Functional analyses of Toxoplasma gondii dihydroorotase reveal a promising anti-parasitic target.

Toxoplasma gondii relies heavily on the de novo pyrimidine biosynthesis pathway for fueling the high uridine-5'-monophosphate (UMP) demand during parasite growth. The third step of de novo pyrimidine biosynthesis is catalyzed by dihydroorotase (DHO), a metalloenzyme that catalyzes the reversible condensation of carbamoyl aspartate to dihydroorotate. Here, functional analyses of TgDHO reveal that tachyzoites lacking DHO are impaired in overall growth due to decreased levels of UMP, and the noticeably growth restriction could be partially rescued after supplementation with uracil or high concentrations of L-dihydroorotate in vitro. When pyrimidine salvage pathway is disrupted, both DHOH35A and DHOD284E mutant strains proliferated much slower than DHO-expressing parasites, suggesting an essential role of both TgDHO His35 and Asp284 residues in parasite growth. Additionally, DHO deletion causes the limitation of bradyzoite growth under the condition of uracil supplementation or uracil deprivation. During the infection in mice, the DHO-deficient parasites are avirulent, despite the generation of smaller tissue cysts. The results reveal that TgDHO contributes to parasite growth both in vitro and in vivo. The significantly differences between TgDHO and mammalian DHO reflect that DHO can be exploited to produce specific inhibitors targeting apicomplexan parasites. Moreover, potential DHO inhibitors exert beneficial effects on enzymatic activity of TgDHO and T. gondii growth in vitro. In conclusion, these data highlight the important role of TgDHO in parasite growth and reveal that it is a promising anti-parasitic target for future control of toxoplasmosis.

Tandem mass tag-based quantitative proteomics analyses of a chicken-original virulent and its attenuated Histomonas meleagridis strain in China.

Introduction: Histomonas meleagridis can cause histomonosis in poultry. Due to the prohibition of effective drugs, the prevention and treatment of the disease requires new strategies. Questions about its pathogenic mechanisms and virulence factors remain puzzling. Methods: To address these issues, a tandem mass tag (TMT) comparative proteomic analysis of a virulent strain and its attenuated strain of Chinese chicken-origin was performed. Results: A total of 3,494 proteins were identified in the experiment, of which 745 proteins were differentially expressed (fold change ≥1.2 or ≤0.83 and p < 0.05), with 192 up-regulated proteins and 553 down-regulated proteins in the virulent strain relative to the attenuated strain. Discussion: Surface protein BspA like, digestive cysteine proteinase, actin, and GH family 25 lysozyme were noted among the proteins up regulated in virulent strains, and these several proteins may be directly related to the pathogenic capacity of the histomonad. Ferredoxin, 60S ribosomal protein L6, 40S ribosomal protein S3, and NADP-dependent malic enzyme which associated with biosynthesis and metabolism were also noted, which have the potential to be new drug targets. The up-regulation of alpha-amylase, ras-like protein 1, ras-like protein 2, and involucrin in attenuated strains helps to understand how it is adapted to the long-term in vitro culture environment. The above results provide some candidate protein-coding genes for further functional verification, which will help to understand the molecular mechanism of pathogenicity and attenuation of H. meleagridis more comprehensively.

Mineralization Reduces the Toxicity and Improves Stability and Protective Immune Response Induced by Toxoplasma gondii.

Vaccination is an ideal strategy for the control and prevention of toxoplasmosis. However, the thermostability and effectiveness of vaccines limit their application. Here, calcium mineralization was used to fabricate Toxoplasma gondii tachyzoites as immunogenic core-shell particles with improved immune response and thermostability. In the current study, T. gondii RH particles coated with mineralized shells were fabricated by calcium mineralization. The mineralized shells could maintain the T. gondii tachyzoites structural integrity for at least 12 months and weaken the virulence. Immunization of mice with mineralized tachyzoites induced high levels of T. gondii-specific antibodies and cytokines. The immunized mice were protected with a 100% survival rate in acute and chronic infection, and brain cyst burdens were significantly reduced. This study reported for the first time the strategy of calcium mineralization on T. gondii and proved that mineralized tachyzoites could play an immune protective role, thus expanding the application of biomineralization in T. gondii vaccine delivery.

