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Abstract Background The protozoan parasite Toxoplasma gondii encodes dozens of phosphatases, among which a plant-like phosphatase absent from mammalian genomes named PPKL, which is involved in regulating brassinosteroid signaling in Arabidopsis, was identified in the genome. Among the Apicomplexa parasites, T. gondii is an important and representative pathogen in humans and animals. PPKL was previously identified to modulate the apical integrity and morphology of the ookinetes and parasite motility and transmission in another important parasite, Plasmodium falciparum. However, the exact function of PPKL in the asexual stages of T. gondii remains unknown. Methods The plant auxin-inducible degron (AID) system was applied to dissect the phenotypes of PPKL in T. gondii. We first analyzed the phenotypes of the AID parasites at an induction time of 24 h, by staining of different organelles using their corresponding markers. These analyses were further conducted for the parasites grown in auxin for 6 and 12 h using a quantitative approach and for the type II strain ME49 of AID parasites. To further understand the phenotypes, the potential protein interactions were analyzed using a proximity biotin labeling approach. The essential role of PPKL in parasite replication was revealed. Results PPKL is localized in the apical region and nucleus and partially distributed in the cytoplasm of the parasite. The phenotyping of PPKL showed its essentiality for parasite replication and morphology. Further dissections demonstrate that PPKL is required for the maturation of daughter parasites in the mother cells, resulting in multiple nuclei in a single parasite. The phenotype of the daughter parasites and parasite morphology were observed in another type of T. gondii strain ME49. The substantial defect in parasite replication and morphology could be rescued by genetic complementation, thus supporting its essential function for PPKL in the formation of parasites. The protein interaction analysis showed the potential interaction of PPKL with diverse proteins, thus explaining the importance of PPKL in the parasite. Conclusions PPKL plays an important role in the formation of daughter parasites, revealing its subtle involvement in the proper maturation of the daughter parasites during division. Our detailed analysis also demonstrated that depletion of PPKL resulted in elongated tubulin fibers in the parasites. The important roles in the parasites are potentially attributed to the protein interaction mediated by kelch domains on the protein. Taken together, these findings contribute to our understanding of a key phosphatase involved in parasite replication, suggesting the potential of this phosphatase as a pharmaceutic target. Graphical Abstract

The apicoplast is a four-membrane plastid found in the apicomplexans, which harbors biosynthesis and organelle housekeeping activities in the matrix. However, the mechanism driving the flux of metabolites, in and out, remains unknown. Here, we used TurboID and genome engineering to identify apicoplast transporters in Toxoplasma gondii. Among the many novel transporters, we show that one pair of apicomplexan monocarboxylate transporters (AMTs) appears to have evolved from a putative host cell that engulfed a red alga. Protein depletion showed that AMT1 and AMT2 are critical for parasite growth. Metabolite analyses supported the notion that AMT1 and AMT2 are associated with biosynthesis of isoprenoids and fatty acids. However, stronger phenotypic defects were observed for AMT2, including in the inability to establish T. gondii parasite virulence in mice. This study clarifies, significantly, the mystery of apicoplast transporter composition and reveals the importance of the pair of AMTs in maintaining the apicoplast activity in apicomplexans.

ABSTRACT In the apicomplexans, endocytosed cargos (e.g., hemoglobin) are trafficked to a specialized organelle for digestion. This follows a unique endocytotic process at the micropore/cytostome in these parasites. However, the mechanism underlying endocytic trafficking remains elusive, due to the repurposing of classical endocytic proteins for the biogenesis of apical organelles. To resolve this issue, we have exploited the genetic tractability of the model apicomplexan Toxoplasma gondii, which ingests host cytosolic materials (e.g., green fluorescent protein[GFP]). We determined an association between protein prenylation and endocytic trafficking, and using an alkyne-labeled click chemistry approach, the prenylated proteome was characterized. Genome editing, using clustered regularly interspaced short palindromic repaet/CRISPR-associated nuclease 9 (CRISPR/Cas9), was efficiently utilized to generate genetically modified lines for the functional screening of 23 prenylated candidates. This identified four of these proteins that regulate the trafficking of endocytosed GFP vesicles. Among these proteins, Rab1B and YKT6.1 are highly conserved but are non-classical endocytic proteins in eukaryotes. Confocal imaging analysis showed that Rab1B and Ras are substantially localized to both the trans-Golgi network and the endosome-like compartments in the parasite. Conditional knockdown of Rab1B caused a rapid defect in secretory trafficking to the rhoptry bulb, suggesting a trafficking intersection role for the key regulator Rab1B. Further experiments confirmed a critical role for protein prenylation in regulating the stability/activity of these proteins (i.e., Rab1B and YKT6.1) in the parasite. Our findings define the molecular basis of endocytic trafficking and reveal a potential intersection function of Rab1B on membrane trafficking in T. gondii. This might extend to other related protists, including the malarial parasites. IMPORTANCE The protozoan Toxoplasma gondii establishes a permissive niche, in host cells, that allows parasites to acquire large molecules such as proteins. Numerous studies have demonstrated that the parasite repurposes the classical endocytic components for secretory sorting to the apical organelles, leaving the question of endocytic transport to the lysosome-like compartment unclear. Recent studies indicated that endocytic trafficking is likely to associate with protein prenylation in malarial parasites. This information promoted us to examine this association in the model apicomplexan T. gondii and to identify the key components of the prenylated proteome that are involved. By exploiting the genetic tractability of T. gondii and a host GFP acquisition assay, we reveal four non-classical endocytic proteins that regulate the transport of endocytosed cargos (e.g., GFP) in T. gondii. Thus, we extend the principle that protein prenylation regulates endocytic trafficking and elucidate the process of non-classical endocytosis in T. gondii and potentially in other related protists.

