIONS: Toxoplasma gondii infection can result in adverse pregnancy outcomes in pregnant women. The pooled rate of vertical transmission was 20% in maternal infection and the incidences of vertical transmission increased in the first, second or third trimester of pregnancy. The pooled rates of transmission in groups treated with spiramycin-only, PSF or PS combined with spiramycin, or other untypical treatments were not significantly different.

Preparation and immunological effectiveness of a Swine influenza DNA vaccine encapsulated in PLGA microspheres.

Optimal preparation conditions of DNA vaccine against swine influenza encapsulated in Poly (D,L)-lactic-co-glycolic acid (PLGA) microspheres were determined. The microspheres were prepared by an emulsion-evaporation method using PLGA as the biodegradable matrix forming polymer. Using the optimal preparation conditions, PLGA microspheres containing the DNA vaccine were produced with good morphology as evident from scanning electron micrographs, high encapsulation rate and high stability. The transfection test indicated that the vaccine could be expressed as an antigen in cells and maintained good bioactivity. Moreover, these results demonstrated that the PLGA microspheres containing DNA vaccine can be used to achieve prolonged release of plasmid DNA. These results have laid a foundation for further development before ultimate industrial application.