A plug and play microfluidic platform for standardized sensitive low-input chromatin immunoprecipitation.

Epigenetic profiling by chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks, and for screening for biomarkers. However, the ChIP-seq protocol for low-input samples is laborious and time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelization. To enable standardized, automated ChIP-seq profiling of low-input samples, we constructed microfluidic PDMS-based plates capable of performing 24 sensitive ChIP reactions within 30 min of hands-on time and 4.5 h of machine-running time. These disposable plates can be conveniently loaded into a widely available controller for pneumatics and thermocycling. In light of the plug and play (PnP) ChIP plates and workflow, we named our procedure PnP-ChIP-seq. We show high-quality ChIP-seq on hundreds to a few thousand of cells for all six post-translational histone modifications that are included in the International Human Epigenome Consortium set of reference epigenomes. PnP-ChIP-seq robustly detects epigenetic differences on promoters and enhancers between naive and more primed mouse embryonic stem cells (mESCs). Furthermore, we used our platform to generate epigenetic profiles of rare subpopulations of mESCs that resemble the two-cell stage of embryonic development. PnP-ChIP-seq allows nonexpert laboratories worldwide to conveniently run robust, standardized ChIP-seq, whereas its high throughput, consistency, and sensitivity pave the way toward large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.

Miniaturised interaction proteomics on a microfluidic platform with ultra-low input requirements.

Essentially all cellular processes are orchestrated by protein-protein interactions (PPIs). In recent years, affinity purification coupled to mass spectrometry (AP-MS) has been the preferred method to identify cellular PPIs. Here we present a microfluidic-based AP-MS workflow, called on-chip AP-MS, to identify PPIs using minute amounts of input material. By using this automated platform we purify the human Cohesin, CCC and Mediator complexes from as little as 4 micrograms of input lysate, representing a 50─100-fold downscaling compared to regular microcentrifuge tube-based protocols. We show that our platform can be used to affinity purify tagged baits as well as native cellular proteins and their interaction partners. As such, our method holds great promise for future biological and clinical AP-MS applications in which sample amounts are limited.

Quantifying crater production and regolith overturn on the Moon with temporal imaging.

Random bombardment by comets, asteroids and associated fragments form and alter the lunar regolith and other rocky surfaces. The accumulation of impact craters over time is of fundamental use in evaluating the relative ages of geologic units. Crater counts and radiometric ages from returned samples provide constraints with which to derive absolute model ages for unsampled units on the Moon and other Solar System objects. However, although studies of existing craters and returned samples offer insight into the process of crater formation and the past cratering rate, questions still remain about the present rate of crater production, the effect of early-stage jetting during impacts and the influence that distal ejecta have on the regolith. Here we use Lunar Reconnaissance Orbiter Camera (LROC) Narrow Angle Camera (NAC) temporal ('before and after') image pairs to quantify the contemporary rate of crater production on the Moon, to reveal previously unknown details of impact-induced jetting, and to identify a secondary impact process that is rapidly churning the regolith. From this temporal dataset, we detected 222 new impact craters and found 33 per cent more craters (with diameters of at least ten metres) than predicted by the standard Neukum production and chronology functions for the Moon. We identified broad reflectance zones associated with the new craters that we interpret as evidence of a surface-bound jetting process. We also observe a secondary cratering process that we estimate churns the top two centimetres of regolith on a timescale of 81,000 years-more than a hundred times faster than previous models estimated from meteoritic impacts (ten million years).

A Combined Fabrication and Instrumentation Platform for Sample Preparation.

While potentially powerful, access to molecular diagnostics is substantially limited in the developing world. Here we present an approach to reduced cost molecular diagnostic instrumentation that has the potential to empower developing world communities by reducing costs through streamlining the sample preparation process. In addition, this instrument is capable of producing its own consumable devices on demand, reducing reliance on assay suppliers. Furthermore, this instrument is designed with an "open" architecture, allowing users to visually observe the assay process and make modifications as necessary (as opposed to traditional "black box" systems). This open environment enables integration of microfluidic fabrication and viral RNA purification onto an easy-to-use modular system via the use of interchangeable trays. Here we employ this system to develop a protocol to fabricate microfluidic devices and then use these devices to isolate viral RNA from serum for the measurement of human immunodeficiency virus (HIV) viral load. Results obtained from this method show significantly reduced error compared with similar nonautomated sample preparation processes.

