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The protozoan parasite Toxoplasma gondii secretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to delete gra17 or gra23 gene in T. gondii Pru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Δgra17 and Pru:Δgra23 mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion of gra17 abrogated acute infection and blocked cyst formation. Although the deletion of gra23 caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Δgra17 induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-γ]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain of T. gondii. These findings show that GRA17 and GRA23 play important roles in T. gondii chronic infection and that irreversible deletion of gra17 in T. gondii type II Pru strain can be a viable option for stimulating protective immunity to T. gondii infection. [ABSTRACT FROM AUTHOR]

Dense granule protein 12 (GRA12) is implicated in a range of processes related to the establishment of Toxoplasma gondii infection, such as the formation of the intravacuolar network (IVN) within the parasitophorous vacuole (PV). This protein is also thought to be important for T. gondii‐host interaction, pathogenesis, and immune evasion, but their exact roles remain unknown. In this study, the contributions of GRA12 to the molecular pathogenesis of T. gondii infection were examined in vitro and in vivo. Deletion of GRA12 in type I RH and type II Pru T. gondii strains did not affect the parasite growth and replication in vitro, however, it caused a significant reduction in the parasite virulence and tissue cyst burden in vivo. T. gondii Δgra12 mutants were more vulnerable to be eliminated by host immunity, without the accumulation of immunity‐related GTPase a6 (Irga6) onto the PV membrane. The ultrastructure of IVN in Δgra12 mutants appeared normal, suggesting that GRA12 is not required for biogenesis of the IVN. Combined deletion of GRA12 and ROP18 induced more severe attenuation of virulence compared to single Δgra12 or Δrop18 mutant strains. These data suggest a functional association between GRA12 and ROP18 that is revealed by the severe attenuation of virulence in a double mutant relative to the single individual mutations. Future studies are needed to define the molecular basis of this putative association. Collectively these findings indicate that although GRA12 is not essential for the parasite growth and replication in vitro, it contributes to the virulence and growth of T. gondii in mice. [ABSTRACT FROM AUTHOR]

• Most antiretroviral compounds had no toxic effect on human foreskin fibroblast cells at 30 µM. • Of 44 tested antiretroviral compounds, 14 showed potency against Toxoplasma gondii. • Antiretroviral compounds did not affect the anti- T. gondii activity of sulfadiazine or pyrimethamine. The obligate intracellular parasite Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. Although infection with this parasite is generally benign, severe illness may occur in infected individuals if their immunity becomes less competent, such as in human immunodeficiency virus (HIV)-infected patients. In this study, the inhibitory activity of 44 commonly used antiretroviral compounds was determined against T. gondii in vitro. Of the 44 tested antiretroviral compounds, 14 showed potency against T. gondii at IC 50 concentrations (concentration inhibiting T. gondii tachyzoite growth by 50%) ranging from 1.18 ± 2.21 µM (nelfinavir) to 18.89 ± 1.87 µM (trovirdine). Of the 14 potent antiretroviral compounds, 7 are HIV-1 protease inhibitors. This study also investigated whether co-administration of these 14 antiretroviral compounds interferes with the anti- T. gondii activity of existing anti- T. gondii drugs, namely sulfadiazine and pyrimethamine. The results showed no significant interaction between any of the 14 tested antiretroviral compounds and pyrimethamine or sulfadiazine. These results warrant investigation of whether administration of the lead antiretroviral drugs with highly potent anti- T. gondii activity to HIV patients may help to limit the occurrence of toxoplasmic encephalitis. [ABSTRACT FROM AUTHOR]

Important progress has been made in understanding how immunity is elicited against Toxoplasma gondii – a complex pathogen with multiple mechanisms of immune evasion. Many vaccine candidates have been tested using various strategies in animal models. However, none of these strategies has delivered as yet, and important challenges remain in the development of vaccines that can eliminate the tissue cysts and/or fully block vertical transmission. In this review, we provide an overview of the current understanding of the host immune response to T. gondii infection and summarize the key limitations for the development of an effective, safe, and durable toxoplasmosis vaccine. We also discuss how the successes and failures in developing and testing vaccine candidates have provided a roadmap for future vaccine development. Highlights There is a compelling need to develop a safe and effective toxoplasmosis vaccine. Successful vaccination of domestic cats is the key step in reducing T. gondii transmission to humans and food-producing animals. An effective toxoplasmosis vaccine must be able to induce both humoral and cellular immune responses, directed against multiple different proteins, at different stages of the parasite life cycle. Live attenuated T. gondii strains offer good protection against toxoplasmosis, but the possibility of reversion to the virulent type remains possible. There is a need to identify more immunogenic antigens, adjuvants, and antigen-delivery systems together with defining robust immunocorrelates of protection. Having a standardized protocol for assessment of vaccine efficacy can facilitate the synergy between the results obtained by various research groups. 'Omics' technologies have revolutionized our understanding of the pathophysiology of toxoplasmosis, paving the way for development of a safe and effective vaccine. [ABSTRACT FROM AUTHOR]

