Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

The obligate intracellular lifestyle of apicomplexan parasites necessitates an invasive phase underpinned by timely and spatially controlled secretion of apical organelles termed micronemes. In Toxoplasma gondii, extracellular potassium levels and other stimuli trigger a signaling cascade culminating in phosphoinositide-phospholipase C (PLC) activation, which generates the second messengers diacylglycerol (DAG) and IP3 and ultimately results in microneme secretion. Here we show that a delicate balance between DAG and its downstream product, phosphatidic acid (PA), is essential for controlling microneme release. Governing this balance is the apicomplexan-specific DAG-kinase-1, which interconverts PA and DAG, and whose depletion impairs egress and causes parasite death. Additionally, we identify an acylated pleckstrin-homology (PH) domain-containing protein (APH) on the microneme surface that senses PA during microneme secretion and is necessary for microneme exocytosis. As APH is conserved in Apicomplexa, these findings highlight a potentially widely used mechanism in which key lipid mediators regulate microneme exocytosis.

SummarySphingosine kinase is a key enzyme in sphingolipid metabolism, catalysing the conversion of sphingosine or dihydrosphingosine into sphingosine‐1‐phosphate or dihydrosphingosine‐1‐phosphate respectively. In mammals, sphingosine‐1‐phosphate is a powerful signalling molecule regulating cell growth, differentiation, apoptosis and immunity. Functions of sphingosine kinase or sphingosine‐1‐phosphate in pathogenic protozoans are virtually unknown. While most organisms possess two closely related sphingosine kinases, only one sphingosine kinase homologue (SKa) can be identified in Leishmania, which are vector‐borne protozoan parasites responsible for leishmaniasis. Leishmania SKa is a large, cytoplasmic enzyme capable of phosphorylating both sphingosine and dihydrosphingosine. Remarkably, deletion of SKa leads to catastrophic defects in both the insect stage and mammalian stage of Leishmania parasites. Genetic and biochemical analyses demonstrate that proper expression of SKa is essential for Leishmania parasites to remove toxic metabolites, to survive stressful conditions, and to cause disease in mice. Therefore, SKa is a pleiotropic enzyme with vital roles throughout the life cycle of Leishmania. The essentiality of SKa and its apparent divergence from mammalian counterparts suggests that this enzyme can be selectively targeted to reduce Leishmania infection.

Exfoliated TiNbO5 nanosheets were obtained by delaminating a layered compound KTiNbO5. Employing a layer-by-layer electrostatic deposition technology, the exfoliated nanosheets were deposited into a multilayer composite film with polyethylenimine as the linker, confirmed by UV–vis absorption spectra and X-ray diffraction. The polymer-free TiNbO5 nanosheet multilayer film was achieved through the subsequent ultraviolet light exposure. The cyclic voltammogram of the resulting TiNbO5 nanosheet multilayer film electrode exhibited a reversible reduction–oxidation process of Ti3+/Ti4+, accompanying with the insertion/deinsertion of Li+ ions into/from the nanosheet galleries. The bandgap energy and flatband potential of TiNbO5 nanosheet were observed to be 3.47eV and −1.01V vs. Ag/AgCl, respectively. The investigation on the photoelectrocatalytic degradation of Rhodamine B with the TiNbO5 nanosheet electrodes revealed that both the oxygen anionic radicals and the dye cationic radical are essential for the mineralization of the dye under visible light-driven photocatalytic conditions, and that the electron separation under an anode bias can suppress the rapid recombination of the photogenerated charge carriers under ultraviolet light irradiation. It is the first time to investigate the mechanism of photoelectrocatalysis for TiNbO5 nanosheet electrode.

