Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single ...cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118 900 chambers/cm2) allows the analysis of 200 single cells per run, for a total of 204 000 PCR reactions using a device footprint of 10 cm2. Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 104. We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.
Magnetizing the fuel in inertial confinement fusion relaxes ignition requirements by reducing thermal conductivity and changing the physics of burn product confinement. Diagnosing the level of fuel ...magnetization during burn is critical to understanding target performance in magneto-inertial fusion (MIF) implosions. In pure deuterium fusion plasma, 1.01 MeV tritons are emitted during deuterium-deuterium fusion and can undergo secondary deuterium-tritium reactions before exiting the fuel. Increasing the fuel magnetization elongates the path lengths through the fuel of some of the tritons, enhancing their probability of reaction. Based on this feature, a method to diagnose fuel magnetization using the ratio of overall deuterium-tritium to deuterium-deuterium neutron yields is developed. Analysis of anisotropies in the secondary neutron energy spectra further constrain the measurement. Secondary reactions also are shown to provide an upper bound for the volumetric fuel-pusher mix in MIF. The analysis is applied to recent MIF experiments M. R. Gomez et al., Phys. Rev. Lett. 113, 155003 (2014) on the Z Pulsed Power Facility, indicating that significant magnetic confinement of charged burn products was achieved and suggesting a relatively low-mix environment. Both of these are essential features of future ignition-scale MIF designs.
The hematopoietic system produces a large number of highly specialized cell types that are derived through a hierarchical differentiation process from a common stem cell population. miRNAs are ...critical players in orchestrating this differentiation. Here, we report the development and application of a high-throughput microfluidic real-time quantitative PCR (RT-qPCR) approach for generating global miRNA profiles for 27 phenotypically distinct cell populations isolated from normal adult mouse hematopoietic tissues. A total of 80,000 RT-qPCR assays were used to map the landscape of miRNA expression across the hematopoietic hierarchy, including rare progenitor and stem cell populations. We show that miRNA profiles allow for the direct inference of cell lineage relations and functional similarity. Our analysis reveals a close relatedness of the miRNA expression patterns in multipotent progenitors and stem cells, followed by a major reprogramming upon restriction of differentiation potential to a single lineage. The analysis of miRNA expression in single hematopoietic cells further demonstrates that miRNA expression is very tightly regulated within highly purified populations, underscoring the potential of single-cell miRNA profiling for assessing compartment heterogeneity.
Summary
The initial appearance of subacute cutaneous lupus erythematosus (SCLE) skin lesions in conjunction with Ro/SS‐A autoantibodies occurring as an adverse reaction to hydrochlorothiazide i.e. ...drug‐induced SCLE (DI‐SCLE) was first reported in 1985. Over the past decade an increasing number of drugs in different classes has been implicated as triggers for DI‐SCLE. The management of DI‐SCLE can be especially challenging in patients taking multiple medications capable of triggering DI‐SCLE. Our objectives were to review the published English language literature on DI‐SCLE and use the resulting summary data pool to address questions surrounding drug‐induced SCLE and to develop guidelines that might be of value to clinicians in the diagnosis and management of DI‐SCLE. A systematic review of the Medline/PubMed‐cited literature on DI‐SCLE up to August 2009 was performed. Our data collection and analysis strategies were prospectively designed to answer a series of questions related to the clinical, prognostic and pathogenetic significance of DI‐SCLE. One hundred and seventeen cases of DI‐SCLE were identified and reviewed. White women made up the large majority of cases, and the mean overall age was 58·0 years. Triggering drugs fell into a number of different classes, highlighted by antihypertensives and antifungals. Time intervals (‘incubation period’) between drug exposure and appearance of DI‐SCLE varied greatly and were drug class dependent. Most cases of DI‐SCLE spontaneously resolved within weeks of drug withdrawal. Ro/SS‐A autoantibodies were present in 80% of the cases in which such data were reported and most remained positive after resolution of SCLE skin disease activity. No significant differences in the clinical, histopathological or immunopathological features between DI‐SCLE and idiopathic SCLE were detected. There is now adequate published experience to suggest that DI‐SCLE does not differ clinically, histopathologically or immunologically from idiopathic SCLE. It should be recognized as a distinct clinical constellation differing clinically and immunologically from the classical form of drug‐induced systemic lupus erythematosus.
Abstract Deformations of the atherosclerotic vascular wall induced by the pulsating blood can be estimated using ultrasound strain imaging. Because these deformations indirectly provide information ...on mechanical plaque composition, strain imaging is a promising technique for differentiating between stable and vulnerable atherosclerotic plaques. This paper first explains 1-D radial strain estimation as applied intravascularly in coronary arteries. Next, recent methods for noninvasive vascular strain estimation in a transverse imaging plane are discussed. Finally, a compounding technique that our group recently developed is explained. This technique combines motion estimates of subsequently acquired focused ultrasound images obtained at various insonification angles. However, because the artery moves and deforms during the multi-angle acquisition, errors are introduced when compounding. Recent advances in computational power have enabled plane wave ultrasound acquisition, which allows 100 times faster image acquisition and thus might resolve the motion artifacts. In this paper the performance of strain imaging using plane wave compounding is investigated using simulations of an artery with a vulnerable plaque and experimental data of a two-layered vessel phantom. The results show that plane wave compounding outperforms 0° focused strain imaging. For the simulations, the root mean squared error reduced by 66% and 50% for radial and circumferential strain, respectively. For the experiments, the elastographic signal-to-noise and contrast-to-noise ratio (SNRe and CNRe ) increased with 2.1 dB and 3.7 dB radially, and 5.6 dB and 16.2 dB circumferentially. Because of the high frame rate, the plane wave compounding technique can even be further optimized and extended to 3D in future.
