Pre Amplification Research Articles

In vitro comparative study of wall portbased high-frequency chest wall oscillation device and internal air-pulse generator device.

Owing to environmental and disease issues, the use of high-frequency chest wall oscillation (HFCWO) devices in hospitals is consistently increasing. This study proposes a cost-effective actuator-less HFCWO device that utilizes an external wall port utility in hospitals to generate the positive and negative pneumatic pressures required for HFCWO treatment instead of an embedded mechanical actuator. The manufactured prototype with the no-amplification (NO-AMP) setting contained an electric pressure regulator to enable intensity level adjustment and two solenoid valves to enable vibration frequency adjustment, whereas the prototype with the pre-amplification (PRE-AMP) setting contained an additional air reservoir and an air-pressure booster. The prototype device was tuned to output average local maximum values in the pressure waveform similar to a commercial VEST-205 device at an 8-12 Hz frequency and 2-4 pressure intensity levels. In vitro comparative experiments demonstrated that the prototype device showed similar local maximum pressures to those of the VEST-205 (mean absolute pressure difference, <3 mmH2O); in contrast, the proposed device showed significantly higher local minimum pressures than those of the VEST-205 (mean absolute pressure difference, >8 mmH2O). Additionally, the driving sound of the proposed device was 17.0-17.8 dB higher than that of VEST-205. We conclude that the proposed device has the potential to substitute for conventional HFCWO devices under the limited but most frequently used operating conditions, although more detailed modifications are necessary in future studies to improve its performance and clinical usability.

Modeling amplified arbitrary filtered microwave photonic links and systems.

Microwave photonic (MWP) links and systems will have more losses as their complexities increase and there will be a need for incorporating optical amplification. Here, we report results of an analytical model developed for amplified arbitrary filtered MWP systems that provides the expressions of the main figures of merit for intensity modulation direct detection. It contemplates the cases of power, intermediate and pre amplification. The model is applied to a long MWP link and then it is evaluated in a MWP reconfigurable filter implemented by means of a programmable waveguide mesh photonic integrated circuit.

Highly Sensitive and Specific Detection of SRY Gene for Non–invasive Prenatal Diagnosis

Abstract Through detecting SRY gene of cell–free fetal DNA (cffDNA) in maternal peripheral blood, the sex of fetuses can be determined, risks of sex–linked genetic disorders can be assessed and birth rate of sick fetuses can be decreased. In this study, a method of real–time polymerase chain reaction (PCR) coupled with invader assay, which was highly sensitive, highly specific and non–contaminated with closed tube detection, was established to detect SRY gene. The optimal reaction conditions were determined to be 250 nM of detection probes, 7.5 U of FEN1 enzyme, 0.5 U of Taq polymerase and 67 °C of annealing temperature in pre–amplification. Finally, it was demonstrated that the simulated samples as low as 4‰ (4 copies μL −1 ) could be detected and two clinical samples with the gestation age of 9 and 10 weeks respectively were also successfully detected. The experimental results indicated that this method could be used to detect SRY gene of cffDNA in maternal peripheral blood, providing an effective technique for clinical non–invasive prenatal diagnosis based on SRY gene.

Abstract AP04: THE MOLECULAR DETECTION OF CIRCULATING TUMOR CELLS IN THE BLOOD OF OVARIAN CANCER PATIENTS

Abstract PURPOSE OF THE STUDY: The detection of circulating tumor cells (CTCs) in the blood of cancer patients has raised considerable interest over the last decade as a promising biomarker of minimal residual disease in various solid tumors. Although hematogenous spread has not been deemed a major issue for ovarian cancer, there are several studies demonstrating the presence and prognostic impact of CTCs in ovarian cancer patients. Recently, we developed a protocol for the enrichment of CTCs from ovarian cancer blood samples, achieving a high purity of the target cells and enabling high specificity. EXPERIMENTAL PROCEDURES: Blood samples were enriched using a density gradient centrifugation (OncoQuick, Greiner Bio–One) followed by a micro–fluidic based isolation of the target cells using the Parsortix™ system (Angle plc.). The captured target cells were lysed, RNA was extracted (RNeasy Micro Kit, Qiagen) and transcribed into cDNA (ssVILO, Life Technologies). After a pre–amplification step (PreAmp Master Mix, Life Technologies), the transcript levels of selected genes (EpCAM, PPIC, TUSC3, EMP2, LAMB1, MAL2, FN1) were measured using RT–qPCR. Additionally, density gradient enriched cells were immuno–fluorescently stained targeting CK8/18/19, PPIC, TP53 and CD45. SUMMARY: By combining a density gradient based pre–enrichment and the micro–fluidic Parsortix™ technology we achieved an up to 106–fold depletion of blood cell contamination. The analysis of CTC–related transcripts in the enriched samples indicated the presence of CTCs in 78% of the ovarian cancer blood samples taken at primary diagnosis (N=15) and in 80% of the samples taken at relapse (N=9). Concordant results of immuno–fluorescent staining and RT–qPCR were obtained in just 30% of ovarian cancer samples. By adding further 22 RT–qPCR markers, 92% of all cancer patients (n=13) and 100% of the ovarian cancer patients (n=7) were classified as being CTC–positive by RT–qPCR. CONCLUSIONS: In conclusion, we developed a protocol which enabled the detection of CTC–related transcripts in the majority of ovarian cancer blood samples, which is a clear improvement in sensitivity as compared to earlier studies employing density gradient centrifugation as the only enrichment step. Our results are currently validated in the large cohort of platinum resistant ovarian cancer patients provided by a multi–center Gannet53 study. Citation Format: Eva Obermayr, Elisabeth Maritschnegg, Paul Speiser, Eva Schuster, Barbara Holzer, Nina Pecha, Robert Zeillinger. THE MOLECULAR DETECTION OF CIRCULATING TUMOR CELLS IN THE BLOOD OF OVARIAN CANCER PATIENTS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP04.

