Toxoplasma MIC2 Is a Major Determinant of Invasion and Virulence

Like its apicomplexan kin, the obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and uses a unique form of gliding motility. The recent identification of several transmembrane adhesive complexes, potentially capable of gripping external receptors and the sub-membrane actinomyosin motor, suggests that the parasite has multiple options for host-cell recognition and invasion. To test whether the transmembrane adhesin MIC2, together with its partner protein M2AP, participates in a major invasion pathway, we utilized a conditional expression system to introduce an anhydrotetracycline-responsive mic2 construct, allowing us to then knockout the endogenous mic2 gene. Conditional suppression of MIC2 provided the first opportunity to directly determine the role of this protein in infection. Reduced MIC2 expression resulted in mistrafficking of M2AP, markedly defective host-cell attachment and invasion, the loss of helical gliding motility, and the inability to support lethal infection in a murine model of acute toxoplasmosis. Survival of mice infected with MIC2-deficient parasites correlated with lower parasite burden in infected tissues, an attenuated inflammatory immune response, and induction of long-term protective immunity. Our findings demonstrate that the MIC2 protein complex is a major virulence determinant for Toxoplasma infection and that MIC2-deficient parasites constitute an effective live-attenuated vaccine for experimental toxoplasmosis

EFL STUDENT’S PROBLEMS WITH PARAGRAPH WRITING AT TAY DO UNIVERSITY, VIETNAM

This paper studied problems in EFL students’ paragraph writing. The research participants were first-year students of English in Tay Do University (TDU). To achieve the desired aims of the current study, the researcher combined both qualitative and quantitative methods, using questionnaires, interviews, and observations as the main instruments. The researcher used the package of social sciences (SPSS) for analysis. Research results showed that the freshmen at TDU often encountered problems in writing paragraphs. These accounted for grammatical structures, vocabulary, and coherence. Basing on the research results, students could recognize their difficulties when writing and find some new ways to overcome them. It is suggested that teachers should find and update some new teaching methods so as to develop students’ paragraph writing skills. Article visualizations

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Diverse distributions and functions of SPARC during Xenopus laevis early development

grantor: University of TorontoSPARC is a highly conserved calcium-binding glycoprotein with dynamic transient patterns of expression during early 'Xenopus laevis' embryonic development. Injection of mature SPARC into the blastocoel cavity of blastula embryos leads to a dose dependent reduction in anterior to posterior structures (ventralization/posteriorization). This phenotype is mimicked by a synthetic peptide (peptide 4.2) corresponding to the C-terminal EF-hand of SPARC. Molecular and morphological studies indicate that SPARC induces involuting chordomesodermal cells to round up, reducing their posterior to anterior migration. This activity on cell-matrix interaction provides the first 'in vivo' evidence of a counter-adhesive activity for SPARC. Although SPARC has been characterized as a secreted extracellular matrix (ECM) glycoprotein, whole mount immunocytochemical analysis indicated that SPARC is associated with cilia of the embryonic/larval ectoderm of ' Xenopus'. SPARC is concentrated in the outer surface layer, particularly in cilia. Immunogold electron microscopy shows that SPARC is associated with the 9 + 2 microtubule arrays of the ciliary axonemes. Tubulin can be immunoprecipitated with antibodies that cross-react with 'Xenopus' SPARC, indicating a direct or indirect interaction of SPARC and tubulin. SPARC is also concentrated circumferentially at the apicolateral plasma membranes of surface ectodermal cells. Injection of anti-sense 'Xenopus ' SPARC morpholinos leads to the dissociation of surface ectoderm by mid-tailbud. A similar dissociation is observed with animal cap explants taken from morpholino injected embryos. These studies indicate that SPARC promotes epithelial cell-cell adhesion and is required for maintaining the integrity of the embryonic epidermis of 'Xenopus' embryos. This represents the first evidence of an adhesive function for SPARC. Collectively, my studies indicate that SPARC functions intracellularly as a calcium-dependent regulator of ciliary movement and that SPARC has adhesive activity in epithelial tissues, in contrast to an anti-adhesive activity in mesenchymal tissues. Since the associations of SPARC with cilia and circumferential enrichment are observed in amphibians and mammals, it is likely that these intracellular and extracellular functions of this glycoprotein are evolutionarily conserved.Ph.D

Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress.

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification

Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80 ▿ †

As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Δku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Δku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Δku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes

Invasion Phenotypes Associated with Reduced MIC2 Expression

<div><p>(A<b>)</b> Illustration of the red-green invasion assay based on differential immunolabeling. Invading parasites (step 2) were counted as green.</p><p>(B) Quantification of the red-green invasion assay: red bars, attached extracellular parasites; green bars, invading and invaded parasites. A single asterisk indicates a statistically significant difference compared to tTA-dhfr; double asterisk indicates statistical difference compared to <i>mic2e/mic2i</i> + ATc (two-tailed Student's <i>t</i>-test). BAPTA-AM-treated parasites were included as a positive control for an attachment/invasion defect. Data are mean values ± s.e.m. of four separate experiments, each with three replicates and counting eight randomly selected fields per well.</p><p>(C) Correlation and linear regression of the percentage of MIC2 expression in cell lysates (left Y-axis and black line) and the percentage of MIC2 secretion (right Y-axis and red dashed line) with the numbers of invaded parasites.</p><p>(D<b>)</b> Attachment to glutaraldehyde-fixed host cells. An asterisk indicates that attachment was significantly lower than tTA-dhfr (<i>p</i> < 0.002, two-tailed Student's <i>t</i>-test). Data were compiled from three separate experiments, counting six fields per well per clone.</p><p>(E) Time-course invasion of tTA-dhfr and Δ<i>mic2e/mic2i</i> parasites ± ATc over an 8 h period. Data represent five individual experiments with three replicates within each experiment.</p></div