Abstract Aim To investigate the influence of gestational age (GA) on the association between completion of the final examination after 10–11 years of basic education and education, financial ...independence and income in early adulthood. Methods A nationwide register‐based study including individuals born in Denmark between 1990 and 1992. Completion of the examination was evaluated at age 18 and education, financial independence and income at age 28. Results Of 165 683 individuals included, 15.7%, 10.8% and 5.5% had low educational level, were not financially independent and had low income. For those who completed the examination odds ratio (OR) ranged from 1.03 at GA = 32–36 weeks to 1.25 at ≤27 weeks for low education, from 1.10 to 0.91 for not being financial independent and from 1.06 to 1.48 for low income. For those who did not complete the examination, OR increased from 7.55 at ≥37 weeks to 15.03 at ≤27 weeks for low education and from 4.68 to 15.31 for not being financial independent. For low income, OR was 2.57 and independent of GA. Conclusion For individuals who completed the examination, the odds of poor socioeconomic outcomes were independent of GA. Individuals who did not complete the examination had increased odds of poor socioeconomic outcomes, particularly as GA decreased.
Cells have evolved biomolecular networks that process and respond to changing chemical environments. Understanding how complex protein interactions give rise to emergent network properties requires ...time-resolved analysis of cellular response under a large number of genetic perturbations and chemical environments. To date, the lack of technologies for scalable cell analysis under well-controlled and time-varying conditions has made such global studies either impossible or impractical. To address this need, we have developed a high-throughput microfluidic imaging platform for single-cell studies of network response under hundreds of combined genetic perturbations and time-varying stimulant sequences. Our platform combines programmable on-chip mixing and perfusion with high-throughput image acquisition and processing to perform 256 simultaneous time-lapse live-cell imaging experiments. Nonadherent cells are captured in an array of 2,048 microfluidic cell traps to allow for the imaging of eight different genotypes over 12 h and in response to 32 unique sequences of stimulation, generating a total of 49,000 images per run. Using 12 devices, we carried out >3,000 live-cell imaging experiments to investigate the mating pheromone response in Saccharomyces cerevisiae under combined genetic perturbations and changing environmental conditions. Comprehensive analysis of 11 deletion mutants reveals both distinct thresholds for morphological switching and new dynamic phenotypes that are not observed in static conditions. For example, kss1Δ, fus3Δ, msg5Δ, and ptp2Δ mutants exhibit distinctive stimulus-frequency-dependent signaling phenotypes, implicating their role in filtering and network memory. The combination of parallel microfluidic control with high-throughput imaging provides a powerful tool for systems-level studies of single-cell decision making.
We have completed a second-generation linkage map that incorporates sequence-based positional information. This new map, the Rutgers Map v.2, includes 28,121 polymorphic markers with physical ...positions corroborated by recombination-based data. Sex-averaged and sex-specific linkage map distances, along with confidence intervals, have been estimated for all map intervals. In addition, a regression-based smoothed map is provided that facilitates interpolation of positions of unmapped markers on this map. With nearly twice as many markers as our first-generation map, the Rutgers Map continues to be a unique and comprehensive resource for obtaining genetic map information for large sets of polymorphic markers.
This Letter presents results from the first fully integrated experiments testing the magnetized liner inertial fusion concept S. A. Slutz et al., Phys. Plasmas 17, 056303 (2010), in which a cylinder ...of deuterium gas with a preimposed 10 Taxial magnetic field is heated by Z beamlet, a 2.5 kJ, 1 TW laser, and magnetically imploded by a 19 MA, 100 ns rise time current on the Z facility. Despite a predicted peak implosion velocity of only 70 km = s, the fuel reaches a stagnation temperature of approximately 3 keV, with T(e) ≈ T(i), and produces up to 2 x 10(12) thermonuclear deuterium-deuterium neutrons. X-ray emission indicates a hot fuel region with full width at half maximum ranging from 60 to 120 μm over a 6 mm height and lasting approximately 2 ns. Greater than 10(10) secondary deuterium-tritium neutrons were observed, indicating significant fuel magnetization given that the estimated radial areal density of the plasma is only 2 mg = cm(2).
Epidemiological studies have revealed a relationship between early growth restriction and the subsequent development of type 2 diabetes. A rat model of maternal protein restriction has been used to ...investigate the mechanistic basis of this relationship. This model causes insulin resistance and diabetes in adult male offspring. The aim of the present study was to determine the effect of early growth restriction on muscle insulin action in late adult life. Rats were fed either a 20% or an isocaloric 8% protein diet during pregnancy and lactation. Offspring were weaned onto a 20% protein diet and studied at 15 Months of age. Soleus muscle from growth restricted offspring (LP) (of dams fed 8% protein diet) had similar basal glucose uptakes compared with the control group (mothers fed 20% protein diet). Insulin stimulated glucose uptake into control muscle but had no effect on LP muscle. This impaired insulin action was not related to changes in expression of either the insulin receptor or glucose transporter 4 (GLUT 4). However, LP muscle expressed significantly less (P<0.001) of the zeta isoform of protein kinase C (PKC zeta) compared with controls. This PKC isoform has been shown to be positively involved in GLUT 4-mediated glucose transport. Expression levels of other isoforms (betaI, betaII, epsilon, theta) of PKC were similar in both groups. These results suggest that maternal protein restriction leads to muscle insulin resistance. Reduced expression of PKC zeta may contribute to the mechanistic basis of this resistance.
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