Background: The threat of Toxoplasma gondii infection in immunocompromised individuals and pregnant women necessitates the development of a safe and effective vaccine. Here, we examined the immune protection conferred by a live attenuated strain of T. gondii.Methods: We tested the efficacy of intraperitoneal vaccination using 500 Ca2+-dependent protein kinase 2 (cdpk2)-deficient tachyzoites of T. gondii Pru strain against acute, chronic, and congenital toxoplasmosis in mice. The kinetics of antibody response, cytokines, and other quantifiable correlates of protection against T. gondii infection were determined.Results: Vaccination with Pru:Δcdpk2 induced a high level of anti-T. gondii immunoglobulin G titer, type 1 T-helper (Th1) response at 28 days postvaccination, and a mixed Th1/type 2 T-helper response at 70 days postvaccination. All vaccinated mice survived a heterologous challenge with 1000 tachyzoites of RH or ToxoDB#9 (PYS or TgC7) strains. Also, vaccination protected against homologous infection with 20 T. gondii Pru cysts, and improved pregnancy outcome by reducing parasite cyst load in the brain, maintaining litter size and body weight of pups born to vaccinated dams challenged with 10 Pru cysts compared to pups born to unvaccinated dams.Conclusions: The use of T. gondii Pru:Δcdpk2 mutant strain represents a promising approach to protection against acute, chronic, and congenital toxoplasmosis in mice. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii , an obligatory intracellular protozoan, can cause serious public health problems and economic losses worldwide. Two novel dense granule proteins (GRA17 and GRA23) were recently identified as T. gondii -secreted proteins which are localized to the parasitophorous vacuole membrane (PVM) and can mediate the movement of small molecules between the host cell and parasitophorous vacuole (PV). In the present study, we evaluated the protective immunity induced by DNA vaccination with genes encoding GRA17 and GRA23 against acute toxoplasmosis in mice. Eukaryotic expressing plasmids pVAX-TgGRA17 and pVAX-TgGRA23 were constructed. Then, BALB/c mice were intramuscularly immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 followed by challenge infection with the highly virulent RH strain of T. gondii . The specific immune responses and protective efficacy against T. gondii were examined by cytokine and serum antibody measurements, lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival time after challenge. Our results showed that mice immunized with pVAX-TgGRA17, pVAX-TgGRA23, or pVAX-TgGRA17 + pVAX-TgGRA23 induced specific humoral and cellular responses, with higher level of IgG antibody, increased levels of Th1-type cytokines IFN-γ and IL-12 (p70), and CD3 + CD4 + CD8 − and CD3 + CD8 + CD4 − T cells, as well as prolonged survival time (9.1 ± 0.32 days for pVAX-TgGRA17, 10.8 ± 0.79 days for pVAX-TgGRA23, and 12.6 ± 2.55 days for pVAX-TgGRA17 + pVAX-TgGRA23) compared to the blank control (7.11 ± 0.33 days), PBS control (7.22 ± 0.44 days), and pVAX I control (7.11 ± 0.33 days). These results demonstrated that both TgGRA17 and TgGRA23 are potential vaccine candidates, TgGRA23 has a better immunogenicity, and co-immunization of pVAX-TgGRA17 and pVAX-TgGRA23 induces better protective efficacy. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a classic model for studying obligate intracellular microorganisms as various genetic manipulation tools have been developed in T. gondii over the past 20 years. Here we summarize the major strategies for T. gondii genetic manipulation including genetic crosses, insertional mutagenesis, chemical mutagenesis, homologous gene replacement, conditional knockdown techniques, and the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system. We evaluate the advantages and limitations of each of these tools in a historical perspective. We also discuss additional applications of modified CRISPR–Cas9 systems for use in T. gondii , such as regulation of gene expression, labeling of specific genomic loci, and epigenetic modifications. These approaches have the potential to revolutionize the analysis of T. gondii biology and help us to better develop new drugs and vaccines. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii, an important protozoan parasite, infects almost all warm-blooded animals and humans. Although treatments in T. gondii are limited by the lack of effective drugs, some calcium-dependent kinases were demonstrated as the promising drug targets to chemotherapy against T. gondii due to their essential roles in T. gondii and absence from their hosts. The objectives of the present study were to investigate the functions of six calcium-dependent protein kinases (CDPK4, CDPK4A, CDPK5, CDPK6, CDPK8, and CDPK9) in T. gondii to assess whether they are suitable for designing as drug targets. We used the CRISPR-Cas9 system to disrupt six CDPK genes successfully by insertion of DHFR* at the guide RNA-targeted region in the six endogenous CDPK loci and successfully obtained the six knockout (KO)-CDPK strains. The biological characteristics of the six strains were evaluated by plaque assays, invasion, egress, replication, and virulence assays, respectively. The results indicated that there was no significant difference between the six KO-CDPK strains and wild-type strain in virulence and the lytic cycle including invasion, egress, and replication. The conclusion was the six CDPKs are not essential for T. gondii lytic cycle and also not virulence factors for mice, suggesting that the six CDPKs may participate in other functions in T. gondii. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii , an obligate intracellular parasite, is able to infect many animal species and humans, and can cause toxoplasmosis of the host. In this study, we examined sequence variation in rhoptry protein 47 (ROP47) gene among T. gondii isolates originating from different hosts and geographical regions. The entire genome region of the ROP47 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ), based on the ROP47 gene sequences. The results of sequence alignments showed that all ROP47 gene sequences were 396 bp in length. There were 19 variable nucleotide positions in the coding region, resulted in 16 amino acid substitutions (12.21%) among all examined T. gondii strains and the existence of polymorphic restriction sites for endonucleases SacI and AflIII, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analysis of ROP47 gene sequences showed that three major clonal lineage types I, II and III were clustered differently, consistent with PCR-RFLP results. These results suggest that ROP47 gene sequence may represent a potential novel genetic marker for population genetic studies of T. gondii isolates. [ABSTRACT FROM AUTHOR]