RT-qPCR-based quantification of small non-coding RNAs.

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate messenger RNA (mRNA) translation into protein. MiRNAs play a key role in gene expression regulation, and their involvement in disease biology is well documented. This has fueled the development of numerous tools for the quantification of miRNA expression levels. These tools are based on three technologies: (microarray) probe hybridization, RNA sequencing, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In this chapter, we describe a quantification system based on RT-qPCR technology, which is currently considered as the most sensitive, flexible, and accurate method for quantification of not only miRNA but also RNA expression in general. To this purpose, we have divided the protocol in three sections: reverse transcription (RT) reaction, optional preamplification (PA), and finally qPCR. Three quality-control (QC) steps are implemented in this workflow for assessment of RNA extraction efficiency, sample purity (e.g., absence of inhibitors), and inter-run variations, by examining the detection level of different spike-in synthetic miRNAs. We conclude by demonstrating raw data preprocessing and normalization using expression data obtained from high-throughput miRNA profiling of human RNA samples.

CD19 mRNA quantification improves rituximab treatment-to-target approach: A proof of concept study

We compared pre-amplification (PA) RT-PCR blood CD19 mRNA quantification with flow cytometry (FC), to personalize rituximab re-treatment in neuromyelitis optica spectrum disorders (NMOSDs) patients. 47 blood samples from 3 NMOSDs patients were studied. PA-RT-PCR quantified CD19 in all samples, and a positivity threshold was defined, whereas CD19+ B cells were under threshold in 31/47 samples by FC. In all samples where CD19+ B cells were above FC threshold, they resulted above the PA-RT-PCR threshold. CD19 mRNA was above threshold in 8 other samples, resulted negative by FC, and preceded the FC positivity in 7/8 samples by 1-3 months, showing major sensitivity.

Compact and Sensitive Millimetre Wave Detectors Based on Low Barrier Schottky Diodes on Impedance Matched Planar Antennas

Compact and highly responsive millimeter wave planar Schottky detectors are proposed for uni-planar and low-cost fabrication. For optimum power transfer, the zero-bias Schottky diodes are impedance matched by the antenna design itself, with an established meander dipole and a new folded dipole type. In particular, up to 200GHz, the folded dipole exhibits a single responsivity peak, notably beneficial for communications. The realized detectors exhibit an outstanding system RF voltage responsivity of up to 16005mV/mW at 87.8GHz without lenses or pre amplification. In addition, an excellent NEP level is demonstrated by the detectors with 0.39pW/ $\sqrt {\text {Hz}}$ .

Validation of high-throughput single cell analysis methodology

High-throughput quantitative polymerase chain reaction (qPCR) approaches enable profiling of multiple genes in single cells, bringing new insights to complex biological processes and offering opportunities for single cell-based monitoring of cancer cells and stem cell-based therapies. However, workflows with well-defined sources of variation are required for clinical diagnostics and testing of tissue-engineered products. In a study of neural stem cell lines, we investigated the performance of lysis, reverse transcription (RT), preamplification (PA), and nanofluidic qPCR steps at the single cell level in terms of efficiency, precision, and limit of detection. We compared protocols using a separate lysis buffer with cell capture directly in RT–PA reagent. The two methods were found to have similar lysis efficiencies, whereas the direct RT–PA approach showed improved precision. Digital PCR was used to relate preamplified template copy numbers to Cq values and reveal where low-quality signals may affect the analysis. We investigated the impact of calibration and data normalization strategies as a means of minimizing the impact of inter-experimental variation on gene expression values and found that both approaches can improve data comparability. This study provides validation and guidance for the application of high-throughput qPCR workflows for gene expression profiling of single cells.