Evaluation of Origanum vulgare Essential Oil and Its Active Ingredients as Potential Drugs for the Treatment of Toxoplasmosis.

Toxoplasma gondii is a serious hazard to public health and animal husbandry. Due to the current dilemma of treatment of toxoplasmosis, it is urgent to find new anti-T. gondii drugs to treat toxoplasmosis. In this study, the anti-T. gondii activity of Origanum vulgare essential oil (Ov EO) was firstly studied, and then, carvanol (Ca), the main ingredient of Ov EO was evaluated using the MTT assay on human foreskin fibroblast (HFF) cells in vitro. The cytotoxicity was evaluated using the MTT assay on HFF cells. The CC50 of Ov EO and Ca was 134.9 and 43.93 µg/ml, respectively. Both of them exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. For the inhibition effect, Ca was better than Ov EO at the same concentration, the IC50 of Ov EO and Ca was 16.08 and 7.688 µg/ml, respectively. In addition, treatment with Ca, was found to change the morphology of T. gondii tachyzoites and made their shapes curl up. These results showed that Ca was able to inhibit the proliferation of T. gondii by reducing invasion, which may be due to its detrimental effect on the mobility of tachyzoites. Our results indicated that Ca could be a potential new and effective drug for treating toxoplasmosis.

In Vitro Evaluation of Lavandula angustifolia Essential Oil on Anti-Toxoplasma Activity.

The current methods of treating toxoplasmosis have a number of side effects, and these therapies are only effective against the acute stage of the disease. Thus, development of new low toxicity and efficient anti-Toxoplasma drugs is extremely important. Natural products are important sources for screening new drugs; among them, essential oils (EOs) have efficacy in anti-bacterial, anti-inflammatory, anti-insect, and other aspects. In this study, 16 EOs were screened for their anti-T. gondii activity. Lavandula angustifolia essential oil (La EO)was found to have an anti-parasitic effect on T. gondii. The cytotoxicity of La EO was firstly evaluated using the MTT assay on human foreskin fibroblast (HFF) cells, and then the anti-T. gondii activity was evaluated by plaque assay. Finally, the invasion experiment and electron microscope observation were used to study the mechanism of La EO in anti-toxoplasma activity. The results indicated that the CC50 of La EO was 4.48 mg/ml and that La EO had activity against T. gondii and the inhibition was in a dose-dependent manner under safe concentrations. La EO was able to reduce T. gondii invasion, which may be due to its detrimental effect on changes of the morphology of tachyzoites. These findings indicated that La EO could be a potential drug for treating toxoplasmosis.

In vitro Anti-parasitic Activity of Pelargonium X. asperum Essential Oil Against Toxoplasma gondii.

Toxoplasmosis is a global zoonotic disease, and one-third of the human population is chronically infected by Toxoplasma gondii. Due to the limited effectiveness and prominent side effects of the existing drugs, there is a dire need for the discovery of new therapeutic options in the treatment of toxoplasmosis. In this study, five essential oils (EO) were screened for their anti-parasitic activity against T. gondii. The cytotoxicity of essential oils was evaluated using the MTT assay on human foreskin fibroblast cells. The CC50 values of Eucalyptus globulus EO, Cupressus sempervirens EO, Citrus aurantifolia EO, Melaleuca alternifolia EO, and Pelargonium X. asperum (Pa) EO were found to be 22.74, 7.25, 15.01, 6.26, and 4.77 mg/mL, respectively. Only PaEO exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. In addition, treatment with PaEO, was found to reduce the volume of T. gondii tachyzoites and make their membrane surfaces rough. These results showed that PaEO was able to inhibit the growth of T. gondii by reducing invasion, which may be due to its detrimental effect on the ability of tachyzoites to move. These findings suggest that PaEO could be a potential anti-T. gondii drug, which may facilitate the development of new and effective treatments against toxoplasmosis.