Abstract: Inspired by the adhesion strategy of marine mussels, self-polymerization of dopamine under alkaline condition has been proven to be a simple and effective method for surface modification of biomaterials. However, this method still has many drawbacks, such as the use of alkaline aqueous medium, low poly(dopamine) deposition rate, and inefficient utilization of dopamine, which greatly hinder its practical application. In the present study, we demonstrate that electropolymerization of dopamine is a facile and versatile approach to surface tailoring of metallic cardiovascular stents, such as small and complex-shaped coronary stent. Electropolymerization of dopamine leads to the formation of a continuous and smooth electropolymerized poly(dopamine) (ePDA) coating on the substrate surface. This electrochemical method exhibits a higher deposition rate and is more efficient in dopamine utilization compared with the typical self-polymerization method. The ePDA coating facilitates the immobilization of biomolecules onto substrates to engineer biomimetic microenvironments. In vitro and in vivo experiments demonstrate that ePDA coating functionalized with vascular endothelial growth factor can greatly enhance the desired cellular responses of endothelial cells and prevent the neointima formation after stent implantation. The proposed methodology may find applications in the area of metallic surface engineering, especially for the cardiovascular stents and potentially all biomedical devices with electroconductive surface as well. [Copyright &y& Elsevier]

Abstract: Chitosan/poly (vinyl pyrollidone) (CHI/PVP) coatings were prepared to improve the antibacterial properties of poly (ethylene terephthalate) (PET) by a simple dip-coating method. The binding capability of CHI/PVP coatings was enhanced by successively pretreatment of PET by polyetherimide and polyacrylic acid and crosslinking. Measurements of water contact angle and atomic force microscope revealed that the coatings created a highly hydrophilic surface with low roughness. Adherences of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) on PET with CHI/PVP coating were significantly reduced. Bactericidal activity of CHI/PVP coatings was good against E. coli and S. aureus and the adding of PVP obviously increased its antiadhesion property. In vitro cytotoxicity tests, cell morphology and activity evaluation of human umbilical vein endothelial cells showed that CHI/PVP coatings had good biocompatibility. [Copyright &y& Elsevier]

Background: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. Methods: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. Results: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. Conclusions: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment. [ABSTRACT FROM AUTHOR]

