(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce i

Keratin 8/18 Regulation of Cell Stiffness-Extracellular Matrix Interplay through Modulation of Rho-Mediated Actin Cytoskeleton Dynamics

Cell mechanical activity generated from the interplay between the extracellular matrix (ECM) and the actin cytoskeleton is essential for the regulation of cell adhesion, spreading and migration during normal and cancer development. Keratins are the intermediate filament (IF) proteins of epithelial cells, expressed as pairs in a lineage/differentiation manner. Hepatic epithelial cell IFs are made solely of keratins 8/18 (K8/K18), hallmarks of all simple epithelia. Notably, our recent work on these epithelial cells has revealed a key regulatory function for K8/K18 IFs in adhesion/migration, through modulation of integrin interactions with ECM, actin adaptors and signaling molecules at focal adhesions. Here, using K8-knockdown rat H4 hepatoma cells and their K8/K18-containing counterparts seeded on fibronectin-coated substrata of different rigidities, we show that the K8/K18 IF-lacking cells lose their ability to spread and exhibit an altered actin fiber organization, upon seeding on a low-rigidity substratum. We also demonstrate a concomitant reduction in local cell stiffness at focal adhesions generated by fibronectin-coated microbeads attached to the dorsal cell surface. In addition, we find that this K8/K18 IF modulation of cell stiffness and actin fiber organization occurs through RhoA-ROCK signaling. Together, the results uncover a K8/K18 IF contribution to the cell stiffness-ECM rigidity interplay through a modulation of Rho-dependent actin organization and dynamics in simple epithelial cells

Multiplexed fluidic plunger mechanism for the measurement of red blood cell deformability

Red blood cell (RBC) deformability plays an important role in the pathology of various diseases, including malaria, hemoglobinopathies, and micronutrient deficiencies. Specifically, in malaria, the analysis of RBC deformability presents new approaches for detecting infections and for rapidly evaluating the response to drugs by patients. A key challenge, however, is that the infected RBCs represent only a small subpopulation of clinical blood specimens. Therefore effective detection of infection and analysis require methods that can measure a large number of individual RBCs. Traditional technologies for measuring RBC deformability either cannot evaluate single cells to identify diseased subpopulations or do not have sufficient measurement throughput to detect rare subpopulations. In additions, they require delicate experiments, expensive equipment, and skilled technicians. To address these issues, we developed a new microfluidic mechanism, known as the Multiplexed Fluidic Plunger (MFP), to measure RBC deformability using many microscale-tapered constrictions in parallel. The deformability of each RBC is determined by the threshold pressure required to squeeze the cell through a constriction. Our mechanism overcomes a key challenge where the pressure applied to each cell is dependent on the presence or absence of other cells and thereby produces in an inconsistent measurement result. We devised a mechanism to avoid this error and showed that a consistent measurement is obtained independent of constrictions occupancy. Furthermore, the sensitivity of the MFP device is comparable or superior to existing techniques since it can distinguish control and 0.0005% glutaraldehyde-treated RBCs (p<0.005). The high sensitivity of this mechanism, potential low production cost, and simplified experimental procedures facilitates its application in a clinical context such as in areas where malaria is endemic. Therefore, we further determined the deformability profiles of RBCs parasitized by P. falciparum (concentrations ranging from 1-16%). The MFP device was able to detect the deformability of RBCs with a parasitemia as low as 1.8% and can therefore potentially be used to evaluate the parasitemia of infected samples.Applied Science, Faculty ofGraduat

H4ev and shK8b cell stiffness as function of FN-gel rigidity.

<p>Mean bead displacement curves of one data set containing 40 independent bead (400 FU beads) measurements for cells plated on (A) 1.8 kPa gel and (B) 3 kPa gel for both H4ev and shK8b cells, along with the numerical fits (dotted line). (C) The corresponding 3- separate experiment averages of the computed elastic constant k_c, showing a differential stiffness increase from 1.8 kPa to 3 kPa in H4ev versus shK8b cells. *, p<0.05 relative to 1.8 kPa gels.</p

Substratum rigidity differentially affects H4ev and shK8b cell shapes.

<p>(A) Phase contrast images of cells one day after seeding on FN-gels of increasing rigidity or in FN-coated dishes, showing a more shK8b round cell shape under low FN-gel rigidity compared to the H4ev spread shape. For higher gel rigidity, both H4ev and shK8b cells show comparable spread shapes. (B) Measurements of H4ev and shK8b cell areas from the corresponding seeding conditions. N = 60. Bars denote SE. *, p<0.05 for H4ev versus shK8b.</p

ROCK involvement in K8/K18 IF modulation of cell stiffness.

<p>(A) Mean bead displacement curves of one data set containing for 400 FU beads attached to a monolayer of cells seeded on a 3 kPa gel, following addition of Y27632 (1 µM, 30 min). (B) The corresponding cell elastic constant k_c obtained in presence of vehicle (Ctrl) or Y27632. The k_c difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.26) (C) Mean bead displacement curves of one data set containing for 125 FU beads attached to a monolayer of cells seeded in a FN-coated dish, following addition of Y27632. (D) The corresponding cell elastic constant k_c obtained in absence (Ctrl) or presence of Y27632. The k_c difference between H4ev and shK8b cell treated with Y27632 is not statistically significant (p = 0.40). The dotted lines correspond to the numerical fits on Y27632-treated cells, while the solid lines correspond to the numerical fit on control cells data. Bars denote SE. *, p<0.05 relative to controls.</p

H4ev and shK8b cell stiffness as function of bead FN-coating density.

<p>(A) Biomechanical model used to evaluate cell elastic and viscous parameters as function of the optical tweezers elastic constant and initial force. H4ev versus shK8b cells are seeded in FN-coated glass bottom dishes, which constitute a very high rigidity substrate (>3 GPa), and are allowed to form monolayers. Thereafter, beads exhibiting a FN coating of (B) 15, (C) 50, (D) 125 and (E) 400 fluorescent units (FU) are allowed to attach for 1 hr on the monolayers, and their displacements measured with the optical tweezers. Average displacement curves are generated from 40 independent bead measurements. The dotted curves present in each graph correspond to the numerical fit obtained from our mechanical model. The cell (F) elastic constant k_c and (G) viscosity constant γ_c are computed according to the model and the average is obtained from 3 separate experiments. Bars denote SE. *, p<0.05 for H4ev versus shK8b.</p

K8/K18 IF modulation of Rho-mediated actin fiber organization.

<p>(A) Confocal images of fibrillar actin at dorsal and ventral cell surfaces, following addition of Y27632 (1 µM, 1 h) on 3-day monolayers serum-starved overnight, showing that ROCK inhibition disrupts H4ev cell actin fiber organization at the basal and apical surface membranes to a greater extent in H4ev cells than in shK8b cells compare to untreated cells. (B) Western blottings of total Rho (A, B, C) and ROCK-1 showing increased Rho level in shK8b versus H4ev cells; Rho-GTP pull-down assay, showing a higher Rho activation in H4ev versus shK8b cells, despite a lower Rho (A, B, C) content. Confocal images of fibrillar actin at the ventral cell surface, showing cells expressing either a (C) RhoA-GFP or (D) a constitutively active mutant myc-RhoA-V17; RhoA-GFP expression induces the formation of dense actin fibers only in H4ev cells, while myc-RhoA-V17 induces the formation of comparable actin fibers in both H4ev and shK8b cells.</p

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