The efficacy and safety of high-dose tranexamic acid in adolescent idiopathic scoliosis: a meta-analysis.

BACKGROUND: This study aimed to evaluate the efficacy and safety of using high-dose intravenous tranexamic acid (TXA) to reduce blood loss in idiopathic scoliosis surgery. METHODS: This study was a meta-analysis, which consisted of retrospective cohort studies (RCSs) and randomized control trials (RCTs) found by searching electronic databases, namely PubMed, Web of Science, The Cochrane Central Register of Controlled Trials (CENTRAL), and the Google Scholar Database, dating from 1960 to 2019. The points of interest included total blood loss, a need for transfusion and transfusion criteria, surgery time, and the evidence of intraoperative and postoperative complications, such as seizures or thromboembolic events. The weighted mean differences (WMD) and 95% confidence interval (CI) of blood loss in the TXA intervention group compared to the control or placebo group were extracted and combined using the random effects model. RESULTS: In this meta-analysis, there was a total of three RCSs and two RCTs, which involved 334 patients. The results showed that blood loss is significantly reduced, with a weighted mean difference in the TXA group (WMD = - 525.14, P = 0.0000, CI ranged from - 839.83, - 210.44, I2 = 82%). Heterogeneity was assessed using the random effects model. CONCLUSIONS: A high dose of intravenous TXA reduced blood loss during adolescent idiopathic scoliosis surgery and did not lead to any significant thromboembolic event. Therefore, a high dose appears to be effective and safe for adolescent idiopathic scoliosis surgery. However, more high-quality research based on larger randomized controlled trials is still needed.

Prevalence and morphological and molecular characteristics of Sarcocystis bertrami in horses in China.

Three cyst-forming Sarcocystis species have been identified in horsemeat; however, there exists considerable confusion concerning their relationships. Here, 74% (34/46) of the examined tissue samples from horses contained sarcocysts based on examination by light microscopy (LM), and the organism was identified as Sarcocystis bertrami based on cyst structure. The S. bertrami cysts were microscopic (up to 6750 µm in length) and exhibited a striated wall with 2.0-5.1 µm villar protrusions (vps) under LM. Transmission electron microscopy (TEM) observations showed that the vps were tightly packed, similar to "type 11c". Four genetic markers (18S, 28S, ITS1 and the mitochondrial cox1 gene) of S. bertrami were sequenced and analyzed. The 28S and ITS1 sequences are the first records for Sarcocystis in horses. The newly obtained sequences of the 18S and cox1 genes both shared the highest similarities with those of S. bertrami and S. fayeri obtained from horses. Phylogenetic analysis based on the 18S, 28S and cox1 sequences revealed that S. bertrami and S. fayeri formed an independent clade within a group comprising Sarcocystis spp. from ruminants and pigs. Therefore, S. bertrami and S. fayeri are considered to represent the same species of Sarcocystis in horses, and S. fayeri is a junior synonym of Sarcocystis bertrami.