[Display omitted] • Dense granular protein GRA76 was more highly expressed in tachyzoites than in bradyzoites of Toxoplasma gondii. • GRA76 is important for the growth and virulence of T. gondii. • Deletion of gra76 in the Pru strain (PruΔ gra76) increased expression of bradyzoite-associated genes. • PruΔ gra76 exhibited increasd propensity for forming bradyzoites in vitro. Pathogenicity of the zoonotic pathogen Toxoplasma gondii largely depends on the secretion of effector proteins into the extracellular milieu and host cell cytosol, including the dense granule proteins (GRAs). The protein-encoding gene TGME49_299780 was previously identified as a contributor to parasite fitness. However, its involvement in parasite growth, virulence and infectivity in vitro and in vivo remains unknown. Here, we comprehensively examined the role of this new protein, termed GRA76, in parasite pathogenicity. Subcellular localization revealed high expression of GRA76 in tachyzoites inside the parasitophorous vacuole (PV). However, its expression was significantly decreased in bradyzoites. A CRISPR-Cas9 approach was used to knock out the gra76 gene in the T. gondii type I RH strain and type II Pru strain. The in vitro plaque assays and intracellular replication showed the involvement of GRA76 in replication of RH and Pru strains. Deletion of the gra76 gene significantly decreased parasite virulence, and reduced the brain cyst burden in mice. Using RNA sequencing, we detected a significant increase in the expression of bradyzoite-associated genes such as BAG1 and LDH2 in the PruΔ gra76 strain compared with the wild-type Pru strain. Using an in vitro bradyzoite differentiation assay, we showed that loss of GRA76 significantly increased the propensity for parasites to form bradyzoites. Immunization with PruΔ gra76 conferred partial protection against acute and chronic infection in mice. These findings show the important role of GRA76 in the pathogenesis of T. gondii and highlight the potential of PruΔ gra76 as a candidate for a live-attenuated vaccine. [ABSTRACT FROM AUTHOR]

Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii. Author summary: Toxoplasma gondii is a protozoan parasite characterized by a highly spatially and temporally coordinated replication process. Some protein phosphatases are conserved among apicomplexan protozoa and regulate numerous cellular and biological processes, such as parasite proliferation, tachyzoite-bradyzoite differentiation and virulence. Here, we identify the role of the putative protein phosphatase 6 of T. gondii (TgPP6), and found two of its subunits, TgPP6C and TgPP6R, to be vital for the parasite replication and virulence. RHΔpp6c and RHΔpp6r exhibited various degrees of asynchronous division and morphological deformation during endodyogeny compared to wild-type parasites. TgPP6C affected the phosphorylation status of proteins involved in the parasite cell division. We also demonstrated the protective efficacy of immunization of mice using RHΔpp6c against acute and chronic infection by wild-type RH, PYS and Pru strains, respectively. These findings reveal novel roles of TgPP6C in the replication and virulence of T. gondii. [ABSTRACT FROM AUTHOR]

Glutaredoxins (Grxs) are ubiquitous antioxidant proteins involved in many molecular processes to protect cells against oxidative damage. Here, we study the roles of Grxs in the pathogenicity of Toxoplasma gondii. We show that Grxs are localized in the mitochondria (Grx1), cytoplasm (Grx2), and apicoplast (Grx3, Grx4), while Grx5 had an undetectable level of expression. We generated Δgrx1‐5 mutants of T. gondii type I RH and type II Pru strains using CRISPR‐Cas9 system. No significant differences in the infectivity were detected between four Δgrx (grx2‐grx5) strains and their respective wild‐type (WT) strains in vitro or in vivo. Additionally, no differences were detected in the production of reactive oxygen species, total antioxidant capacity, superoxide dismutase activity, and sensitivity to external oxidative stimuli. Interestingly, RHΔgrx1 or PruΔgrx1 exhibited significant differences in all the investigated aspects compared to the other grx2‐grx5 mutant and WT strains. Transcriptome analysis suggests that deletion of grx1 altered the expression of genes involved in transport and metabolic pathways, signal transduction, translation, and obsolete oxidation–reduction process. The data support the conclusion that grx1 supports T. gondii resistance to oxidative killing and is essential for the parasite growth in cultured cells and pathogenicity in mice and that the active site CGFS motif was necessary for Grx1 activity. [ABSTRACT FROM AUTHOR]

