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The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.

Plasmodium falciparum ( Pf ) parasite development in liver represents the initial step of the life-cycle in the human host after a Pf- infected mosquito bite. While an attractive stage for life-cycle interruption, understanding of parasite-hepatocyte interaction is inadequate due to limitations of existing in vitro models. We explore the suitability of hepatocyte organoids (HepOrgs) for Pf -development and show that these cells permitted parasite invasion, differentiation and maturation of different Pf strains. Single-cell messenger RNA sequencing (scRNAseq) of Pf- infected HepOrg cells has identified 80 Pf- transcripts upregulated on day 5 post-infection. Transcriptional profile changes are found involving distinct metabolic pathways in hepatocytes with Scavenger Receptor B1 (SR-B1) transcripts highly upregulated. A novel functional involvement in schizont maturation is confirmed in fresh primary hepatocytes. Thus, HepOrgs provide a strong foundation for a versatile in vitro model for Pf liver-stages accommodating basic biological studies and accelerated clinical development of novel tools for malaria control. Suitable in vitro models allowing to assess Plasmodium liver stage development are still limited. Here, Yang et al. show that hepatocytes derived from human hepatocyte organoids (HepOrgs) can support P. falciparum development. This allowed for the identification and validation of the importance of the host factor, scavenger receptor B1 (SRB1), in parasite development.

After infection of the human host, the first stage of the Plasmodium falciparum (Pf) lifecycle takes place in the liver. Understanding of host-parasite interactions during liver stage development is compromised by the rapid loss of functionality and Pf permissiveness of cultured primary human hepatocytes (PHHs). Here, we substantially delay the loss of Pf permissiveness by using a medium containing serum-replacement and signal transduction inhibitors. We analyzed and integrated transcriptomic profiles of cultured PHHs with the phenotypic presentation of developing Pf liver stages, revealing a number of host signaling pathways that contributed to dedifferentiation of hepatocytes and influenced Pf liver stage development. In particular, inhibition of the Wnt pathway showed a significant positive impact on size and maturity of Pf liver stage schizonts, while retaining the metabolic activity and epithelial nature of PHHs. Therefore, our study provides insights into hepatocyte characteristics that are important for Pf permissiveness and an improved in vitro liver stage model. This should facilitate identification and development of novel therapeutic strategies for Pf liver stages.

Background Sporozoite invasion of hepatocytes is an essential step in the Plasmodium life-cycle and has similarities, at the cellular level, to merozoite invasion of erythrocytes. In the case of the Plasmodium blood-stage, efforts to identify host–pathogen protein–protein interactions have yielded important insights including vaccine candidates. In the case of sporozoite-hepatocyte invasion, the host–pathogen protein–protein interactions involved are poorly understood. Methods To gain a better understanding of the protein–protein interaction between the sporozoite ligands and host receptors, a systematic screen was performed. The previous Plasmodium falciparum and human surface protein ectodomain libraries were substantially extended, resulting in the creation of new libraries comprising 88 P. falciparum sporozoite protein coding sequences and 182 sequences encoding human hepatocyte surface proteins. Having expressed recombinant proteins from these sequences, a plate-based assay was used, capable of detecting low affinity interactions between recombinant proteins, modified for enhanced throughput, to screen the proteins for interactions. The novel interactions identified in the screen were characterized biochemically, and their essential role in parasite invasion was further elucidated using antibodies and genetically manipulated Plasmodium parasites. Results A total of 7540 sporozoite-hepatocyte protein pairs were tested under conditions capable of detecting interactions of at least 1.2 µM K D . An interaction between the human fibroblast growth factor receptor 4 (FGFR4) and the P. falciparum protein Pf34 is identified and reported here, characterizing its affinity and demonstrating the blockade of the interaction by reagents, including a monoclonal antibody. Furthermore, further interactions between Pf34 and a second P. falciparum rhoptry neck protein, PfRON6, and between human low-density lipoprotein receptor (LDLR) and the P. falciparum protein PIESP15 are identified. Conditional genetic deletion confirmed the essentiality of PfRON6 in the blood-stage, consistent with the important role of this protein in parasite lifecycle. Pf34 was refractory to attempted genetic modification. Antibodies to Pf34 abrogated the interaction and had a modest effect upon sporozoite invasion into primary human hepatocytes. Conclusion Pf34 and PfRON6 may be members of a functionally important invasion complex which could be a target for future interventions. The modified interaction screening assay, protein expression libraries and P. falciparum mutant parasites reported here may be a useful tool for protein interaction discovery and antigen candidate screening which could be of wider value to the scientific community.

