Centrifugal Microfluidics Traps for Parallel Isolation and Imaging of Single Cells.

Analysis at the single cell level has becoming an increasingly important procedure to diagnose cancer tissue biopsies. These tissue samples are often heterogeneous and consist of 1000-15,000 cells. We study the use of centrifugal microfluidics to isolate single cells into micro chambers. We describe the optimization of our microfluidics flow device, characterize its performance using both polystyrene beads as a cell analogue and MCF-7 breast cancer cells, and discuss potential applications for the device. Our results show rapid isolation of ~2000 single cell aliquots in ~20 min. We were able to occupy 65% of available chambers with singly occupied cancer cells, and observed capture efficiencies as high as 80% using input samples ranging from 2000 to 15,000 cells in 20 min. We believe our device is a valuable research tool that addresses the unmet need for massively parallel single cell level analysis of cell populations.

Microfluidic Immiscible Phase Filtration System for the Isolation of Small Numbers of Cells from Whole Blood.

Isolation of circulating tumor cells (CTCs) has generated clinical and academic interest due to the important role that CTCs play in cancer metastasis and diagnosis. Here, we present a PDMS and glass prototype of a microfluidic device for the immunomagnetic, immiscible phase filtration based capture, and isolation of MCF-7 breast cancer cells, from various sample matrices including PBS-based buffer, blood plasma, and unprocessed whole blood. Following optimization of surface energy of an oil-water interface, microfluidic geometry, and bead-binding kinematics, our microfluidic device achieved 95 ± 4% recovery of target cells from PBS-based buffer with 95% purity, 90 ± 3% recovery of target cells from blood plasma and recovery of ~70 ± 5% from unprocessed whole blood with purity >99% with 1 ml blood samples with 1,000 spiked target cells. From quantitative studies to assess the nonspecific carryover of contaminants from whole blood, we found that our system accomplishes a >175 fold depletion in platelets, >900 fold depletion in erythrocytes, and >1,700 fold depletion in leukocytes with respect to unprocessed whole blood, enabling us to avoid sample pre-processing. In addition, we found that ~95% of the isolated target cells were viable, making them suitable for subsequent molecular and cellular studies. We quantify and propose mechanisms for the carryover of platelet, erythrocyte, and leukocyte contamination in purified samples, rather than relying on sample pre-processing. These results validate the continued study of our platform for extraction of CTCs from patient samples and other rare cell isolation applications. © 2019 International Society for Advancement of Cytometry.

A Microfluidics Workflow for Sample Preparation for Next-Generation DNA Sequencing.

Next-generation sequencing technology requires amplified, short DNA fragments with known end sequences. Samples must undergo processing steps, including extraction and purification of genomic DNA (gDNA), fragmentation, end repair, adapter ligation, and amplification, to prepare a sequencing library. The process of sample preparation requires careful control of temperature and buffer conditions, as well as the stringent removal of contaminants. As a result, library preparation methods are often plagued by sample loss, long protocol times, numerous manual steps, and high cost. We attempt to understand and optimize each step of sample preparation on a microfluidic platform using magnetic bead motion through channels containing immiscible phases. Our platform integrates all steps associated with library preparation with no buffer exchanges and utilizes just 30-60 µL of reagents. Our chip shows a sixfold improvement in yield compared with an affinity spin column when capturing gDNA from samples of ~50 ± 4 MCF-7 cells. Finally, we show whole-genome shotgun sequencing results from 660 pg of human gDNA, in which >93 ± 1% of reads map to a reference genome at or above 99.9% confidence, matching a commercially available sample preparation kit optimized for low-cell-count samples.

A talin mutant that impairs talin-integrin binding in platelets decelerates αIIbß3 activation without pathological bleeding.