Several calcium-binding proteins including calcium-dependent protein kinases play important roles in several facets of the intracellular infection cycle of the apicomplexan protozoan parasite Toxoplasma gondii. However, the role of the calcium-binding epidermal growth factor (EGF) domain-containing proteins (CBDPs) remains poorly understood. In this study, we examined the functions of four CBDP genes in T. gondii RH strain of type I by generating knock-out strains using CRISPR-Cas9 system. We investigated the ability of mutant strains deficient in CBDP1, CBDP2, CBDP3, or CBDP4 to form plaques, replicate intracellularly, and egress from the host cells. The results showed that no definite differences between any of these four CBDP mutant strains and the wild-type strain in terms of their ability to form plaques, intracellular replication, and egress. Additionally, CBDP mutants did not exhibit any significant attenuated virulence compared to the wild-type strain in mice. The expression profiles of CBDP2-4 genes were conserved among T. gondii strains of different genotypes, life cycle stages, and developmental forms. Whether other CBDP genes play any roles in the pathogenicity of T. gondii strains of different genotypes remains to be elucidated. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an obligate intracellular apicomplexan parasite which is able to infect almost all warm-blooded animals. There is no information about the prevalence and genetic characterization of T. gondii in badgers ( Melogale moschata ) in China. Here, a total of 367 badgers were captured from different cities in Jiangxi province, Southern China. Genomic DNA was extracted from brain tissues of each badgers, and 57 (15.45%) of them were positive for T. gondii by semi-nested PCR of the B1 gene. The positive DNA samples were typed at 11 genetic markers, including 10 nuclear loci (SAG1, 5′-SAG2 and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1) and an apicoplast locus (Apico), with multilocus polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. Among them, 4 were completely typed at all loci, and 2 was genotyped for 9 loci, showing that they belong to ToxoDB#9. This is the first report of prevalence and genetic characterization of T. gondii isolates from badgers in China, which contributes to broader understanding of population structure of T. gondii in China. It is important for the prevention and control of T. gondii infection in wild animals. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a worldwide prevalent pathogen that infects most of the warm-blood vertebrates. To investigate the regulation network of splenic miRNAs altered by acute infection with T. gondii , we herein investigated the changes of miRNA profile in mouse spleen via next generation sequencing and bioinformatics analysis. A total of 379 miRNAs was identified, 131 miRNAs of them were differentially expressed (including 97 upregulated and 34 downregulated miRNAs). 48 differentially expressed miRNAs had validated targets in the miRWalk2.0 database. Gene Ontology (GO) enrichment analysis revealed that the validated targets of differently expressed miRNAs were significantly enriched in gene transcription regulation. It suggested that T. gondii can modulate host gene expression through targeting to trans-regulation factors. The genes involved in apoptosis or anti-apoptosis were both targeted by differentially expressed miRNAs. The change of power balance between the miRNAs targeting host apoptosis genes and those regulating host anti-apoptosis genes contributes to the fate of host apoptosis process. Twelve pathways were significantly enriched in KEGG analysis with most of them being cancer related, including pathways in cancer, pancreatic cancer, colorectal cancer, axon guidance, MAPK signaling pathway, focal adhesion, chronic myeloid leukemia, renal cell carcinoma, prostate cancer, glioma, regulation of actin cytoskeleton, and Wnt signaling pathway. Our study showed a changed miRNA regulation network in mouse spleen infected by T. gondii . These findings will be helpful for better understanding of miRNA regulation network in host– T. gondii interaction, revealing the relationship among T. gondii infection, gene regulation, apoptosis and cancer process alterations in infected spleen. [ABSTRACT FROM AUTHOR]

Abstract: The non-biorecognition of general biomaterials and inherent biospecificity of biological systems pose key challenges to the optimal functions of medical devices. In this study, we constructed the surface-mediated functional gene delivery through layer-by-layer self-assembly of protamine sulfate (PrS) and plasmid DNA encoding hepatocyte growth factor (HGF), aiming at specific enhancing endothelial cells (EC) compeititiveness over smooth muscle cells (SMC). Characterizations of the (PrS/HGF-pDNA) multilayered films present the linear buildup with homogeneous and flat topographical feature. The amount of DNA can be easily controlled. By using these multilayered films, both human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (HUASMC) can be directly transfected when they contact with the multilayered films. On transfection, increasing secretion of HGF has been detected in both HUVEC and HUASMC culture, which leads to selective promotion of HUVEC proliferation. In the co-culture experiment, we also exhibit the promoted and hindered growth of HUVEC and HUASMC, respectively, which could be attributed to the inverse influence of HUVEC on HUASMC. These results collectively demonstrate that our system can be served as a powerful tool for enhancing competitiveness of EC over SMC, which opens perspectives for the regulation of intercellular competitiveness in the field of interventional therapy. [Copyright &y& Elsevier]

