REDUCED DEFORMABILITY OF PARASITIZED RED BLOOD CELLS AS A BIOMARKER FOR ANTIMALARIAL DRUG EFFICACY

Background Malaria remains a challenging and fatal infectious disease throughout the developing world. Malaria progressively induces structural and functional changes causing rigidification (loss of deformability) of infected red blood cells. Antimalarials may accelerate this process, thereby allowing the infected erythrocyte to be removed from the circulation earlier. The rapid spread of antimalarial drug resistance increases the urgency for the development of new drugs. Many biomarkers, including malaria specific genes and parasite synthesized proteins and metabolites, have been developed to evaluate drug efficacy as well as to screen new drugs. Methods Recently, we developed a microfluidic mechanism, called the multiplexed fluidic plunger that provides sensitive and rapid measurement of single red blood cell deformability. Here, we systematically evaluated the deformability changes of late stage trophozoite infected-RBCs after treatment with various well known as well as unknown antimalarials. Results We show a concentration and time - dependent response relationship that exists between chloroquine treatment and iRBCs deformability change. We determined that rigidification of trophozoite-infected RBCs is a universal property of almost all clinical antimalarial drug treatments. Spiroindolone compounds (+)-SJ733 and NITD246, inhibitors to a Plasmodium falciparum cation-transporting ATPase ATP4, induced the highest rigidified trophozoite-infected RBCs. Collectively, these results suggest that changes in the deformability of iRBCs could be used as a biomarker for antimalarial drug treatments. Therefore, as a proof-of-principle, we tested a group of bisindole alkaloids. The results revealed that cladoniamide A, which has a rare scaffold and lower IC50 value than that of chloroquine may be a promising antimalarial drug candidate. Conclusions Our results demonstrate that rigidification of infected-RBCs may be used as a biomarker for antimalarial drug efficacy as well as for new drug screening. As a proof of principle, we successfully discovered a potential antimalarial drug

Cell-­phoresis o fRed Blood Cells Revealing Biophysical Signatures in Falciparum Malaria

We describe the cell-phoresis mechanism for massively parallel analysis of red blood cell (RBC) deformability by transporting single cells through microstructures to measure their spatial dispersion. Analogous to gel electrophoresis, which transport molecules through nanostructures to measure their length, the spatial dispersion of RBCs within microstructures indicate their deformability. Similar to gel electrophoresis, cell-phoresis require minimal instrumentation, provide a simple image-based readout, and could be performed simultaneously on multiple samples as part of a biophysical assay. We applied the cell-phoresis mechanism to study the biophysical signatures of falciparum malaria where we demonstrate label-­‐free and calibration-­‐free detection of ring-­‐stage infection, as well as in vitro assessment of antimalarial drug efficacy. We show that all clinical antimalarial drugs rigidify RBCs infected P. falciparum and that recently discovered PfATP4 inhibitors show a distinct biophysical signature. We anticipate cell-phoresis to be a functional assay for screening new antimalarials and adjunctive agents, as well as for validating their mechanisms of action

Reduced deformability of parasitized red blood cells as a biomarker for anti-malarial drug efficacy

Background: Malaria remains a challenging and fatal infectious disease in developing nations and the urgency for the development of new drugs is even greater due to the rapid spread of anti-malarial drug resistance. While numerous parasite genetic, protein and metabolite biomarkers have been proposed for testing emerging anti-malarial compounds, they do not universally correspond with drug efficacy. The biophysical character of parasitized cells is a compelling alternative to these conventional biomarkers because parasitized erythrocytes become specifically rigidified and this effect is potentiated by anti-malarial compounds, such as chloroquine and artesunate. This biophysical biomarker is particularly relevant because of the mechanistic link between cell deformability and enhanced splenic clearance of parasitized erythrocytes. Methods: Recently a microfluidic mechanism, called the multiplexed fluidic plunger that provides sensitive and rapid measurement of single red blood cell deformability was developed. Here it was systematically used to evaluate the deformability changes of late-stage trophozoite-infected red blood cells (iRBCs) after treatment with established clinical and pre-clinical anti-malarial compounds. Results: It was found that rapid and specific iRBC rigidification was a universal outcome of all but one of these drug treatments. The greatest change in iRBC rigidity was observed for (+)-SJ733 and NITD246 spiroindolone compounds, which target the Plasmodium falciparum cation-transporting ATPase ATP4. As a proof-of-principle, compounds of the bisindole alkaloid class were screened, where cladoniamide A was identified based on rigidification of iRBCs and was found to have previously unreported anti-malarial activity with an IC50 lower than chloroquine. Conclusion: These results demonstrate that rigidification of iRBCs may be used as a biomarker for anti-malarial drug efficacy, as well as for new drug screening. The novel anti-malarial properties of cladoniamide A were revealed in a proof-of-principle drug screen.Applied Science, Faculty ofMedicine, Faculty ofScience, Faculty ofNon UBCChemistry, Department ofMechanical Engineering, Department ofUrologic Sciences, Department ofReviewedFacult

