Characterization of functions in parasite growth and virulence of four Toxoplasma gondii genes involved in lipid synthesis by CRISPR-Cas9 system.

Fatty acid uptake is extremely important for the survival and growth of the intracellular parasite Toxoplasma gondii. In this study, CRISPR-Cas9 gene editing technology was used to investigate the role of four lipid synthesis enzymes, namely, glycerol-3-phosphate dehydrogenase (G3PDH), malonyl CoA-acyl carrier protein transacylase (FabD), acyl-ACP thiolesterase (TE), and diacylglycerol acyltransferase (DGAT), in the virulence and infectivity of Type I RH and Type II Prugniaud (Pru) strains of T. gondii. Immunofluorescence analysis of the tachyzoite stage showed that FabD protein was located in the apicoplast; however, the expression level of the other three proteins was undetectable. Compared with wild-type (WT) strains, the growth of RHΔG3PDH, RHΔTE, and RHΔDGAT in vitro and their virulence in vivo were not significantly different. However, RHΔFabD exhibited a significantly reduced growth rate, compared with the WT strain. The deletion of FabD attenuated the virulence of Type II Pru strain and reduced the formation of cysts in vivo. These data improved our understanding of the role of lipid synthesis enzymes in the pathogenesis of T. gondii.

The Role of Type II Fatty Acid Synthesis Enzymes FabZ, ODSCI, and ODSCII in the Pathogenesis of Toxoplasma gondii Infection.

Toxoplasma gondii is an obligate intracellular protozoan parasite, which has a worldwide distribution and can infect a large number of warm-blooded animals and humans. T. gondii must colonize and proliferate inside the host cells in order to maintain its own survival by securing essential nutrients for the development of the newly generated tachyzoites. The type II fatty acid biosynthesis pathway (FASII) in the apicoplast is essential for the growth and survival of T. gondii. We investigated whether deletion of genes in the FASII pathway influences the in vitro growth and in vivo virulence of T. gondii. We focused on beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) and oxidoreductase, short chain dehydrogenase/reductase family proteins ODSCI and ODSCII. We constructed T. gondii strains deficient in FabZ, ODSCI, and ODSCII using CRISPR-Cas9 gene editing technology. The results of immunofluorescence assay, plaque assay, proliferation assay and egress assay showed that in RHΔFabZ strain the apicoplast was partly lost and the growth ability of the parasite in vitro was significantly inhibited, while for RHΔODSCI and RHΔODSCII mutant strains no similar changes were detected. RHΔFabZ exhibited reduced virulence for mice compared with RHΔODSCI and RHΔODSCII, as shown by the improved survival rate. Deletion of FabZ in the PRU strain significantly decreased the brain cyst burden in mice compared with PRUΔODSCI and PRUΔODSCII. Collectively, these findings suggest that FabZ contributes to the growth and virulence of T. gondii, while ODSCI and ODSCII do not contribute to these traits.

ROP18-Mediated Transcriptional Reprogramming of HEK293T Cell Reveals New Roles of ROP18 in the Interplay Between Toxoplasma gondii and the Host Cell.

Toxoplasma gondii secretes a number of virulence-related effector proteins, such as the rhoptry protein 18 (ROP18). To further broaden our understanding of the molecular functions of ROP18, we examined the transcriptional response of human embryonic kidney cells (HEK293T) to ROP18 of type I T. gondii RH strain. Using RNA-sequencing, we compared the transcriptome of ROP18-expressing HEK293T cells to control HEK293T cells. Our analysis revealed that ROP18 altered the expression of 750 genes (467 upregulated genes and 283 downregulated genes) in HEK293T cells. Gene ontology (GO) and pathway enrichment analyses showed that differentially expressed genes (DEGs) were significantly enriched in extracellular matrix- and immune-related GO terms and pathways. KEGG pathway enrichment analysis revealed that DEGs were involved in several disease-related pathways, such as nervous system diseases and eye disease. ROP18 significantly increased the alternative splicing pattern "retained intron" and altered the expression of 144 transcription factors (TFs). These results provide new insight into how ROP18 may influence biological processes in the host cells via altering the expression of genes, TFs, and pathways. More in vitro and in vivo studies are required to substantiate these findings.

