Microfluidic label-free bioprocessing of human reticulocytes from erythroid culture.

In vitro erythroid cultures from human hematopoietic stem cells produce immature red blood cells (RBCs) called reticulocytes, which are important for RBCs production, and are widely used in scientific studies of malaria pathology, hematological diseases and protein translation. However, in vitro reticulocyte cultures contain expelled cell nuclei and erythroblasts as undesirable by-products and current purification methods such as density gradient centrifugation and fluorescence-activated cell sorting (FACS) are not optimal for integrated bioprocessing and downstream therapeutic applications. Developments in Dean flow fractionation (DFF) and deterministic lateral displacement (DLD) microfluidic sorting methods are ideal alternatives due to label-free size sorting, throughput scalability and low manufacturing cost. DFF sorting of reticulocytes from whole erythroid culture showed a 2.4-fold increase in cell recovery compared to FACS albeit with a lower purity; DLD sorting showed comparable cell recovery and purity with FACS using an inverse-L pillar structure to emphasize size and deformability sorting of reticulocytes. The viability and functional assurance of purified reticulocytes showed conserved cell deformability and supported the propagation of malaria parasites. Collectively, our study on label-free RBCs isolation represents a significant technical advancement towards developing in vitro generated viable human RBCs, opening opportunities for close-loop cell manufacturing, downstream therapeutic and research purposes.

Temperature-Induced Catch-Slip to Slip Bond Transit in Plasmodium falciparum-Infected Erythrocytes.

Plasmodium falciparum malaria-infected red blood cells (IRBCs), or erythrocytes, avoid splenic clearance by adhering to host endothelium. Upregulation of endothelial receptors intercellular adhesion molecule-1 (ICAM-1) and cluster of differentiation 36 (CD36) are associated with severe disease pathology. Most in vitro studies of IRBCs interacting with these molecules were conducted at room temperature. However, as IRBCs are exposed to temperature variations between 37°C (body temperature) and 41°C (febrile temperature) in the host, it is important to understand IRBC-receptor interactions at these physiologically relevant temperatures. Here, we probe IRBC interactions against ICAM-1 and CD36 at 37 and 41°C. Single bond force-clamp spectroscopy is used to determine the bond dissociation rates and hence, unravel the nature of the IRBC-receptor interaction. The association rates are also extracted from a multiple bond flow assay using a cellular stochastic model. Surprisingly, IRBC-ICAM-1 bond transits from a catch-slip bond at 37°C toward a slip bond at 41°C. Moreover, binding affinities of both IRBC-ICAM-1 and IRBC-CD36 decrease as the temperature rises from 37 to 41°C. This study highlights the significance of examining receptor-ligand interactions at physiologically relevant temperatures and reveals biophysical insight into the temperature dependence of P. falciparum malaria cytoadherent bonds.

Nanoscale Architecture of the Cortical Actin Cytoskeleton in Embryonic Stem Cells.

Mechanical cues influence pluripotent stem cell differentiation, but the underlying mechanisms are not well understood. Mouse embryonic stem cells (mESCs) exhibit unusual cytomechanical properties, including low cell stiffness and attenuated responses to substrate rigidity, but the underlying structural basis remains obscure. Using super-resolution microscopy to investigate the actin cytoskeleton in mESCs, we observed that the actin cortex consists of a distinctively sparse and isotropic network. Surprisingly, the architecture and mechanics of the mESC actin cortex appear to be largely myosin II-independent. The network density can be modulated by perturbing Arp2/3 and formin, whereas capping protein (CP) negatively regulates cell stiffness. Transient Arp2/3-containing aster-like structures are implicated in the organization and mechanical homeostasis of the cortical network. By generating a low-density network that physically excludes myosin II, the interplay between Arp2/3, formin, and CP governs the nanoscale architecture of the actin cortex and prescribes the cytomechanical properties of mESCs.

A reference document on Permissible Limits for solvents and buffers during in vitro antimalarial screening.

Antimalarial drug discovery expands on targeted and phenotype-based screening of potential inhibitory molecules to ascertain overall efficacy, phenotypic characteristics and toxicity, prior to exploring pharmacological optimizations. Candidate inhibitors may have varying chemical properties, thereby requiring specific reconstitution conditions to ensure solubility, stability or bioavailability. Hence, a variety of solvents, buffers, detergents and stabilizers become part of antimalarial efficacy assays, all of which, above certain threshold could interfere with parasite viability, invasion or red blood cell properties leading to misinterpretation of the results. Despite their routine use across malaria research laboratories, there is no documentation on non-toxic range for common constituents including DMSO, glycerol, ethanol and methanol. We herein constructed a compatibility reference guide for 14 such chemicals and estimated their Permissible Limit against P. falciparum asexual stages at which viability and replication of parasites are not compromised. We also demonstrate that at the estimated Permissible Limit, red blood cells remain healthy and viable for infection by merozoites. Taken together, this dataset provides a valuable reference tool for the acceptable concentration range for common chemicals during in vitro antimalarial tests.

Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device.

The development of personalized cancer therapy depends on a robust system to monitor the patient's individual response to anticancer treatment. Anticancer drug efficacy has been tested on circulating tumor cells (CTCs) derived from patient blood samples after ex vivo expansion into CTC clusters. Current attempts to culture these primary cancer cells focus on long-term maintenance under growth factor supplements into cell lines, which usually takes >6 months and results in a CTC expansion efficiency of <20%. We recently developed a simple but unique microfluidics-based culture approach that requires minimal preprocessing (∼30 min) and does not require prior enrichment of CTCs or depend on the use of growth factor supplements. The approach capitalizes on co-culture of immune cells from the same patient blood sample within specially designed microwells that promote CTC cluster formation within 2 weeks, with an overall cluster formation success rate of ∼50%. Drug screening is facilitated by the incorporation of a gradient generator for parallel exposure to two or more drugs at various concentrations. Owing to the cost-effectiveness and less-invasive nature of this procedure, routine monitoring of disease progression can be achieved. The described microfluidics system can be operated with a single syringe pump to introduce drug compounds (which takes ∼6 min), followed by incubation of the CTC clusters for 48 h before analysis. In addition to its applications in biomedical research, the rapid readout of our platform will enable clinicians to assess or predict a patient's response to various therapeutic strategies, so as to enable personalized or precision therapy.

Graphene oxide inhibits malaria parasite invasion and delays parasitic growth in vitro.

The interactions between graphene oxide (GO) and various biological entities have been actively investigated in recent years, resulting in numerous potential bioapplications of these nanomaterials. Despite this, the biological interactions between GO and disease-causing protozoan parasites have not been well elucidated and remain relatively unexplored. Here, we investigate the in vitro interactions between GO nanosheets and a particular species of malaria parasites, Plasmodium falciparum (P. falciparum). We hypothesize that GO nanosheets may exhibit antimalarial characteristic via action mechanisms of physical obstruction of P. falciparum parasites as well as nutrient depletion. To ascertain this, we characterize the physical interactions between GO nanosheets, red blood cells (RBCs), and malarial parasites as well as the adsorption of several biomolecules necessary for parasitic survival and growth on GO nanosheets. Subsequent to establishing the origin of this antimalarial behavior of GO nanosheets, their efficiency in inhibiting parasite invasion is evaluated. We observe that GO nanosheets at various tested concentrations significantly inhibit the invasion of malaria parasites into RBCs. Furthermore, GO nanosheets delay parasite progression from the ring to the trophozoite stage. Overall, this study may further shed light on the graphene-parasite interactions and potentially facilitate the development of nanomaterial-based strategies for combating malaria.

Single molecule and multiple bond characterization of catch bond associated cytoadhesion in malaria.

The adhesion of malaria infected red blood cells (iRBCs) to host endothelial receptors in the microvasculature, or cytoadhesion, is associated with severe disease pathology such as multiple organ failure and cerebral malaria. Malaria iRBCs have been shown to bind to several receptors, of which intercellular adhesion molecule 1 (ICAM-1) upregulation in brain microvasculature is the only one correlated to cerebral malaria. We utilize a biophysical approach to study the interactions between iRBCs and ICAM-1. At the single molecule level, force spectroscopy experiments reveal that ICAM-1 forms catch bond interactions with Plasmodium falciparum parasite iRBCs. Flow experiments are subsequently conducted to understand multiple bond behavior. Using a robust model that smoothly transitions between our single and multiple bond results, we conclusively demonstrate that the catch bond behavior persists even under flow conditions. The parameters extracted from these experimental results revealed that the rate of association of iRBC-ICAM-1 bonds are ten times lower than iRBC-CD36 (cluster of differentiation 36), a receptor that shows no upregulation in the brains of cerebral malaria patients. Yet, the dissociation rates are nearly the same for both iRBC-receptor interactions. Thus, our results suggest that ICAM-1 may not be the sole mediator responsible for cytoadhesion in the brain.

YAP Regulates Actin Dynamics through ARHGAP29 and Promotes Metastasis.

Yes-associated protein (YAP) is regulated by mechanical cues via the interaction of the Hippo pathway with cytoskeleton. Previous studies showed that YAP plays a role in regulating the actomyosin network by suppressing Rho GTPase in medaka fish. Here, we identify Rho GTPase activating protein 29 (ARHGAP29) as a transcriptional target of YAP in a human gastric cancer cell line. YAP promotes the expression of ARHGAP29 to suppress the RhoA-LIMK-cofilin pathway, destabilizing F-actin. The overexpression of YAP causes cytoskeletal rearrangement by altering the dynamics of F-actin/G-actin turnover, thus promoting migration. In a mouse model, circulating tumor cells (CTCs) exhibit an increased ARHGAP29 RNA level compared with cells at primary tumor sites, and the metastatic potential of CTCs is positively correlated with ARHGAP29 expression. Moreover, increased ARHGAP29 expression is correlated with shortened survival of human gastric cancer patients. Our study provides a model to understand YAP's contribution to cancer metastasis via regulation of actin dynamics.

Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway.

The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.

Expression dynamics and physiologically relevant functional study of STEVOR in asexual stages of Plasmodium falciparum infection.

The extensive modification of Plasmodium falciparum-infected erythrocytes by variant surface antigens plays a major role in immune evasion and malaria-induced pathology. Here, using high-resolution microscopy, we visualize the spatio-temporal expression dynamics of STEVOR, an important variant surface antigens family, in a stage-dependent manner. We demonstrate that it is exported to the cell surface where protein molecules cluster and preferentially localize in proximity to knobs. Quantitative evidence from our force measurements and microfluidic assays reveal that STEVOR can effectively mediate the formation of stable, robust rosettes under static and physiologically relevant flow conditions. Our results extend previously published studies in P. falciparum and emphasize the role of STEVOR in rosetting, an important contributor to disease pathology.

Liquid biopsy and therapeutic response: Circulating tumor cell cultures for evaluation of anticancer treatment.

The lack of a robust anticancer drug screening system to monitor patients during treatment delays realization of personalized treatment. We demonstrate an efficient approach to evaluate drug response using patient-derived circulating tumor cell (CTC) cultures obtained from liquid biopsy. Custom microfabricated tapered microwells were integrated with microfluidics to allow robust formation of CTC clusters without pre-enrichment and subsequent drug screening in situ. Rapid feedback after 2 weeks promotes immediate intervention upon detection of drug resistance or tolerance. The procedure was clinically validated with blood samples (n = 73) from 55 patients with early-stage, newly diagnosed, locally advanced, or refractory metastatic breast cancer. Twenty-four of these samples were used for drug evaluation. Cluster formation potential correlated inversely with increased drug concentration and therapeutic treatment. This new and robust liquid biopsy technique can potentially evaluate patient prognosis with CTC clusters during treatment and provide a noninvasive and inexpensive assessment that can guide drug discovery development or therapeutic choices for personalized treatment.

High-throughput malaria parasite separation using a viscoelastic fluid for ultrasensitive PCR detection.

A novel microfluidic device for high-throughput particle separation using a viscoelastic fluid, which enables the rapid detection of extremely rare malaria parasites by using PCR analysis, is proposed. Our device consists of two segments: the 1st stage for sheathless pre-alignment and the 2nd stage for separation based on size-dependent viscoelasticity-induced lateral migration. The use of a high-aspect ratio channel and a viscoelastic polymer solution with low viscosity enables high-throughput processing. The device performance was first optimized using synthetic particles. A mixture of 2 and 10 µm particles was focused at the center plane in the 1st stage. The smaller particles, serving as surrogates for malaria parasites, were subsequently separated in the 2nd stage with a recovery rate of ∼96% at 400 µl min(-1). Finally, separation of the malaria parasites from the white blood cells was performed. At 400 µl min(-1), almost all white blood cells were removed and the malaria parasites were separated with a ∼94% recovery rate and ∼99% purity. Although the initial concentration of the malaria parasites was too low to be detected by PCR analysis, WBC depletion and buffer removal increased the parasite concentration sufficiently such that PCR detection was possible.

Investigation of the biomechanical effect of variable stiffness shoe on external knee adduction moment in various dynamic exercises.

BACKGROUND: The growing ageing population and high prevalence of knee osteoarthritis (OA) in athletes across nations have created a strong demand for improved non-invasive therapeutic alternatives for knee OA. The aim of this study is to investigate the effect of the variable stiffness shoe (VSS), a new non-invasive therapeutic approach, on external knee adduction moment (EKAM) in various dynamic exercises. EKAM is believed to have positive correlation with the progression and development of knee OA. METHODS: Thirty young participants (16 male and 14 female; age 22.6 ± 1.9 years) from National University of Singapore were enrolled in this study. The tested activities were walking, running, drop-landing, and lateral hopping. All the dynamic exercises were recorded simultaneously by the 8-camera VICON Motion Systems (Oxford Metric, UK) with a sampling rate of 100 Hz. RESULTS: The results showed that the EKAM was reduced in all the dynamic exercises with the use of VSS. The VSS produced significant reductions in the peak EKAM during walking (4.97%, p = 0.039), running (11.15%, p = 0.011), drop-landing (11.18%, p = 0.038) and lateral hopping (17.34%, p = 0.023) as compared to the control shoe. CONCLUSIONS: The reduction of EKAM with the use of VSS in various dynamic exercises demonstrates its potential in delaying the onset and the progression of knee OA in early stage of knee OA patients.