TITLE: Prévalence et caractéristiques morphologiques et moléculaires de Sarcocystis bertrami chez les chevaux en Chine. ABSTRACT: Trois espèces de Sarcocystis formant des kystes ont été identifiées dans la viande de cheval, mais il existe une confusion considérable concernant leurs relations. Ici, 74 % (34/46) des échantillons de tissus examinés provenant de chevaux contenaient des sarcocystes selon l'examen par microscopie optique (LM), et l'organisme a été identifié comme Sarcocystis bertrami selon la structure du kyste. Les kystes de S. bertrami étaient microscopiques (jusqu'à 6 750 µm de longueur) et présentaient une paroi striée avec des saillies villaires (vp) de 2,0 à 5,1 µm sous LM. Les observations en microscopie électronique à transmission ont montré que les vp étaient très serrées, similaires au « type 11c ¼. Quatre marqueurs génétiques (18S, 28S, ITS1 et le gène mitochondrial cox1) de S. bertrami ont été séquencés et analysés. Les séquences de 28S et ITS1 sont les premières de Sarcocystis chez les chevaux. Les séquences nouvellement obtenues de 18S et des gènes mitochondriaux cox1 partagent les similitudes les plus élevées avec celles de S. bertrami et S. fayeri obtenues à partir de chevaux. Une analyse phylogénétique basée sur les séquences de 18S, 28S et cox1 a révélé que S. bertrami et S. fayeri formaient un clade indépendant au sein d'un groupe comprenant les Sarcocystis spp. des ruminants et des porcs. Par conséquent, S. bertrami et S. fayeri sont considérés comme représentant la même espèce de Sarcocystis chez les chevaux, et S. fayeri est un synonyme plus récent de Sarcocystis bertrami.

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To understand the influence of roots of understory plant entering litter layer on litter decomposition in forest ecosystems, we examined the effects of different treatments of Lolium multiflorum root biomass on microorganisms and enzyme activities during leaf litter decomposition of Symplocos setchuensis, a dominant species in a mid-subtropical evergreen broad-leaved forest, through a litter bag simulation experiment. Results showed that diversity index of bacterial and fungal communities of leaf litter surface under three treatments, i.e. no root (N), less roots (L), more roots (M), in a 240-day decomposition process showed the following pattern: M > L > N. The effects of these different root biomass treatments on the composition and quantity of fungal community were more significant than those on bacterial community. The biomass of living roots growing in the litter bag gradually decreased at the end of the growing season of L. multiflorum. The impacts of root growth on the composition of the fungal community gradually decreased during decomposition. At the same decomposition stage, the activities of acid phosphatase, ß-glucosidase, polyphenol oxidase, and peroxidase on the litter surface were higher in the treatments with roots than that without roots. These results indicated that root growth could change the composition and quantity of microbial communities and increase the extracellular enzyme activities of microbes, and thus stimulating litter decomposition.

[Effect of Toxoplasma gondii Infection in Female Mice on Dopamine Level in the Brain of the Male Offspring].

OBJECTIVE: To investigate the effect of Toxoplasma gondii infection in female mice on dopamine level in the brain of male offspring. METHODS: Thirty-six ICR female mice were randomly divided into control group and infection group, 18 mice in each group. Each mouse in infection group was orally infected with 10 cysts of T. gondii Prugniaud strain. On the 90th day after infection, the infected female mice were mated with normal male ICR mice at 1:1 ratio. On the 20th day of pregnancy, 2 mice in each group were delivered for fetal mice by cesarean section, and the brain of male fetal mice (n = 6) in each group were collected. On the 14th and 63rd day after birth, 6 male offspring mice in each group were sacrificed, and the brain were collected. Dopamine levels in the cortex, cerebellum, hippocampus, and striatum were analyzed by high-performance liquid chromatography-electrochemical detection (HPLC-ECD). RESULTS: Three mice in infection group died during the experiment, and 6 out of 15 female mice mated successfully. The number of fetal mice and F1 generation mice in infection group was 12 (male: 7) and 21 (male: 15), respectively. All the mice in control group mated successfully. The number of fetal mice and F1 generation mice was 23 (male: 12) and 179 (male: 92), respectively. The dopamine level in the cerebellum of fetal mice of infection group and control group was (413.25 ± 21.78) ng/g and (346.30 ± 51.83) ng/g, respectively (P < 0.01). No significant difference was found in dopamine content in the cortex between the two groups (P > 0.05). Compared with the control group, on the 14th day and 63rd day after birth, the dopamine content in cortical areas [(462.50 ± 24.80) ng/g and (1215.77 ± 113.64) ng/g], cerebellum area [(271.55 ± 26.19) ng/g and (1328.82 ± 39.62) ng/g], hippocampus area [(225.78 ± 24.17) ng/g and (1322.70 ± 58.34) ng/g], and striatum area [(455.23 ± 61.53) ng/g and (991.32 ± 54.31) ng/g] of the male offspring in infection group were significantly higher than that of the control (P < 0.05, P < 0.01). CONCLUSION: T. gondii infection in female mice causes an increase of dopamine level in the brain of F1 generation male mice.