Diffuse primary extra-gastrointestinal stromal tumor of the peritoneum: A rare case report.

RATIONALE: Gastrointestinal stromal tumors that present outside the gastrointestinal tract are known for extra-gastrointestinal stromal tumors (EGISTs) and they share the same morphological and immunohistochemical characteristics with gastrointestinal stromal tumors. Here we report a rare case of diffuse primary EGIST arising at peritoneum. PATIENT CONCERNS: A 57-year-old male presented to the hospital with abdominal pain and right lower abdominal tenderness. DIAGNOSIS: The core needle puncture biopsy showed epithelial-like cells and the nuclei were ovoid and focally elongated. Immunohistochemical examination was consistent with a primary EGIST of the peritoneum. INTERVENTIONS: The patient was treated with Imatinib mesylate. OUTCOMES: Five months later, there is no complication resulting from treatment. The follow-up abdominal contrast-enhanced CT showed the lesion was significantly decreased in size, and was evaluated as partial response. The patient continued the treatment with Imatinib as prescribed by the oncologist. LESSONS: EGISTs are rare and should be considered in the differential diagnosis of the peritoneal tumors and immunohistochemistry helps to confirm the diagnosis. Further study with longer follow-up is desired to characterize these uncommon tumors.

Initial chest CT findings in COVID-19: correlation with clinical features.

In December 2019, coronavirus disease 2019 (COVID-19), a new de novo infectious disease, was first identified in Wuhan, China and quickly spread across China and around the world. The etiology was a novel betacoronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Lu et al., 2020). On Mar. 11, 2020, World Health Organization (WHO) characterized COVID-19 as a global pandemic. As of Mar. 22, 2020, over 292 000 confirmed COVID-19 cases have been reported globally. To date, COVID-19, with its high infectivity, has killed more people than severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) combined (Wu and McGoogan, 2020).

Evaluation of immune protection against Toxoplasma gondii infection in mice induced by a multi-antigenic DNA vaccine containing TgGRA24, TgGRA25 and TgMIC6.

Toxoplasma gondii infection is prevalent in humans and animals worldwide. In this study, recombinant eukaryotic expression plasmids (pVAX-GRA24, pVAX-GRA25 and pVAX-MIC6) were constructed, and then injected into Kunming mice intramuscularly, as cocktailed plasmids or as single-gene plasmids. We evaluated immune protective responses by detecting the titer of antibodies and cytokine production of IFN-γ, IL-2, IL-4, IL-10, IL-12 and IL-23, the percentages of the subclasses of T lymphocytes, as well as the records of the survival time and cyst decrement in the brain of the mouse model after challenge with the T. gondii RH and Pru strains, respectively. Compared with the control groups, antibody and cytokine production were significantly increased, while the survival times of mice in all immunized groups were also prolonged, and the number of T. gondii cysts in their brains were decreased significantly (29.03% for pVAX-GRA24; 40.88% for pVAX-GRA25; 37.70% for pVAX-MIC6; 48.06% for pVAX-GRA24 + pVAX-GRA25; and 55.37% for pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6). The mouse group immunized with the three-gene cocktail (TgGRA24 + TgGRA25 + TgMIC6) had better performance in each detection index than the mouse groups immunized with the two-gene cocktail of TgGRA24 + TgGRA25, which was better than that in the group immunized with the single gene vaccine of TgGRA24, TgMIC6 or TgGRA25. In conclusion, TgGRA24 or TgGRA25 may be good vaccine candidates against T. gondii infection, but the three-gene cocktail of TgGRA24, TgMIC6 and TgGRA25 may induce the strongest protective immunity. Further studies of multi-antigenic DNA vaccines or cocktailed vaccines against T. gondii infection are necessary.