Tight regulation of integrin affinity is critical for hemostasis. A final step of integrin activation is talin binding to 2 sites within the integrin ß cytoplasmic domain. Binding of talin to a membrane-distal NPxY sequence facilitates a second, weaker interaction of talin with an integrin membrane-proximal region (MPR) that is critical for integrin activation. To test the functional significance of these distinct interactions on platelet function in vivo, we generated knock-in mice expressing talin1 mutants with impaired capacity to interact with the ß3 integrin MPR (L325R) or NPLY sequence (W359A). Both talin1(L325R) and talin1(W359A) mice were protected from experimental thrombosis. Talin1(L325R) mice, but not talin(W359A) mice, exhibited a severe bleeding phenotype. Activation of αIIbß3 was completely blocked in talin1(L325R) platelets, whereas activation was reduced by approximately 50% in talin1(W359A) platelets. Quantitative biochemical measurements detected talin1(W359A) binding to ß3 integrin, albeit with a 2.9-fold lower affinity than wild-type talin1. The rate of αIIbß3 activation was slower in talin1(W359A) platelets, which consequently delayed aggregation under static conditions and reduced thrombus formation under physiological flow conditions. Together our data indicate that reduction of talin-ß3 integrin binding affinity results in decelerated αIIbß3 integrin activation and protection from arterial thrombosis without pathological bleeding.

Talin and kindlin: the one-two punch in integrin activation.

Abstract Proper cell-cell and cell-matrix contacts mediated by integrin adhesion receptors are important for development, immune response, hemostasis and wound healing. Integrins pass trans-membrane signals bidirectionally through their regulated affinities for extracellular ligands and intracellular signaling molecules. Such bidirectional signaling by integrins is enabled by the conformational changes that are often linked among extracellular, transmembrane and cytoplasmic domains. Here, we review how talin-integrin and kindlin-integrin interactions, in cooperation with talin-lipid and kindlin-lipid interactions, regulate integrin affinities and how the progress in these areas helps us understand integrin-related diseases.

Loss of Purkinje cells in the PKCgamma H101Y transgenic mouse.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal, dominant neurodegenerative disorder caused by mutations in PKCgamma. The objective of this study was to determine effects of PKCgamma H101Y SCA14 mutation on Purkinje cells in the transgenic mouse. Results demonstrated that wild type PKCgamma-like Purkinje cell localization of HA-tagged PKCgamma H101Y mutant proteins, altered morphology and loss of Purkinje cells were observed in the PKCgamma H101Y SCA14 transgenic mouse at four weeks of age. Failure of stereotypical clasping responses in the hind limbs of transgenic mice was also observed. Further, PKCgamma H101Y SCA14 mutation caused lack of total cellular PKCgamma enzyme activity, loss of connexin 57 phosphorylation on serines, and activation of caspase-12 in the PKCgamma H101Y SCA14 transgenic mouse. Results clearly demonstrate a need for PKCgamma control of gap junctions for maintenance of Purkinje cells. This is the first transgenic mouse to our knowledge which models a human SCA14 mutation.

Is telemetry monitoring necessary in low-risk suspected acute chest pain syndromes?

BACKGROUND: Non-ICU telemetry monitoring has proven to be a valuable resource for patients suspected of having an acute myocardial infarction. While a significant number of patients are admitted to these units, the actual incidence of events or interventions is low. OBJECTIVE: To identify a subset of patients in whom telemetry monitoring does not alter management. DESIGN: Prospective observational study. SETTING: Large tertiary care facility. PATIENTS: A total of 414 patients consecutively admitted from the emergency department for suspected acute coronary syndromes were studied. Patients were excluded if they presented with ST-segment elevations, were revascularized on hospital admission, were admitted to a surgical service, were transferred from another floor or unit, or remained in the emergency department for the course of the stay. OUTCOMES: Events were defined as development of myocardial infarction, episodes of chest pain, new or rapid atrial arrhythmias, ventricular arrhythmias, any form of AV nodal block, and asystole. Intervention or change in management was any increase, decrease, or change in medication, cardioversion, electrophysiology study, or transfer to the ICU. RESULTS: Patients who had atypical chest pain and normal ECG findings were significantly less likely to have both intervention and events (4 interventions vs 23 interventions [p < 0.0001], 12 events vs 45 events [p < 0.0001]), compared to those with typical chest pain and abnormal ECG findings. When normal laboratory values were added, only four telemetry events were observed. CONCLUSION: Patients with atypical chest pain and normal ECG findings represent a subset of patients with low risk for life-threatening arrhythmia. Use of telemetry monitoring in this subset of patients should be reevaluated.