Nucleic acid sample preparation using spontaneous biphasic plug flow.

Nucleic acid (NA) extraction and purification has become a common technique in both research and clinical laboratories. Current methods require repetitive wash steps with a pipet that are laborious and time-consuming, making the procedure inefficient for clinical settings. We present here a simple technique that relies on spontaneous biphasic plug flow inside a capillary to achieve sample preparation. By filling the sample with oil, aqueous contaminants were displaced from the captured NA without pipetting wash buffers or use of external force and equipment. mRNA from mammalian cell culture was purified, and polymerase chain reaction (PCR) amplification showed similar threshold cycle values as those obtained from a commercially available kit. Human immunodeficiency virus (HIV) viral-like particles were spiked into serum and a 5-fold increase in viral RNA extraction yield was achieved compared to the conventional wash method. In addition, viral RNA was successfully purified from human whole blood, and a limit of detection of approximately 14 copies of RNA extracted per sample was determined. The results demonstrate the utility of the current technique for nucleic acid purification for clinical purposes, and the overall approach provides a potential method to implement nucleic acid testing in low-resource settings.

Regulating oxygen levels in a microfluidic device.

Microfluidic devices made from poly(dimethylsiloxane) (PDMS) are gas permeable and have been used to provide accurate on-chip oxygen regulation. However, pervaporation in PDMS devices can rapidly lead to dramatic changes in solution osmotic pressure. In the present study, we demonstrate a new method for on-chip oxygen control using pre-equilibrated aqueous solutions in gas-control channels to regulate the oxygen content in stagnant microfluidic test chambers. An off-chip gas exchanger is used to equilibrate each control solution prior to entering the chip. Using this strategy, problems due to pervaporation are considerably reduced. An integrated PDMS-based oxygen sensor allows accurate real-time measurements of the oxygen within the microfluidic chamber. The measurements were found to be consistent with predictions from finite-element modeling.

EPOXI at comet Hartley 2.

Understanding how comets work--what drives their activity--is crucial to the use of comets in studying the early solar system. EPOXI (Extrasolar Planet Observation and Deep Impact Extended Investigation) flew past comet 103P/Hartley 2, one with an unusually small but very active nucleus, taking both images and spectra. Unlike large, relatively inactive nuclei, this nucleus is outgassing primarily because of CO(2), which drags chunks of ice out of the nucleus. It also shows substantial differences in the relative abundance of volatiles from various parts of the nucleus.

Evidence of recent thrust faulting on the Moon revealed by the Lunar Reconnaissance Orbiter Camera.

Lunar Reconnaissance Orbiter Camera images reveal previously undetected lobate thrust-fault scarps and associated meter-scale secondary tectonic landforms that include narrow extensional troughs or graben, splay faults, and multiple low-relief terraces. Lobate scarps are among the youngest landforms on the Moon, based on their generally crisp appearance, lack of superposed large-diameter impact craters, and the existence of crosscut small-diameter impact craters. Identification of previously known scarps was limited to high-resolution Apollo Panoramic Camera images confined to the equatorial zone. Fourteen lobate scarps were identified, seven of which are at latitudes greater than +/-60 degrees, indicating that the thrust faults are globally distributed. This detection, coupled with the very young apparent age of the faults, suggests global late-stage contraction of the Moon.

A revised approach to patient selection for cardiac resynchronization treatment using multiple asynchrony parameters in Narrow- and Wide-QRS cardiomyopathy causes cardiac reverse remodelling: a single centre non-randomized prospective study.

AIMS: Conflicting data exists on the benefit of cardiac resynchronization treatment (CRT) in patients with narrow QRS (Narrow-QRS) cardiomyopathy (CMP). We determined the effect of CRT in patients with CMP and mechanical asynchrony based on a comprehensive assessment by multiple echocardiographic criteria. METHODS AND RESULTS: Ninety patients, 65 +/- 16 years, 32 with Narrow-QRS <120 ms and 58 with wide QRS >or=120 ms (Wide-QRS) CMP who met criteria for significant mechanical asynchrony by 15 criteria before CRT were studied. Responders were patients in whom end-systolic volume (ESV) reduced by >or=15% post-CRT. There was no difference in the response to CRT in the Narrow-QRS (ESV 132 +/- 60 to 120 +/- 60 mL, P = 0.02) or Wide-QRS (123 +/- 54 to 102 +/- 50 mL, P < 0.01) groups at 1 +/- 2 month follow-up. A difference of >or=40% in time to peak contraction in a cardiac cycle on tissue velocity imaging between the earliest and the most delayed segment had the best area under curve for response to CRT, 0.71 (0.55-0.85), P = 0.02. Using logistic regression model, delay in mid-posterolateral segment of >or=20% in a cardiac cycle compared with remaining 10 segments was the only predictor of response to CRT in the overall study population. Conclusion In patients with CMP and mechanical asynchrony by multiple criteria, response to CRT in Narrow-QRS group is similar to those with Wide-QRS. Greater than or equal to 40% delay in systolic contraction between 12 left ventricular (LV) segments or >or=20% delay of posterolateral segment to other LV segments predicted CRT response.