TITLE: Évaluation de la protection immunitaire contre l'infection par Toxoplasma gondii chez la souris, induite par un vaccin à ADN multi-antigénique contenant TgGRA24, TgGRA25 et TgMIC6. ABSTRACT: L'infection à Toxoplasma gondii est répandue chez les humains et les animaux dans le monde entier. Dans cette étude, des plasmides d'expression eucaryotes recombinants (pVAX-GRA24, pVAX-GRA25 et pVAX-MIC6) ont été construits, puis injectés à des souris Kunming par voie intramusculaire, comme cocktails de plasmides ou comme plasmides à un seul gène. Nous avons évalué les réponses de protection immunitaire en détectant le titre des anticorps et la production de cytokines IFN-γ, IL-2, IL-4, IL-10, IL-12 et IL-23, les pourcentages des sous-classes des lymphocytes T, ainsi qu'en mesurant les temps de survie et de décrément des kystes dans le cerveau du modèle souris après challenge par les souches RH et Pru de T. gondii, respectivement. Comparativement aux groupes témoins, la production d'anticorps et de cytokines était significativement accrue, tandis que le temps de survie des souris de tous les groupes immunisés était également prolongé et que le nombre de kystes de T. gondii dans leur cerveau diminuait de manière significative (29,03 % pour pVAX-GRA24 ; 40,88 % pour pVAX-GRA25 ; 37,70 % pour pVAX-MIC6 ; 48,06 % pour pVAX-GRA24 + pVAX-GRA25 ; 55,37 % pour pVAX-GRA24 + pVAX-GRA25 + pVAX-MIC6). Le groupe de souris immunisées avec les cocktails à trois gènes (TgGRA24 + TgGRA25 + TgMIC6) présentait la meilleure performance dans chaque indice de détection par rapport aux groupes de souris immunisées avec des cocktails à deux gènes de TgGRA24 + TgGRA25, qui était supérieur à ceux immunisés avec les vaccins monogéniques TgGRA24, TgMIC6 ou TgGRA25. En conclusion, TgGRA24 ou TgGRA25 peuvent être de bons candidats au vaccin contre l'infection à T. gondii, mais un cocktail à trois gènes de TgGRA24, TgMIC6 et TgGRA25 peut induire la plus forte immunité protectrice. Des études supplémentaires sur les vaccins à ADN multi-antigéniques ou les vaccins en cocktail contre l'infection à T. gondii sont nécessaires.

Brain connectomic associations with traditional Chinese medicine diagnostic classification of major depressive disorder: a diffusion tensor imaging study.

BACKGROUND: Major depressive disorder (MDD) is highly heterogeneous in pathogenesis and manifestations. Further classification may help characterize its heterogeneity. We previously have shown differential metabolomic profiles of traditional Chinese medicine (TCM) diagnostic subtypes of MDD. We further determined brain connectomic associations with TCM subtypes of MDD. METHODS: In this naturalistic study, 44 medication-free patients with a recurrent depressive episode were classified into liver qi stagnation (LQS, n = 26) and Heart and Spleen Deficiency (HSD, n = 18) subtypes according to TCM diagnosis. Healthy subjects (n = 28) were included as controls. Whole-brain white matter connectivity was analyzed on diffusion tensor imaging. RESULTS: The LQS subtype showed significant differences in multiple network metrics of the angular gyrus, middle occipital gyrus, calcarine sulcus, and Heschl's gyrus compared to the other two groups. The HSD subtype had markedly greater regional connectivity of the insula, parahippocampal gyrus, and posterior cingulate gyrus than the other two groups, and microstructural abnormalities of the frontal medial orbital gyrus and middle temporal pole. The insular betweenness centrality was strongly inversely correlated with the severity of depression and dichotomized the two subtypes at the optimal cutoff value with acceptable sensitivity and specificity. CONCLUSIONS: The LQS subtype is mainly characterized by aberrant connectivity of the audiovisual perception-related temporal-occipital network, whereas the HSD subtype is more closely associated with hyperconnectivity and microstructural abnormalities of the limbic-paralimbic network. Insular connectivity may serve a biomarker for TCM-based classification of depression.Trial registration Registered at http://www.clinicaltrials.gov (NCT02346682) on January 27, 2015.

MIC16 gene represents a potential novel genetic marker for population genetic studies of Toxoplasma gondii.