GRIM­19­mediated Stat3 activation is a determinant for resveratrol­induced proliferation and cytotoxicity in cervical tumor­derived cell lines.

Resveratrol is a natural phenol, produced from red grapes, berries and peanuts. Previous studies have suggested that resveratrol exerts anticancer effects. Activation of the signal transducer and activator of transcription 3 (Stat3) is important in cancer. However, the mechanisms by which resveratrol suppresses the Stat3 signaling pathway remain to be elucidated. The aim of the present study was to investigate the effects of resveratrol on GRIM­19­Stat3 signaling in HeLa cells, derived from a cervical tumor. HeLa cells were divided into experimental groups and treated with resveratrol. Western blotting was used to analyze the expression levels of p­Stat3, Stat3, GRIM­19 and ß­actin. Cell viability was determined using an MTT assay. The results showed that 100 µM resveratrol suppressed the proliferation and Stat3 phosphorylation in HeLa cells, and induced the expression of the gene associated with retinoid­IFN­induced mortality 19 (GRIM­19) protein. Overexpression of GRIM­19 suppressed the Stat3 signaling pathway in HeLa cells. The Stat3 signaling pathway was activated following the downregulation of GRIM­19 expression using short interfering RNAs (siRNAs). Resveratrol suppressed cell proliferation, however, this effect was decreased through the use of siRNAs. The suppression of Stat3 phosphorylation by resveratrol decreased following treatment with siRNAs. To the best of our knowledge, the present study is among the first to identify GRIM­19­Stat3 signaling as a target of resveratrol, and further elucidates the mechanisms underlying the antitumor activity of resveratrol.

[Impairment of learning and memory ability in mice with latent infection of Toxoplasma gondii].

OBJECTIVE: To detect the learning and memory ability in mice model of latent Toxoplasma gondii infection with object recognition test and Morris water maze test. METHODS: Thirty-six Kunming mice were divided into control group, infection group with 6 cysts each mouse (low infection group), and infection group with 12 cysts each mouse (high infection group) averaged. Mice in the two infection groups were orally infected with T. gondii Prugniaud (PRU) low virulence strain. Object recognition test was conducted at the 63rd day after infection. After the first day of adaptation and the second day of familiarization in the test, the time expended on exploring new and familiar objects was recorded on the third day and the discrimination index (DI) was calculated. Morris water maze test was conducted at the 66th day. The ability of spatial learning, spatial memory retention and working memory capacity was evaluated by place navigation test, spatial probe test, and working memory test, respectively. The mice were sacrificed at the 74th day after infection. The left cerebral hemisphere of mice was fixed, sliced, and stained with eosin-hematoxylin for pathological examination. The right hemisphere was used to detect the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) content. RESULTS: The results of object recognition test showed that the discrimination index of high infection group and low infection group was (14.3 +/- 5.2)% and (17.5 +/- 5.6)%, respectively, significantly lower than the control [(28.9 +/- 7.1)%] (P < 0.01). In the place navigation test, the latency to find the platform in the two infection groups was longer than the control, with significant difference on the second and third day (P<0.05). In the spatial probe test, the percentage of the distance across the platform quadrant in the total swimming distance of high infection group and low infection group were (19.9 +/- 5.0)% and (23.9 +/- 6.8)%, respectively, significantly lower than the control [(27.4 +/- 3.6)%] (P < 0.05). In the working memory test, at the fourth day of test the latency of high infection group and low infection group [(365 +/- 14.2) s and (35.3 +/- 13.7) s] was significantly longer than the control [(30.4 +/- 12.5) s] (P<0.05). In all the tests, there was no statistical significance between low infection group and high infection group (P > 0.05). The brain sections of two infection groups showed cysts of T. gondii, proliferation of glial cells, widened gap around small blood vessels, and a phenomenon of "vascular cuff". The activity of SOD in the mice brains of two infection groups was significantly lower than the control, while MDA level was significantly higher (P < 0.05). SOD and MDA showed no significant difference between two infection groups (P > 0.05). CONCLUSION: Latent infection of T. gondii may lead to learning and memory impairment in mice.

