Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates.

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.

Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231.

Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (-50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism.

2,8-Disubstituted-1,5-naphthyridines as Dual Inhibitors of Plasmodium falciparum Phosphatidylinositol-4-kinase and Hemozoin Formation with In Vivo Efficacy.

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum (Pf) phosphatidylinositol-4-kinase ß (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the PfPI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf. It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.

Defining multiplicity of vector uptake in transfected Plasmodium parasites.

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part because the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species.

Plasmodium knowlesi as a model system for characterising Plasmodium vivax drug resistance candidate genes.

Plasmodium vivax causes the majority of malaria outside Africa, but is poorly understood at a cellular level partly due to technical difficulties in maintaining it in in vitro culture conditions. In the past decades, drug resistant P. vivax parasites have emerged, mainly in Southeast Asia, but while some molecular markers of resistance have been identified, none have so far been confirmed experimentally, which limits interpretation of the markers, and hence our ability to monitor and control the spread of resistance. Some of these potential markers have been identified through P. vivax genome-wide population genetic analyses, which highlighted genes under recent evolutionary selection in Southeast Asia, where chloroquine resistance is most prevalent. These genes could be involved in drug resistance, but no experimental proof currently exists to support this hypothesis. In this study, we used Plasmodium knowlesi, the most closely related species to P. vivax that can be cultured in human erythrocytes, as a model system to express P. vivax genes and test for their role in drug resistance. We adopted a strategy of episomal expression, and were able to express fourteen P. vivax genes, including two allelic variants of several hypothetical resistance genes. Their expression level and localisation were assessed, confirming cellular locations conjectured from orthologous species, and suggesting locations for several previously unlocalised proteins, including an apical location for PVX_101445. These findings establish P. knowlesi as a suitable model for P. vivax protein expression. We performed chloroquine and mefloquine drug assays, finding no significant differences in drug sensitivity: these results could be due to technical issues, or could indicate that these genes are not actually involved in drug resistance, despite being under positive selection pressure in Southeast Asia. These data confirm that in vitro P. knowlesi is a useful tool for studying P. vivax biology. Its close evolutionary relationship to P. vivax, high transfection efficiency, and the availability of markers for colocalisation, all make it a powerful model system. Our study is the first of its kind using P. knowlesi to study unknown P. vivax proteins and investigate drug resistance mechanisms.

Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two -omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three -omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.

Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort.

Several cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however, these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17-18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.