Plasmodium species, the causative agent of malaria, have a complex life cycle involving two hosts. The sporozoite life stage is characterized by an extended phase in the mosquito salivary glands followed by free movement and rapid invasion of hepatocytes in the human host. This transmission stage has been the subject of many transcriptomics and proteomics studies and is also targeted by the most advanced malaria vaccine. We applied Bayesian data integration to determine which proteins are not only present in sporozoites but are also specific to that stage. Transcriptomic and proteomic Plasmodium data sets from 26 studies were weighted for how representative they are for sporozoites, based on a carefully assembled gold standard for Plasmodium falciparum (Pf) proteins known to be present or absent during the sporozoite life stage. Of 5418 Pf genes for which expression data were available at the RNA level or at the protein level, 975 were identified as enriched in sporozoites and 90 specific to them. We show that Pf sporozoites are enriched for proteins involved in type II fatty acid synthesis in the apicoplast and GPI anchor synthesis, but otherwise appear metabolically relatively inactive in the salivary glands of mosquitos. Newly annotated hypothetical sporozoite-specific and sporozoite-enriched proteins highlight sporozoite-specific functions. They include PF3D7_0104100 that we identified to be homologous to the prominin family, which in human has been related to a quiescent state of cancer cells. We document high levels of genetic variability for sporozoite proteins, specifically for sporozoite-specific proteins that elicit antibodies in the human host. Nevertheless, we can identify nine relatively well-conserved sporozoite proteins that elicit antibodies and that together can serve as markers for previous exposure. Our understanding of sporozoite biology benefits from identifying key pathways that are enriched during this life stage. This work can guide studies of molecular mechanisms underlying sporozoite biology and potential well-conserved targets for marker and drug development.

Vibrio harveyi is a normal flora present in seawater, but in recent years, with large density aquaculture, V. harveyi has become one of the causative organisms in fish and shrimp. In this study, V. harveyi VieA gene was used as the target to design primers and establish the loop-mediated isothermal amplification ( LAMP) combined with visual lateral flow test strip (LFD). The optimal reaction conditions were optimized as 60°C, 45 min, 1.2 mM Mg 2+ , 0.64 mM dNTPs, 0.25 mM betaine, and 16:1 ratio of internal/external primers. The results showed that the established LAMP assay was able to specifically detect V. harveyi with a sensitivity of 3.4 × 10 –5 ng/µL. Clinical applicability analysis revealed that only V. harveyi was detected in the samples for pearl gentian grouper and whiteleg shrimp artificially infected by Vibrio . Therefore, we established a visual, reliable, rapid and sensitive LAMP-LFD method for detecting V. harveyi in aquatic animals.