The angiogenic and prognostic implications of VEGF, Ang-1, Ang-2, and MMP-9 for hepatocellular carcinoma with background of hepatitis B virus.

The objective of this study was to investigate the expressions of angiogenic factors and elucidate their angiogenic and prognostic roles in hepatocellular carcinoma (HCC) with background of hepatitis B virus (HBV). We evaluated microvessel density (MVD) of HCC, and investigated immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietins (Ang-1 and Ang-2), and matrix metalloproteinases-9 (MMP-9) in 67 specimens of surgically resected HCC, which were all positive for hepatitis B surface antigen. We investigated the relationship between their expressions and clinicopathological factors or prognosis. The microvessel density (MVD) of tumor tissue and surrounding normal liver tissue was 93.1 +/- 43.8/mm2 and 30.4 +/- 14.8/mm2, respectively. The MVD of well-differentiated HCC was significantly less than that of poorly differentiated HCC. MVD was positively correlated with VEGF and Ang-2 expression (P = 0.0023 and 0.0265, respectively). There was less tumor recurrence in low Ang-2 and low MMP-9 group than high Ang-2 and/or high MMP-9 group (P = 0.002). In Cox regression model, portal vein thrombus and intrahepatic metastasis was the risk factors of tumor recurrence (P = 0.003 and 0.001, respectively). Our study showed that the expression of VEGF and Ang-2 were positively correlated with MVD. Ang-2 expression and/or MMP-9 expression might be a significant predictive factor for recurrence after resection in HCC patients with the background of HBV.

Genomic analysis of a Chinese isolate of Getah-like virus and its phylogenetic relationship with other Alphaviruses.

An alphavirus, M-1 strain, was isolated from a pool of culicine mosquitoes collected in Hainan island of China during an arbovirus survey in 1964. In the present study, we determined the complete nucleotide sequence of the M-1 strain using RT-PCR and RACE techniques. The M-1 genome is 11,690 nucleotides (nt) in length and contains two open reading frames (ORFs) encoding four nonstructural proteins and five structural proteins, respectively. Searches using Blast and comparison analyses suggested that M-1 is closely linked to Sagiyama virus (SAGV, AB032553) with 98% identity and Getah viruse (GETV, AY702913) with 97.8% identity in the full-length nucleotide sequence. However, compared with SAGV, there is 1 deletion (3 nucleotides in length) in the Capsid region, a deletion in the 3' untranslated region (10 nucleotides in length) and 2 insertions in the 3' untranslated region involving a total of 5 nucleotides. Interestingly, from the 5' UTR to the end of coding region, M-1 share the highest identity with GETV, even though the identity of 3' UTR drops dramatically to 76.2%. Furthermore, phylogenetic analysis based on the complete genomic sequences and sequences for structural or non-structural proteins of M-1 and 15 alphaviruses showed that M-1 grouped with GETV first and then grouped together with SAGV. Based on the comparison analysis and phylogenetic analysis, we conclude that M-1 strain can be considered as a strain that is a Chinese isolate of Getah-like virus.