Background: Little information about the prevalence of gastrointestinal parasites in yaks (Bos grunniens) in northwest China is available. Therefore, the objective of the study was to quantify faecal egg counts of gastrointestinal parasites (helminths and coccidia) in free-range yaks from Gannan Tibetan Autonomous Prefecture, Gansu Province, Northwest China. Results: Parasites were detected in 290 of 733 (39.56%) faecal samples. The results showed that Strongylidae, Trichuris spp. and Eimeria spp. were detected all year round, Strongyloides papillosus was detected in autumn and summer, and Nematodirus spp. was detected in both autumn and spring. In contrast, Fasciola spp. was only detected in spring. The prevalence rates of parasitic infections in different seasons were significantly different. Conclusions: To our knowledge, this is the first investigation of gastrointestinal parasites in yaks (Bos grunniens) in Gansu, China. The results demonstrated a high prevalence of gastrointestinal parasitic infections, specifically GN infections, in yaks in GTAP and these infections can cause economic losses to the local cattle industry. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an intracellular zoonotic parasite that causes toxoplasmosis, which can cause economic losses and serious public health problems worldwide. A member of the T. gondii calcium-dependent protein kinases family, TgCDPK1 was recently identified as an essential regulator of exocytosis in T. gondii, and participated in direct parasite motility, host-cell invasion and egress. In the present study, the protective immunity of recombinant TgCDPK1 protein (rTgCDPK1) was evaluated against acute toxoplasmosis in mice. rTgCDPK1 were expressed and purified, BABL/c mice were intraperitoneally immunized with rTgCDPK1 and challenged with the highly virulent RH strain of T. gondii. The specific immune responses were analyzed by measuring the cytokine and serum antibody, and lymphocyte proliferation assays, flow cytometry of lymphocytes and the survival curve were employed to evaluate the protective efficacy. From the results we found that special humoral and cellular responses could be elicited in vaccine mice, and higher level of IgG antibody, and the significant increased levels of Th1-type cytokines IFN-γ, IL-12 (p70), IL10 and CD3+CD4+CD8− and CD3+CD8+CD4− T cells could also be detected comparing to control mice (P < 0.05). All vaccinated mice prolonged survival time (14.90 ± 2.89 days) challenge with 1000 tachyzoites of RH, while the control mice died within 8 days. These results indicated that TgCDPK1 protein was a potential vaccine candidate against acute toxoplasmosis. • The rTgCDPK1 could elicited special humoral and cellular responses in vaccine mice. • All vaccinated mice prolonged survival time challenge with tachyzoites of RH. • TgCDPK1 protein is a potential vaccine candidate against acute toxoplasmosis. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an obligatory intracellular protozoan, which infects most of the warm-blooded animals, causing serious public health problems and enormous economic losses worldwide. The rhoptry effector protein 54 (ROP54) has been indicated as a virulence factor that promotes Toxoplasma infection by modulating GBP2 loading onto parasite-containing vacuoles, which can modulate some aspects of the host immune response. In order to evaluate the immuno-protective value of ROP54, we constructed a eukaryotic recombinant plasmid expressing T. gondii ROP54 and intramuscularly immunized Kunming mice with this recombinant plasmid against acute and chronic toxoplasmosis. All mice immunized with pVAX-ROP54 elicited a high level of specific antibody responses, a significant increase of lymphocyte proliferation, and a significant level of Th1-type cytokines (IFN-γ, IL-2 and IL-12p70), in addition to an increased production of Th2-type cytokines (IL-4 and IL-10). These results demonstrated that pVAX-ROP54 induced significant cellular and humoral (Th1/Th2) immune responses, which extended the survival time (13.0 ± 1.15 days for pVAX-ROP54 vs 6.7 ± 0.48 days for pVAX I, 6.8 ± 0.42 days for PBS and 6.5 ± 0.53 for blank control) and significantly reduced cyst burden (35.9% for pVAX-ROP54, 1% for pVAX I and 2% for PBS, compared with blank control) of immunized mice. These results indicate that the recombinant ROP54 plasmid can provide partial protection and might be a potential vaccine candidate against acute and chronic toxoplasmosis. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii can infect all warm-blooded animals including human beings. T. gondii dense granule protein 16 (TgGRA16) as a crucial virulence factor could modulate the host gene expression. Here, a DNA vaccine expressing TgGRA16 was constructed to explore the protective efficacy against T. gondii infection in Kunming mice. The immune responses induced by pVAX-GRA16 were also evaluated. Mice immunized with pVAX-GRA16 could elicit higher levels of specific IgG antibody and strong cellular response compared to those in controls. The DNA vaccination significantly increased the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) and the percentages of CD4+ and CD8+ T cells in mice. After lethal challenge, mice immunized with pVAX-GRA16 (8.4±0.78 days) did not show a significant longer survival time than that in controls (7.1±0.30 days) (p>0.05). However, in chronic toxoplasmosis model (administration of 10 brain cysts of PRU strain orally), numbers of tissue cysts in mice immunized with pVAX-GRA16 were significantly reduced compared to those in controls (p<0.05) and the rate of reduction could reach 43.89%. The results indicated that the TgGRA16 would be a promising vaccine candidate for further development of effective epitope-based vaccines against chronic T. gondii infection in mice. [ABSTRACT FROM AUTHOR]