Iapetus: unique surface properties and a global color dichotomy from Cassini imaging.

Since 2004, Saturn's moon Iapetus has been observed repeatedly with the Imaging Science Subsystem of the Cassini spacecraft. The images show numerous impact craters down to the resolution limit of approximately 10 meters per pixel. Small, bright craters within the dark hemisphere indicate a dark blanket thickness on the order of meters or less. Dark, equator-facing and bright, poleward-facing crater walls suggest temperature-driven water-ice sublimation as the process responsible for local albedo patterns. Imaging data also reveal a global color dichotomy, wherein both dark and bright materials on the leading side have a substantially redder color than the respective trailing-side materials. This global pattern indicates an exogenic origin for the redder leading-side parts and suggests that the global color dichotomy initiated the thermal formation of the global albedo dichotomy.

A noninvasive thin film sensor for monitoring oxygen tension during in vitro cell culture.

Oxygen tension in mammalian cell culture can profoundly affect cellular differentiation, viability, and proliferation. However, precise measurement of dissolved oxygen in real time remains difficult. We report a new noninvasive sensor that can accurately measure oxygen concentration during cell culture while being compatible with live-cell imaging techniques such as fluorescence and phase contrast microscopy. The sensor is prepared by integrating the porphyrin dye, Pt(II) meso-tetrakis(pentafluorophenyl)porphine (PtTFPP) into polydimethylsiloxane (PDMS) thin films. Response of the sensor in the presence of oxygen can be characterized by the linear Stern-Volmer relationship with high sensitivity (K(SV) = 584 +/- 71 atm(-1)). A multilayer sensor design, created by sandwiching the PtTFPP-PDMS with a layer of Teflon AF followed by a second PDMS layer, effectively mitigates against dye cytotoxicity while providing a substrate for cell attachment. Using this sensor, changes in oxygen tension could be monitored in real-time as attached cells proliferated. The oxygen tension was found to decrease due to oxygen consumption by the cells, and the data could be analyzed using Fick's law to obtain the per-cell oxygen consumption rate. This sensor is likely to enable new studies on the effects of dissolved oxygen on cellular behavior.

The equatorial ridges of Pan and Atlas: terminal accretionary ornaments?

In the outer regions of Saturn's main rings, strong tidal forces balance gravitational accretion processes. Thus, unusual phenomena may be expected there. The Cassini spacecraft has recently revealed the strange "flying saucer" shape of two small satellites, Pan and Atlas, located in this region, showing prominent equatorial ridges. The accretion of ring particles onto the equatorial surfaces of already-formed bodies embedded in the rings may explain the formation of the ridges. This ridge formation process is in good agreement with detailed Cassini images showing differences between rough polar and smooth equatorial terrains. We propose that Pan and Atlas ridges are kilometers-thick "ring-particle piles" formed after the satellites themselves and after the flattening of the rings but before the complete depletion of ring material from their surroundings.

Amalthea's density is less than that of water.

Radio Doppler data from the Galileo spacecraft's encounter with Amalthea, one of Jupiter's small inner moons, on 5 November 2002 yield a mass of (2.08 +/- 0.15) x 10(18) kilograms. Images of Amalthea from two Voyager spacecraft in 1979 and Galileo imaging between November 1996 and June 1997 yield a volume of (2.43 +/- 0.22) x 10(6) cubic kilometers. The satellite thus has a density of 857 +/- 99 kilograms per cubic meter. We suggest that Amalthea is porous and composed of water ice, as well as rocky material, and thus formed in a cold region of the solar system, possibly not at its present location near Jupiter.