Rapid quantification of microRNAs in plasma using a fast real time PCR system

The ability to rapidly detect circulating small RNAs, in particular microRNAs (miRNAs), would further increase their already established potential as biomarkers in a range of conditions. One rate-limiting factor is the time taken to perform quantitative real time PCR amplification. We therefore evaluated the ability of a novel thermal cycler to perform this step in less than 10 minutes. Quantitative PCR was performed on an xxpress® thermal cycler (BJS Biotechnologies, Perivale, UK), which employs a resistive heating system and forced air cooling to achieve thermal ramp rates of 10 °C/s, and a conventional peltier-controlled LightCycler 480 system (Roche, Basel, Switzerland) ramping at 4.8 °C/s. The threshold cycle (Ct) for detection of 18S rDNA from a standard genomic DNA sample was significantly more variable across the block (F-test, p=2.4x10-25) for the xxpress (20.01±0.47SD) than the LightCycler (19.87±0.04SD). RNA was extracted from human plasma, reverse transcribed and a panel of miRNAs amplified and detected using SYBR green (Kapa Biosystems, Wilmington, Ma, USA). The sensitivity of both systems was broadly comparable and both detected a panel of miRNAs reliably and indicated similar relative abundances. The xxpress thermal cycler facilitates rapid qPCR detection of small RNAs and brings point-of care diagnostics based upon circulating miRNAs a step closer to reality.

Real-time PCR demonstrates high prevalence of Schistosoma japonicum in the Philippines:implications for surveillance and control

BACKGROUND: The Philippines has a population of approximately 103 million people, of which 6.7 million live in schistosomiasis-endemic areas with 1.8 million people being at risk of infection with Schistosoma japonicum. Although the country-wide prevalence of schistosomiasis japonica in the Philippines is relatively low, the prevalence of schistosomiasis can be high, approaching 65% in some endemic areas. Of the currently available microscopy-based diagnostic techniques for detecting schistosome infections in the Philippines and elsewhere, most exhibit varying diagnostic performances, with the Kato-Katz (KK) method having particularly poor sensitivity for detecting low intensity infections. This suggests that the actual prevalence of schistosomiasis japonica may be much higher than previous reports have indicated.

METHODOLOGY/PRINCIPAL FINDINGS: Six barangay (villages) were selected to determine the prevalence of S. japonicum in humans in the municipality of Palapag, Northern Samar. Fecal samples were collected from 560 humans and examined by the KK method and a validated real-time PCR (qPCR) assay. A high S. japonicum prevalence (90.2%) was revealed using qPCR whereas the KK method indicated a lower prevalence (22.9%). The geometric mean eggs per gram (GMEPG) determined by the qPCR was 36.5 and 11.5 by the KK. These results, particularly those obtained by the qPCR, indicate that the prevalence of schistosomiasis in this region of the Philippines is much higher than historically reported.

CONCLUSIONS/SIGNIFICANCE: Despite being more expensive, qPCR can complement the KK procedure, particularly for surveillance and monitoring of areas where extensive schistosomiasis control has led to low prevalence and intensity infections and where schistosomiasis elimination is on the horizon, as for example in southern China.

Surface alloying and mixing at the Mn/Fe(001) interface: Real-time photoelectron spectroscopy and modified embedded atom simulations

Structural and magnetic properties of thin Mn films on the Fe(001) surface have been investigated by a combination of photoelectron spectroscopy and computer simulation in the temperature range 300 Kless than or equal toTless than or equal to750 K. Room-temperature as deposited Mn overlayers are found to be ferromagnetic up to 2.5-monolayer (ML) coverage, with a magnetic moment parallel to that of the iron substrate. The Mn atomic moment decreases with increasing coverage, and thicker samples (4-ML and 4.5-ML coverage) are antiferromagnetic. Photoemission measurements performed while the system temperature is rising at constant rate (dT/dtsimilar to0.5 K/s) detect the first signs of Mn-Fe interdiffusion at T=450 K, and reveal a broad temperature range (610 Kless than or equal toTless than or equal to680 K) in which the interface appears to be stable. Interdiffusion resumes at Tgreater than or equal to680 K. Molecular dynamics and Monte Carlo simulations allow us to attribute the stability plateau at 610 Kless than or equal toTless than or equal to680 K to the formation of a single-layer MnFe surface alloy with a 2x2 unit cell and a checkerboard distribution of Mn and Fe atoms. X-ray-absorption spectroscopy and analysis of the dichroic signal show that the alloy has a ferromagnetic spin structure, collinear with that of the substrate. The magnetic moments of Mn and Fe atoms in the alloy are estimated to be 0.8mu(B) and 1.1mu(B), respectively.

Design Methodology for Real-Time FPGA-Based Sound Synthesis

Explicit finite difference (FD) schemes can realise highly realistic physical models of musical instruments but are computationally complex. A design methodology is presented for the creation of FPGA-based micro-architectures for FD schemes which can be applied to a range of applications with varying computational requirements, excitation and output patterns and boundary conditions. It has been applied to membrane and plate-based sound producing models, resulting in faster than real-time performance on a Xilinx XC2VP50 device which is 10 to 35 times faster than general purpose and DSP processors. The models have developed in such a way to allow a wide range of interaction (by a musician) thereby leading to the possibility of creating a highly realistic digital musical instrument.

