Label-free DNA sequencing using Millikan detection.

A label-free method for DNA sequencing based on the principle of the Millikan oil drop experiment was developed. This sequencing-by-synthesis approach sensed increases in bead charge as nucleotides were added by a polymerase to DNA templates attached to beads. The balance between an electrical force, which was dependent on the number of nucleotide charges on a bead, and opposing hydrodynamic drag and restoring tether forces resulted in a bead velocity that was a function of the number of nucleotides attached to the bead. The velocity of beads tethered via a polymer to a microfluidic channel and subjected to an oscillating electric field was measured using dark-field microscopy and used to determine how many nucleotides were incorporated during each sequencing-by-synthesis cycle. Increases in bead velocity of approximately 1% were reliably detected during DNA polymerization, allowing for sequencing of short DNA templates. The method could lead to a low-cost, high-throughput sequencing platform that could enable routine sequencing in medical applications.

Single-molecule enzymology based on the principle of the Millikan oil drop experiment.

The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity.

VA-ECMO-Facilitated Clot Debulking and PFO Closure in a Patient With Recurrent Pulmonary Thromboses.

A 27-year-old female with stage IIIc cervical cancer presented with dyspnea and stroke symptoms. Work-up revealed bilateral pulmonary embolisms, acute/subacute strokes, and a patent foramen ovale. After multidisciplinary team discussion, the patient underwent patent foramen ovale closure, complicated by cardiogenic shock requiring venoarterial extracorporeal membrane oxygenation. She successfully underwent pulmonary thromboendarterectomy, extracorporeal membrane oxygenation decannulation, and hospital discharge.

Nucleic acid amplification of individual molecules in a microfluidic device.

A microfluidic device was developed that enabled rapid polymerase chain reaction (PCR) analysis of individual DNA molecules. The device combined a means for accessing samples serially from a microtiter plate, channels for assembling eight parallel PCR reactions, and integrated resistive heaters for rapid thermocycling (>5 degrees C/s heating, >7 degrees C/s cooling) of samples as they flowed continuously through PCR channels. Amplification was monitored by fluorescence detection of Taqman probes. The long, narrow channels (10 microm x 180 microm x 40 mm) allowed sufficient separation between neighboring DNA templates to enable amplification of discreet DNA molecules. The functionality of the device was demonstrated by reproducibly amplifying a 2D6.6 CYP450 template and distinguishing between wild-type and mutant sequences using Taqman probes. A comparison of the rate of individual amplification events to the expected Poisson distribution confirmed that the device could reliably analyze individual DNA molecules. This work establishes the feasibility of rapid, single-molecule interrogation of nucleic acids.

Comparison of on-chip and off-chip microfluidic kinase assay formats.

Kinases represent an important class of targets for pharmaceutical drug development. Microfluidic devices capable of running kinase assays with either an on-chip or an off-chip enzymatic reaction have been developed. For the on-chip assay, reagent addition, mixing, enzymatic reaction, and electrophoretic separation and detection of substrate and product all take place in the channels of the microfluidic chip. For the off-chip assay, the reaction takes place in a microtiter plate, whereas the electrophoretic separation and detection of substrate and product take place in the channels of the chip. To probe differences between the on-chip and off-chip assays, a panel of commercially available kinase inhibitors was assayed at 10 microM against cyclic AMP-dependent protein kinase A, glycogen synthase kinase 3beta, mitogen- and stress-activated protein kinase, and Akt1 using both the off-chip and on-chip assays. Good correlation was observed between inhibition measured by the two methods, with most of the differences in measured inhibition being attributed to compound solubility and enzyme concentration effects. Microfluidic devices represent an attractive platform for kinase assays due to high data quality and the possibility of on-chip assay integration, leading to reagent and labor savings.

Lesiones penetrantes en el cerebro por cuerpos extraños intracraneales. Presentación de tres casos / Penetrating injuries in the brain by intracranial foreign bodies. Presentation of three cases

Fundamento: Las lesiones penetrantes cerebrales causadas por objetos extraños, incluidos proyectiles son comúnmente vistas en situaciones de guerra. Las lesiones no causadas por proyectiles son raras en la práctica neuroquirúrgica en la vida civil en tiempo de paz. Objetivo: Ilustrar formas clínicas y la evaluación de lesiones penetrantes del cerebro no causadas por municiones de armas de fuego en tiempo de paz, a través de la presentación de tres casos. Presentación de casos: Presentamos tres pacientes con cuerpos extraños intracraneales, el primero que durante intento suicida se introdujo a través de agujero de trépano objeto metálico (alambre de cobre); el segundo caso, paciente que al sufrir trauma de cráneo se le realizó tomografía axial computarizada de cráneo y se detectó la presencia de hematoma subdural agudo y cuerpo extraño intracraneal (pedazo de alambre); el tercer caso se trata de recluso que durante una riña sufrió herida con penetración de un cuerpo extraño intracraneal (clavo). Conclusiones: La intervención quirúrgica de estos pacientes de manera urgente y su estado neurológico al entrar al quirófano repercutió de manera decisiva en su evolución.

Background: Penetrating brain injuries due to foreign objects including gunshot wounds are commonly seen in war times. Injuries not caused by gunshot are rare in neurosurgical practice in civil lifetime in peace times. Objective: To illustrate the clinical forms and the evaluation of penetrating brain injuries not caused by ammunitions of firearms in peacetime, through the presentation of three cases. Cases presentation: We report three patients with intracranial foreign bodies, the first during suicide attempt was introduced via burr hole metal object (copper wire); the second case, the patient suffered head injury and underwent computed tomography of the skull and the presence of acute subdural hematoma and intracranial foreign body (piece of wire) was detected; the third case involves prisoner who suffered injury during a fight with intracranial penetration of a foreign body (nail). Conclusions: Urgently surgical intervention in these patients and their neurological status entering the operating room impacted decisively in their evolution.

Agonist-induced calcium response in single human platelets assayed in a microfluidic device.

To facilitate drug discovery directed toward platelet-specific targets, we developed a platelet isolation and fluorophore-loading method that yields functionally responsive platelets in which we were able to detect agonist-induced calcium flux using a microfluidics-based screening platform. The platelet preparation protocol was designed to minimize preparation-induced platelet activation and to optimize signal strength. Measurement of platelet activation, as monitored by ratiometric determination of agonist-induced calcium flux in fluor-loaded human platelets, was optimized in a macrosample cuvette format in preparation for detection in a microfluidic chip-based assay. For the microfluidic device used in these studies, a cell density of 1 to 2 x 10(6) platelets per milliliter and a nominal flow rate of 5 to 10 nl per second provided optimal event resolution of 5 to 20 platelets traversing the detection volume per unit time. Platelets responded in a dose-dependent manner to adenosine diphosphate and protease-activating peptide (PAR) 1 thrombin receptor-activating peptide (TRAP). The work presented here constitutes proof-of-principle experiments demonstrating the enabling application of a microfluidic device to conduct high-throughput signaling studies and drug discovery screening against human platelet targets.