Fasciola hepatica is a helminth parasite with a worldwide distribution, which can cause chronic liver disease, fasciolosis, leading to economic losses in the livestock and public health in many countries. Control is mostly reliant on the use of drugs, and as a result, drug resistance has now emerged. The identification of F. hepatica genes involved in interaction between the parasite and host immune system is utmost important to elucidate the evasion mechanisms of the parasite and develop more effective strategies against fasciolosis. In this study, we aimed to identify molecules in F. hepatica excretory and secretory products (FhESPs) interacting with the host peripheral blood mononuclear cells (PBMCs), Th1-like cytokines (IL2 and IFN-γ), and Th17-like cytokines (IL17) by Co-IP combined with tandem mass spectrometry. The results showed that 14, 16, and 9 proteins in FhESPs could bind with IL2, IL17, and IFN-γ, respectively, which indicated that adult F. hepatica may evade the host immune responses through directly interplaying with cytokines. In addition, nine proteins in FhESPs could adhere to PBMCs. Our findings provided potential targets as immuno-regulators, and will be helpful to elucidate the molecular basis of host-parasite interactions and search for new potential proteins as vaccine and drug target candidates. [ABSTRACT FROM AUTHOR]

In-stent restenosis is one of the most serious modes of failure of cardiovascular stent implant. Although drug-eluting stents have been proven to reduce in-stent restenosis, the nonspecific inhibitory effects of anti-proliferative drugs, such as rapamycin, result in delayed re-endothelialization and fatal late stent thrombosis. Although many studies have focused on promoting rapid re-endothelialization, a feasible method of reducing excessive extracellular matrix (ECM) production and cell proliferation might provide a promising way to efficiently inhibit the restenosis in vivo. In this study, we constructed a surface-mediated gene delivery system through a layer-by-layer assembly of protamine sulfate (PrS) and a functional plasmid DNA (pDNA) encoding short hairpin RNA to downregulate the expression of transforming growth factor-β1 (TGF-β1), aiming to inhibit cell proliferation and reduce excessive ECM production. We demonstrated that (PrS/pDNA) films were successfully constructed with good stability under physiological conditions. The (PrS/pDNA) films were able to transfect fibroblasts, thus reducing the secretion of fibronectin and collagen and inhibiting cell proliferation in vitro . Further in vivo experiments showed that the transfection of arterial tissue led to significant local downregulation of TGF-β1 and ECM proteins and inhibited neointimal hyperplasia. These functional gene delivery films avoid the use of non-specific drugs and may serve as part of a new strategy for targeting in-stent restenosis in the field of cardiovascular disease. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a global pathogen that infects a wide range of animals and humans. During T. gondii infection, the spleen plays an important role in coordinating the adaptive and innate immune responses. However, there is little information regarding the changes in global gene expression within the spleen following T. gondii infection. To address this gap in knowledge, we examined the transcriptome of the mouse spleen following T. gondii infection. We observed differential expression of 2310 transcripts under these conditions. Analysis of KEGG and GO enrichment indicated that T. gondii alters multiple immune signaling cascades. Most of differentially expressed GO terms and pathways were downregulated, while immune-related GO terms and pathways were upregulated with response to T. gondii infection in mouse spleen. Most cytokines were upregulated in infected spleens, and all differentially expressed chemokines were upregulated which enhanced the immune cells chemotaxis to promote recruitment of immune cells, such as neutrophils, eosinophils, monocytes, dendritic cells, macrophages, NK cells, basophils, B cells, and T cells. Although IFN-γ-induced IDO (Ido1) was upregulated in the present study, it may not contribute a lot to the control of T. gondii because most differentially expressed genes involved in tryptophan metabolism pathway were downregulated. Innate immunity pathways, including cytosolic nucleic acid sensing pathway and C-type lectins-Syk-Card9 signaling pathways, were upregulated. We believe our study is the first comprehensive attempt to define the host transcriptional response to T. gondii infection in the mouse spleen. [ABSTRACT FROM AUTHOR]