BACKGROUND: The zoonotic agent Toxoplasma gondii is distributed world-wide, and can infect a broad range of hosts including humans. Microneme protein 16 of T. gondii (TgMIC16) is responsible for binding to aldolase, and is associated with rhomboid cleavage and presence of trafficking signals during invasion. However, little is known of the TgMIC16 sequence diversity among T. gondii isolates from different hosts and geographical locations. RESULTS: In this study, we examined sequence variation in MIC16 gene among T. gondii isolates from different hosts and geographical regions. The entire genomic region of the MIC16 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using Bayesian inference (BI) and maximum parsimony (MP) based on the MIC16 gene sequences. The results of sequence alignments showed two lengths of the sequence of MIC16 gene among all the examined 12 T. gondii strains: 4391 bp for strains TgCatBr5 and MAS, and 4394 bp for strains RH, TgPLH, GT1, PRU, QHO, PTG, PYS, GJS, CTG and TgToucan. Their A+T content ranged from 50.30 to 50.59 %. A total of 107 variable nucleotide positions (0.1-0.9 %) were identified, including 29 variations in 10 exons and 78 variations in 9 introns. Phylogenetic analysis of MIC16 sequences showed that typical genotypes (Type I, II and III) were able to be grouped into their respective genotypes. Moreover, the three major clonal lineages (Type I, II and III) can be differentiated by PCR-RFLP using restriction enzyme Pst I. CONCLUSIONS: Phylogenetic analysis and PCR-RFLP of the MIC16 locus among T. gondii isolates from different hosts and geographical regions allowed the differentiation of three major clonal lineages (Type I, II and III) into their respective genotypes, suggesting that MIC16 gene may provide a novel potential genetic marker for population genetic studies of T. gondii isolates.

[Morphological Characteristics and Molecular Identification of Trichuris sp. from Giraffe].

Objective: To investigate the morphological characteristics of Trichuris sp. from giraffe in Hefei wild zoo and identify its species using molecular techniques. Methods: Morphological characteristics of Trichuris collected from giraffe were analyzed. The internal transcribed spacer 1（ITS-1） was amplified by PCR and the PCR product was sequenced. The resulting sequence was homology analysis in GenBank and its heredity evolution tree was constructed by MEGA 4.0 software. Results: The male worms had a body length of 35.89-58.56 mm, an esophagus to body length ration of 0.29-0.40, and a spicule length of 1.96-3.89 mm. The thick and thin proportions of body were 7.02-23.45 mm and 28.05-40.05 mm respectively. These data showed different degrees of variation with previous reports. The PCR resulted in a product of 491 bp, comprising part of 18S rRNA and full length ITS-1. Sequence alignment showed that the identified Trichuris was most homologous（98.6%） with T. bos taurus HE608848, T. capreolus JX218218, and T. japanese AB367795, but it was only 46.0% homologous with T. discolor AB367794. In the heredity evolution tree, it was not located on the same branch as T. discolor, T. ovis and T. bos taurus. Conclusions: The Trichuris sp. collected from giraffe is different from previous reports in morphology and ITS-1 sequence. Further research is needed to determine if it is a new species.

Regulation of the oncogenic function of distal-less 4 by microRNA-122 in hepatocellular carcinoma.

Distal-less 4 (DLX4) is a member of the DLX family of homeobox genes. Recent reports have suggested that abnormal expression of DLX4 is present in several types of human tumors, including breast cancer, leukemia and colon cancer. However, the function and the mechanistic regulation of DLX4 in hepatocellular carcinoma (HCC) are elusive. In the present study, a proportion of hepatocellular carcinomas were identified to exhibit upregulated DLX4 expression. This study proposed that the overexpression of DLX4 is associated with the downregulation of miR-122, an underexpressed miRNA in human HCC. Functional studies have demonstrated that the downregulation of DLX4 in hepatocellular carcinoma cell lines is regulated by miR-122 through binding to its 3'UTR. Furthermore, a DLX4 overexpression vector lacking the 3'UTR was shown to abolish miR-122-induced inhibition of proliferation in the HCC cell line Hep3B. These results gave new insight into the mechanism of the miR-122/DLX4 axis in HCC.