La0.90Dy0.05Nb2O7 nanosheets were achieved by exfoliating a layered compound HLa0.90Dy0.05Nb2O7. Multilayer films composed of the exfoliated nanosheets were prepared by a layer-by-layer deposition technology, which were subsequently heat-treated at 450°C and exposed under ultraviolet light to obtain the polymer-free nanosheet films, respectively. The resulting La0.90Dy0.05Nb2O7 nanosheet suspension and its films exhibit intense emission by the host excitation and negligibly low emission by the direct Dy3+ excitation, whereas the photoluminescence emissions of the bulk precursors are largely dominated by the direct Dy3+ excitation rather than the host excitation. The comparison between the excitation spectra and the bandgap absorption spectra indicates that the enhanced host excitation-mediated photoluminescence of La0.90Dy0.05Nb2O7 nanosheet results from the efficient energy transfer from the O–Dy charge-transfer transition to Dy3+ within the nanosheet, and that the intensive emission of the multilayer films is attributed to the energy transfer from both of the O–Dy charge-transfer transition and the O–Nb network to Dy3+. Dy3+ in the La0.90Dy0.05Nb2O7 nanosheet and the nanosheet-based films gives two emission peaks at around 480 and 576nm and the blue emission is prominent in the film form.

Mesoporous anatase TiO2-pillared hexaniobate has been successfully prepared by an exfoliation–restacking route. The resulting nanocomposite was characterized by powder X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscope, thermogravimetric analysis, UV–Vis spectroscopy and N2 adsorption–desorption measurements. It was reveal that the obtained material has a gallery height of about 2nm, a specific surface area of about 170m2/g and a wide pore size distribution with two extrema at about 2 and 3nm. The mesoporous material exhibits an enhanced photocatalytic activity in the degradation of acid red G under ultraviolet light irradiation, attributed to its high surface area, mesoporosity and the electronic coupling between the host and the guest components. A photoexcitation model in the semiconductor–semiconductor pillared photocatalyst was proposed based on the results of XPS and UV–Vis.

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl(-)) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(-) mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(-) promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-). These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.

Background: Human leishmaniasis is caused by more than 20 Leishmania species and has a wide range of symptoms. Our recent studies have demonstrated the essential role of sphingolipid degradation in the virulence of Leishmania (Leishmania) major, a species responsible for localized cutaneous leishmaniasis in the Old World. In this study, we investigated the function of sphingolipid degradation in Leishmania (Leishmania) amazonensis, an etiological agent of localized and diffuse cutaneous leishmaniasis in South America.Methodology/Principal Findings: First, we identified the enzyme LaISCL which is responsible for sphingolipid degradation in L. amazonensis. Primarily localized in the mitochondrion, LaISCL shows increased expression as promastigotes progress from replicative log phase to non-replicative stationary phase. To study its function, null mutants of LaISCL (Laiscl(-)) were generated by targeted gene deletion and complemented through episomal gene add-back. In culture, loss of LaISCL leads to hypersensitivity to acidic pH and poor survival in murine macrophages. In animals, Laiscl(-) mutants exhibit severely attenuated virulence towards C57BL6 mice but are fully infective towards BALB/c mice. This is drastically different from wild type L. amazonensis which cause severe pathology in both BALB/c and C57BL 6 mice.Conclusions/Significance: A single enzyme LaISCL is responsible for the turnover of sphingolipids in L. amazonensis. LaISCL exhibits similar expression profile and biochemical property as its ortholog in L. major. Deletion of LaISCL reduces the virulence of L. amazonensis and the outcome of Laiscl(-)-infection is highly dependent on the host's genetic background. Therefore, compared to L. major, the role of sphingolipid degradation in virulence is substantially different in L. amazonensis. Future studies may reveal whether sphingolipid degradation is required for L. amazonensis to cause diffuse cutaneous infections in humans.

An effective active heterostructured photocatalyst of porous SnO(2)-pillared tetratitanate nanocomposite is synthesized by assembling tetratitanate nanosheets with SnO(2) nanoparticles via an exfoliation-restacking route. The nanocomposite was characterized by powder X-ray diffraction, high-resolution transmission electron microscope, thermogravimetric analysis, UV-vis DRS, X-ray photoelectron spectroscopy, and N(2) adsorption-desorption measurements. It was found that the pillared nanaocomposite is mesoporous with a gallery height of about 2 nm and a specific surface area of 154 m(2)/g. The pillared nanaocomposite exhibited enhanced photocatalytic activity in the photodegradation of Rhodamine B under UV light irradiation. The improved performance is attributed to the electronic coupling between the host and the guest components, as well as its high surface area and mesoporosity.