Spraying layer-by-layer (LbL) assembly technique has been developed for rapid and efficient fabrication of functional films. The spraying method generally requires relatively fast formation of stable interactions among the building components. In this study, inspired by nature, the fast and strong coordination bond between catechol groups (polydopamine, PDA) and metal ions (Fe 3 + ) was employed to fabricate (poly(ethylenimine)/PDA-coated carbon nanotubes) (PEI/CNTs@PDA) multilayer film. The coordination bond significantly enhanced the films' crosslinking during the spraying LbL procedures. Ellipsometry results confirmed the successful assembly of the [PEI/(CNTs@PDA + Fe 3 + )] multilayer film. The hierarchical micro/nano-structures were observed on the multilayer surface. As a potential functionalization, the multilayer film was then coated with a low surface energy silane compound via thermal chemical vapor deposition method, leading to a superhydrophobic surface. By taking advantages of the specific rapid coordination bond of PDA and Fe 3 + , this study provides an efficient and fast strategy to fabricate functional surfaces through spraying LbL assembly. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a worldwide zoonosis caused by Toxoplasma gondii , which can infect warm-blooded animals and humans. A serological survey was undertaken to examine the seroprevalence and risk factors associated with T. gondii infection in sika deer in northeastern China. 114 (13.46%, 95% CI 11.16–15.76) out of 847 serum samples were positive to T. gondii by modified agglutination test (MAT) at a 1:25 cut-off, with titers of 1:25 in 44, 1:50 in 32, 1:100 in 17, 1:500 in 11, 1:1500 or higher in 10. These samples were collected between November 2012 and October 2013 from Inner Mongolia, Jilin and Heilongjiang provinces in China. However, statistically significant differences were not observed between T. gondii seroprevalence and genders or regions of sika deer in the logistic regression analysis ( P > 0.05) and left out of the final model. Seroprevalence of T. gondii infection in male sika deer was 14.07% (95% CI 11.14–17.01), slightly higher than that in the female (12.38%) (95% CI 8.69–16.06) and seroprevalence of T. gondii infection in Harbin, Changchun city, Jilin city and Chifeng city were 12.02% (95% CI 7.60–16.44), 15.51% (95% CI 11.52–19.50), 12.27% (95% CI 7.23–17.31) and 12.50% (95% CI 7.38–17.63), respectively. Seasons of sampling were considered as main risk factors associated with T. gondii infection, autumn (15.32%) were more than two times (OR = 1.98, 95% CI = 1.18–3.33, P = 0.01) at risk of acquiring T. gondii infection compared to winter (8.37%). Our results indicated a widespread exposure to T. gondii among sika deer in China. To our knowledge, this is the first report of T. gondii seroprevalence in sika deer in China. [ABSTRACT FROM AUTHOR]

Abstract: Reversibly light-responsive and biocompatible micelles with an appropriate size were constructed from an amphiphilic spiropyran-containing hyperbranched polyphosphate (denoted as HPHEEP-SP). The polymer was conveniently synthesized based on the modification of a biodegradable hyperbranched polyphosphate with carboxyl-containing spiropyran molecules. HPHEEP-SP can self-assemble to biocompatible micelles with an average diameter of 186.3 nm and a critical micelle concentration of 0.052 mg mL−1. After 5 min of UV irradiation, the diameter of the micelles decreased gradually to about 100 nm, which is ascribed to the transformation of hydrophobic spiropyran to hydrophilic merocyanine. Subsequent exposure the micelles to visible light, the diameter of the micelles was restored. Model drug coumarin 102 was then encapsulated into the micelles successfully. Light-controlled release and re-encapsulation behaviours were lastly demonstrated by fluorescence spectroscopy. This study provides a convenient way to construct smart nanocarriers for controlled release and re-encapsulation of hydrophobic drugs. [Copyright &y& Elsevier]