As an adjunctive treatment of chronic periodontitis, it seems that the application of periocline or the other antimicrobials is effective against periodontopathogens. In this study, nanoliposomes were investigated as carriers of minocycline hydrochloride and the inhibition effects of minocycline hydrochloride nanoliposomes on the proliferation and lipopolysaccharide (LPS)-stimulated production of tumor necrosis factor-α (TNF-α) of macrophages were elucidated. After stimulation with 10 μg/mL LPS, murine macrophages (ANA-1) were treated with 10, 20, 40, 50 and 70 μg/mL 2% minocycline hydrochloride nanoliposomes, minocycline hydrochloride solution, and periocline for 6, 12, 24, 48 and 60 hours, respectively. A tetrazolium (MTT) assay was used to evaluate macrophages cell proliferation rate and the levels of TNF-α mRNA were measured by SYBR Green Real Time PCR. Ten to 70 μg/mL 2% minocycline hydrochloride nanoliposomes, minocycline hydrochloride solution, and periocline showed dose- and time-dependent inhibition of ANA-1 proliferation. Minocycline hydrochloride nanoliposomes showed dose- and ratio-dependent inhibition of LPS-stimulated TNF-α secretion of ANA-1. The inhibition effect of 10 μg/mL minocycline hydrochloride nanoliposomes was significantly better than that of two positive control groups, and equated to that of 60 or 70 μg/mL periocline. The expression of TNF-α mRNA in experimental group continued to reduce linearly with time. All three preparations of minocycline hydrochloride showed dose- and time-dependent inhibition of proliferation of ANA-1. Minocycline hydrochloride nanoliposomes have stronger and longer inhibition effect on LPS-stimulated TNF-α secretion of macrophages cell than minocycline hydrochloride solution and periocline.

The association between Hashimoto Thyroiditis (HT) and papillary thyroid cancer (PTC) remains uncertain. HT, the most common inflammatory condition of the thyroid, is postulated to increase the risk of PTC and yet confer cancer-retarding effects. In this study, we aim to evaluate the prevalence of HT in patients surgically treated for PTC and evaluate the long-term prognostic implications. This is a retrospective study of 521 patients with PTC who underwent hemi- or total thyroidectomy between January 2000 and December 2018 at a tertiary referral centre. Patients were categorised into two group: group A ( =402) consists of patients with PTC without HT, whereas group B ( =119) consists of patients with PTC and HT. Demographic and clinicopathological details, recurrence rates and overall survival were collected. Univariate and multivariate analyses were performed to evaluate for clinical factors associated with HT. A total of 521 patients with a mean age of 46.7 years were evaluated. HT was detected in 22.8% of patients with PTC. On multivariate analysis, presence of HT was associated with a lower incidence of extrathyroidal extension (hazard ratio: 0.59, 95%confidence interval 0.37-0.95). Also, patients with HT tend to have fewer cycles of radioactive iodine and correspondingly have excellent response to treatment. However, no differences in recurrence rates and overall survival were detected. One-fifth of patients with PTC have coexisting HT. These patients tend to have less-aggressive tumour features such as extrathyroidal extension. However, the effect of HT on recurrence and overall survival appears to be inconsequential clinically.

Background Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi , providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR). Results Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the C t values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed. Conclusions Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.

O-glycosylation of the Plasmodium sporozoite surface proteins CSP and TRAP was recently identified, but the role of this modification in the parasite life cycle and its relevance to vaccine design remain unclear. Here, we identify the Plasmodium protein O-fucosyltransferase (POFUT2) responsible for O-glycosylating CSP and TRAP. Genetic disruption of POFUT2 in Plasmodium falciparum results in ookinetes that are attenuated for colonizing the mosquito midgut, an essential step in malaria transmission. Some POFUT2-deficient parasites mature into salivary gland sporozoites although they are impaired for gliding motility, cell traversal, hepatocyte invasion, and production of exoerythrocytic forms in humanized chimeric liver mice. These defects can be attributed to destabilization and incorrect trafficking of proteins bearing thrombospondin repeats (TSRs). Therefore, POFUT2 plays a similar role in malaria parasites to that in metazoans: it ensures the trafficking of Plasmodium TSR proteins as part of a non-canonical glycosylation-dependent endoplasmic reticulum protein quality control mechanism. The role of O-glycosylation in the malaria life cycle is largely unknown. Here, the authors identify a Plasmodium protein O-fucosyltransferase and show that it is important for normal trafficking of a subset of surface proteins, particularly CSP and TRAP, and efficient infection of mosquito and vertebrate hosts.