A prospective clinical trial of a real-time polymerase chain reaction assay for the diagnosis of candidemia in nonneutropenic, critically ill adults

Background. Invasive Candida infection among nonneutropenic, critically ill adults is a clinical problem that has received increasing attention in recent years. Poor performance of extant diagnostic modalities has promoted risk-based, preemptive prescribing in view of the poor outcomes associated with inadequate or delayed antifungal therapy; this risks unnecessary overtreatment. A rapid, reliable diagnostic test could have a substantial impact on therapeutic practice in this patient population.

Methods. Three TaqMan-based real-time polymerase chain reaction assays were developed that are capable of detecting the main medically important Candida species, categorized according to the likelihood of fluconazole susceptibility. Assay 1 detected Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida dubliniensis. Assays 2 and 3 detected Candida glabrata and Candida krusei, respectively. The clinical performance of these assays, applied to serum, was evaluated in a prospective trial of nonneutropenic adults in a single intensive care unit.

Results. In all, 527 specimens were obtained from 157 participants. All 3 assays were run in parallel for each specimen; they could be completed within 1 working day. Of these, 23 specimens were obtained from 23 participants categorized as having proven Candida infection at the time of sampling. If a single episode of Candida famata candidemia was excluded, the estimated clinical sensitivity, specificity, and positive and negative predictive values of the assays in this trial were 90.9%, 100%, 100% and 99.8%, respectively.

Conclusions. These data suggest that the described assays perform well in this population for enhancing the diagnosis of candidemia. The extent to which they may affect clinical outcomes, prescribing practice, and cost-effectiveness of care remains to be ascertained.

A Novel Packet Marking Function for Real-Time Interactive MPEG-4 Video Applications in a Differentiated Services Network

The future convergence of voice, video and data applications on the Internet requires that next generation technology provides bandwidth and delay guarantees. Current technology trends are moving towards scalable aggregate-based systems where applications are grouped together and guarantees are provided at the aggregate level only. This solution alone is not enough for interactive video applications with sub-second delay bounds. This paper introduces a novel packet marking scheme that controls the end-to-end delay of an individual flow as it traverses a network enabled to supply aggregate- granularity Quality of Service (QoS). IPv6 Hop-by-Hop extension header fields are used to track the packet delay encountered at each network node and autonomous decisions are made on the best queuing strategy to employ. The results of network simulations are presented and it is shown that when the proposed mechanism is employed the requested delay bound is met with a 20% reduction in resource reservation and no packet loss in the network.

Host-based P2P flow identification and use in real-time

Data identification is a key task for any Internet Service Provider (ISP) or network administrator. As port fluctuation and encryption become more common in P2P traffic wishing to avoid identification, new strategies must be developed to detect and classify such flows. This paper introduces a new method of separating P2P and standard web traffic that can be applied as part of a data mining process, based on the activity of the hosts on the network. Unlike other research, our method is aimed at classifying individual flows rather than just identifying P2P hosts or ports. Heuristics are analysed and a classification system proposed. The accuracy of the system is then tested using real network traffic from a core internet router showing over 99% accuracy in some cases. We expand on this proposed strategy to investigate its application to real-time, early classification problems. New proposals are made and the results of real-time experiments compared to those obtained in the data mining research. To the best of our knowledge this is the first research to use host based flow identification to determine a flows application within the early stages of the connection.

Real-time imaging of novel spatial and temporal responses to photodynamic stress.

Cells subjected to various forms of stress have been shown to induce bystander responses in nontargeted cells, thus extending the stress response to a larger population. However, the mechanism(s) of bystander responses remains to be clearly identified, particularly for photodynamic stress. Oxidative stress and cell viability were studied on the spatial and temporal levels after photodynamic targeting of a subpopulation of EMT6 murine mammary cancer cells in a multiwell plate by computerized time-lapse fluorescence microscopy. In the targeted population a dose-dependent loss of cell viability was observed in accordance with increased oxidative stress. This was accompanied by increased oxidative stress in bystander populations but on different time scales, reaching a maximum more rapidly in targeted cells. Treatment with extracellular catalase, or the NADPH oxidase inhibitor diphenyleneiodinium, decreased production of reactive oxygen species (ROS) in both populations. These effects are ascribed to photodynamic activation of NADPH-oxidase in the targeted cells, resulting in a rapid burst of ROS formation with hydrogen peroxide acting as the signaling molecule responsible for initiation of these photodynamic bystander responses. The consequences of increased oxidative stress in bystander cells should be considered in the overall framework of photodynamic stress.

Real time monitoring of the polymerisation of PMMA bone cement using Raman spectroscopy

In this investigation Raman spectroscopy was shown to be a method that could be used to monitor the polymerisation of PMMA bone cement. Presently there is no objective method that orthopaedic surgeons can use to quantify the curing process of cement during surgery. Raman spectroscopy is a non-invasive, non-destructive technique that could offer such an option. Two commercially available bone cements (Palacos® R and SmartSet® HV) and different storage conditions (4 and 22°C) were used to validate the technique. Raman spectroscopy was found to be repeatable across all conditions with the completion of the polymerisation process particularly easy to establish. All tests were benchmarked against current temperature monitoring methods outlined in ISO and ASTM standards. There was found to be close agreement with the standard methods and the Raman spectroscopy used in this study.