Power Line Carrier Communication

Communication is the backbone for all modes of development in this modern world. Without communication the whole world is virtually deaf and dumb. In order to minimise the cost spent on additional wires, we are using the same existing power lines as a medium of communication in this project. The data are mixed with radio frequency carrier (40-500 kHz) and then injected into high voltage power line using a suitable Coupling Capacitor.The power line has a rigid, long conductor parallel to the ground that guides the carrier waves to travel along the transmission line. This system which is economic and reliable for inter grid message transfer is mainly used for telecommunication, tele-protection and tele-monitoring between electrical substations through power lines at high voltages such as 110KV, 220KV, 440KV. I. INTRODUCTION The PLCC has a transmitter block and receiver block. In the transmitter block ,we use audio signal as an input which should be amplified with a pre amplifier before getting modulated. After pre amplification the audio signal is modulated with the carrier signal by FM modulator. Before transmitting the signal, it should be amplified once again using RF amplifier. Now the modulated and amplified signal is given to the transformer in which isolates RF signal with the 230V AC supply. Finally the signal is coupled to the AC line with the help of coupling capacitor, which allows high frequency modulated signal simultaneously providing high impedance to the power frequency (50 Hz). In receiver block the signal is received from the 230V power line through the coupling capacitor. Then the received signal is given to transformer which is used for isolation purpose and then the RF signal is amplified using RF amplifier. The amplified output is given to PLL FM demodulator for the extraction of audio signal from the modulated signal. Then demodulated output is power amplified and is given to the loud speaker. II. COMPONENTS A. I.Ne 555 Timer (Modulator) The NE555 is a highly stable controller capable of producing accurate timing pulses. It is an 8pin timer IC and has mainly two modes of operation: monostable and astable. With monostable operation, IC 555 is commonly used for generating time delays and pulses.In the time-delay or monostable mode of operation, the timed interval is controlled by a single external resistor and capacitor network. In the astable mode of operation, the frequency and duty cycle can be controlled independently with two external resistors and a single external capacitor.The threshold and trigger levels normally are two-thirds and one-third, respectively, of VCC. This IC consists of 23 transistors, 2 diodes and 16 resistors. The explanation of terminals coming out of the 555 timer IC is as follows. The pin number used in the following discussion refers to the 8-pin DIP and 8-pin metal can packages.The NE555 is characterized for operation from 0°C to 70°C

Analyses of the immunologically relevant gene transcript by qRT-PCR in primary cutaneous melanoma by the young and the elderly.

e21135 Background: To understand the dynamics of anti tumor immune responses (IR) in the setting of the ageing immune system, we examined immunologically relevant gene transcripts in formalin fixed paraffin embedded (FFPE) primary cutaneous melanoma (PCM) samples derived from five young (age &lt;50 years) and five elderly (age &gt;or=70 years) patients by qRT-PCR. Methods: A total of 10 FFPE tumor blocks were collected between 2004-2007 of patients with PCM at the University of Connecticut Health Center. Total RNA was extracted from FFPE tissue sections and random primed cDNA was generated. Pre amplification using pooled Taqman assays and Universal PCR Master Mix (Applied Biosystems, USA) was performed for 14 cycles to ensure an adequate amount of starting material for qPCR. Using 15 ng total RNA, the quantification of human IR genes (IL-2, INFg, TNFa, TGFb1, IL-10, Foxp3, and IDO-1) and transcripts of succinate dehydrogenase (SDHA) (as endogenous control) was accomplished by qRT-PCR amplification of a cDNA using TaqMan Universal PCR Master Mix and TaqMan gene expressions assay probes. All reaction assays including that of SDHA were performed in triplicate on an ABI 7500 Fast system (Applied Biosystems). Cycle threshold (Ct) values for each mRNA were normalized to SDHA (DCT) and represented as RQ=2-DCT. The average RQ was calculated for all ten samples in both cohorts and Student’s T-test was performed to evaluate the statistical significance of the IR gene expression. Results: While no significant difference of expression of Th1 cytokines (IL-2, IFNg, TNFa) or Th2/3 cytokines (IL-10, TGFb) could be detected within the two groups, FOXp3 expression was significantly decreased in PCM in the elderly. Conclusions: Our results suggest ageing may be associated with less regulatory responses in situ in PCM and that more expanded analyses of the nature of the IR in PCM are likely to provide information with implications on prognosis and immune based treatment options for elderly PCM subjects.

Establishment of AFLP Fingerprinting in Genetic Diversity Analysis of Pine Caterpillars*

In order to establish AFLP technique system by using the genomic DNA of Dendrolimus tabulaeformis Tsai et Liu as test material,different dosages of T4 DNA ligase,concentration of Mg2+,dNTP and primers were optimized in preamplification reaction system,and the dilute multiple of preamplif ication production and the concentration of Mg2+,dNTP,primers and different dosages of Taq DNA polymerase were also optimized in selective amplification reaction systerm. Very steady results were achieved. In the linking reaction,T4 DNA ligase 2 U was added to the ligation system. The preamplif ication system included Mg2+ 1.5 mmol/L,dNTP 0.2 mmol/L,PstI primer and TaqI primer 5.0×10-7 μmol/L,and 40 dilute multiple of preamplification production. The selective amplif ication systerm included Mg2+ 1.5 mmol/L,dNTP 0.2 mmol/L,PstI primer and TaqI primer 5.0× 10-7 μmol/L,Taq DNA polymerase 1 U. By using D. tabulaeformis Tsai et Liu,D. spectabilis Butler and D. punctatus (Walker) as test materials,ten pairs of informative primers were identified from eighty-one pairs of primers by optimizing the AFLP reaction system. The result established a strong foundation for the AFLP analysis of pine caterpillar. Fig 9,Ref 17