Positivity constraints on the LECs of O(p6) χPT lagrangian are discussed. We demonstrate that the constraints are automatically satisfied inside the Mandelstam triangle for ππ scatterings, when NC is large. Numerical tests are made in the NC = 3 case, and it is found that these constraints are also well respected.

Fe-doped titania hollow spheres(Fe-TiO2) with different dopant contents have been prepared by a hydrothermal precipitation method with carbon spheres as the template.The phase structure,morphology,composition,and crystalline grain size were characterized by X-ray powder diffraction,scanning electron microscopy,infrared spectroscopy,thermogravimetry/differential scanning calorimetry.The as-prepared hollow spheres were anatase with 0.5–3.20 μm in diameter,30–60 nm in thickness,and 150–300 m2/g in specific surface area.With increasing dopant content,Fe-TiO2 exhibited an intense absorption in the visible light region.Fe-TiO2 exhibited photocatalytic activity for the degradation of methylene blue under visible light irradiation,and the degradation rate of methylene blue over 0.5% Fe-TiO2 hollow spheres was about 75% in 80 min.The catalytic reaction mechanism was discussed.

We estimate the diphoton coupling of f(0)(600), f(0)(980), and f(2)(1270) resonances in a coupled channel dispersive approach. The f(0)(600) diphoton coupling is also reinvestigated using a single channel T matrix for pi pi scattering with better analyticity property, and it is found to be significantly smaller than that of a qq state. Especially we also estimate the diphoton coupling of the third-sheet pole located near KK threshold, denoted as f(0)(III)(980). It is argued that this third-sheet pole may be originated from a coupled channel Breit-Wigner description of the f(0)(980) resonance.

An analysis including most recent Belle data on X(3872) is performed, using coupled channel Flatté formula. A third sheet pole close to but below D0D∗0 threshold is found, besides the bound state/virtual state pole discussed in previous literature. The co-existence of two poles near the D0D∗0 threshold indicates that the X(3872) may be of ordinary cc¯ 23P1 state origin, distorted by strong coupled channel effects. The latter manifests itself as a molecular bound state (or a virtual state).

In eukaryotes, sphingolipids (SLs) are important membrane components and powerful signaling molecules. In Leishmania, the major group of SLs is inositol phosphorylceramide (IPC), which is common in yeast and Trypanosomatids but absent in mammals. In contrast, sphingomyelin is not synthesized by Leishmania but is abundant in mammals. In the promastigote stage in vitro, Leishmania use SL metabolism as a major pathway to produce ethanolamine (EtN), a metabolite essential for survival and differentiation from non-virulent procyclics to highly virulent metacyclics. To further probe SL metabolism, we identified a gene encoding a putative neutral sphingomyelinase (SMase) and/or IPC hydrolase (IPCase), designated ISCL (Inositol phosphoSphingolipid phospholipase C-Like). Despite the lack of sphingomyelin synthesis, L. major promastigotes exhibited a potent SMase activity which was abolished upon deletion of ISCL, and increased following over-expression by episomal complementation. ISCL-dependent activity with sphingomyelin was about 20 fold greater than that seen with IPC. Null mutants of ISCL (iscl(-)) showed modest accumulation of IPC, but grew and differentiated normally in vitro. Interestingly, iscl(-) mutants did not induce lesion pathology in the susceptible BALB/c mice, yet persisted indefinitely at low levels at the site of infection. Notably, the acute virulence of iscl(-) was completely restored by the expression of ISCL or heterologous mammalian or fungal SMases, but not by fungal proteins exhibiting only IPCase activity. Together, these findings strongly suggest that degradation of host-derived sphingomyelin plays a pivotal role in the proliferation of Leishmania in mammalian hosts and the manifestation of acute disease pathology.

By utilizing SPORT5 and aerial images, Great Wall of Ming Dynasty in Beijing has been investigated in detail and current distribution of Great Wall has been reported. In this paper, DEM is assisted to extract Great Wall information and the length of Great Wall has also been calculated. It studies the relationship between Great Wall and the surrounding land use, villages, roads, terrain by using GIS spatial analysis method. Finally, the influence weight of every relevant factor is calculated by AHP(Analytic Hierarchy Process) method and this thesis sets up buffer model and confirms the protection zone, all these has proposed scientific basis for the protection, management, and investigation of Great Wall in Beijing.