Abstract Background The Lisfranc joint has complex structures, and articular surfaces overlap on conventional X-ray radiographs. Hence, there is no available auxiliary examination for diagnosing related injuries. At present, few studies on the imaging of Lisfranc ligaments have been reported, and related imaging data are rare. Therefore, no imaging reference can be used for related diagnosis and repair operations. This study aims to observe and describe the morphology and structure of Lisfranc ligaments using magnetic resonance imaging (MRI), in order to provide imaging reference for the diagnosis and repair of Lisfranc joint injuries. Methods MRI scanning was performed on 60 sides of normal feet of 30 healthy adult volunteers. In the MRI scanning on the Lisfranc joint, sagittal scanning was focused on the area between the lateral margin and medial margin of the Lisfranc joint, while oblique coronal scanning was focused on the area parallel to the Lisfranc joint clearance. After acquisition of MRI images, data were burned into a CD, and the morphology and structure of the Lisfranc ligament on the MRI image were observed and described. Hence, the imaging parameters of the Lisfranc ligament were acquired, providing an imaging reference for the diagnosis and repair of Lisfranc joint injuries. Results By observing the obtained images of the Lisfranc ligament through appropriate MRI scanning, it was found that the Lisfranc ligament originates at the site 12.63 ± 1.20 mm from the lateral side of the base of the medial cuneiform bone, with a length of 8.02 ± 1.5 mm, a width of 2.53 ± 0.61 mm, a height of 6.96 ± 1.01 mm, forms an included angle of 46.79 ± 3.47° with the long axis of the first metatarsal bone, and finally ends at the base of the second phalanx. Detailed imaging parameters of the Lisfranc joint and ligament were obtained from the present imaging experiment, providing an imaging reference for the diagnosis and repair of Lisfranc joint injuries. Conclusions On the MRI images, the sagittal section can clearly display the corresponding situation of the Lisfranc joint bone and longitudinal arch of the foot, tolerably display the Lisfranc joint dorsal ligaments and metatarsal ligaments, and poorly display the Lisfranc ligament. The oblique coronal section can clearly display the transverse arch of the foot and clearly display the cross-section of the Lisfranc ligament. The oblique crosssection can clearly display the horizontal arch of the Lisfranc joint and more clearly display its surrounding ligaments and tendons, especially the entire Lisfranc ligament and its attachment points. This is an important section for the diagnosis of Lisfranc ligament injuries. This study provides a certain imaging reference for the MRI scanning, diagnosis, and repair of Lisfranc joint injuries. Further research with large sample size is still needed to confirm the conclusions.

Abstract In the original publication of this article [1], the spelling of the first author's name A. Ablimit was incorrect.

AbstractAim:To investigate the mode of action of WSS45, one sulfated derivative of an α-D-glucan from the Gastrodia elata Bl, on the multiplication cycle of dengue virus serotype 2 (DV2), including initial infection and intracellular replication.Methods:Virus multiplication in BHK cells were monitored by qRT-PCR, plaque reduction assay, and flow cytometry. Initial virus infection was dissected into adsorption and penetration steps by converting temperature and treating by acid glycine. Surface bound virions were detected by immunofluorescence staining for Evelope protein.Results:WSS45 effectively inhibited DV2 infection in BHK cells with an EC50 value of 0.68±0.17 μg/mL , mainly interfered with virus adsorption, in a very early stage of the virus cycle. However, WSS45 showed no viricidal effect. Moreover, WSS45 could increase the detaching of virus from cell surface in BHK cell line.Conclusion:WSS45 exerted potent inhibitory effect on DV2 through interfering with the interaction between viruses and targeted cells. This activity was related to its molecular size. [ABSTRACT FROM AUTHOR]