Mapping QTLs for enhancing early biomass derived from Aegilops tauschii in synthetic hexaploid wheat.

Strong early vigour plays a crucial role in wheat yield improvement by enhancing resource utilization efficiency. Synthetic hexaploid wheat (SHW) combines the elite genes of tetraploid wheat with Aegilops tauschii and has been widely used in wheat genetic improvement for its abundant genetic variation. The two SHWs Syn79 and Syn80 were derived from the crossing of the same tetraploid wheat DOY1 with two different Ae. tauschii accessions, AT333 and AT428, respectively. The Syn80 possessed better early vigour traits than Syn79, theretically caused by their D genome from Ae. tauschii. To dissect their genetic basis in a hexaploid background, 203 recombinant inbred lines (RILs) derived from the cross of Syn79 x Syn80 were developed to detect quantitative trait loci (QTL) for four early biomass related traits: plant height (PH), tiller number (TN), shoot fresh weight (SFW) and shoot dry weight (SDW) per plant, under five different environmental conditions. Determined from the data of SNP markers, two genome regions on 1DS and 7D were stably associated with the four early biomass related traits showing pleiotropic effects. Four stable QTLs QPh.saas-1DS, QTn.saas-1DS, QSfw.saas-1DS and QSdw.saas-1DS explaining 7.92, 15.34, 9.64 and 10.15% of the phenotypic variation, respectively, were clustered in the region of 1DS from AX-94812958 to AX-110910133. Meanwhile, QPh.saas-7D, QTn.saas-7D, QSfw.saas-7D and QSdw.saas-7D were flanked by AX-109917900 and AX-110605376 on 7D, explaining 16.12, 24.35, 15.25 and 13.37% of the phenotypic variation on average, respectively. Moreover, these genomic QTLs on 1DS and 7D enhancing biomass in the parent Syn80 were from Ae. tauschii AT428. These findings suggest that these two QTLs from Ae. tauschii can be expressed stably in a hexaploid background at the jointing stage and be used for wheat improvement

Intratumoral tertiary lymphoid structure (TLS) maturation is influenced by draining lymph nodes of lung cancer

Background Tertiary lymphoid structure (TLS) is an organized infiltration of immune cells, showing features of germinal center (GC) commonly seen in secondary lymphoid organs. However, its relationship with tumor-draining lymph nodes (TDLNs) has not been studied and we hypothesized that TDLN may influence maturation of intratumoral TLS in non-small cell lung cancer (NSCLC).Methods Tissue slides of 616 patients that had undergone surgeries were examined. Cox proportional hazard regression model was used to assess risk factors of patients’ survival, and logistic regression model was used for their relationship with TLS. Single-cell RNA-sequencing (scRNA-seq) was employed to explore transcriptomic features of TDLNs. Immunohistochemistry, multiplex immunofluorescence and flow cytometry were performed to analyze cellular composition. Cellular components of NSCLC samples from The Cancer Genome Atlas database were inferred with Microenvironment Cell Populations-counter (MCP-counter) method. Murine NSCLC models were used to dissect underlying mechanisms for relationship between TDLN and TLS maturation.Results While GC+ TLS was associated with better prognosis, GC− TLS was not. TDLN metastasis reduced the prognostic relevance of TLS, and was associated with less GC formation. Primary tumor sites showed reduced B cell infiltration in TDLN-positive patients, and scRNA-seq revealed diminished memory B cell formation in tumor-invaded TDLNs, together with an emphasis on weakened interferon (IFN)-γ response. Murine NSCLC models revealed that IFN-γ signaling is involved in memory B cell differentiation in TDLNs and GC formation in primary tumors.Conclusions Our research emphasizes the influence of TDLN on intratumoral TLS maturation and suggests a role of memory B cells and IFN-γ signaling in this communication