Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification

Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins.

Development of a sequential injection-square wave voltammetry method for determination of paraquat in water samples employing the hanging mercury drop electrode

This work describes the development and optimization of a sequential injection method to automate the determination of paraquat by square-wave voltammetry employing a hanging mercury drop electrode. Automation by sequential injection enhanced the sampling throughput, improving the sensitivity and precision of the measurements as a consequence of the highly reproducible and efficient conditions of mass transport of the analyte toward the electrode surface. For instance, 212 analyses can be made per hour if the sample/standard solution is prepared off-line and the sequential injection system is used just to inject the solution towards the flow cell. In-line sample conditioning reduces the sampling frequency to 44 h(-1). Experiments were performed in 0.10 M NaCl, which was the carrier solution, using a frequency of 200 Hz, a pulse height of 25 mV, a potential step of 2 mV, and a flow rate of 100 mu L s(-1). For a concentration range between 0.010 and 0.25 mg L(-1), the current (i(p), mu A) read at the potential corresponding to the peak maximum fitted the following linear equation with the paraquat concentration (mg L(-1)): ip = (-20.5 +/- 0.3) Cparaquat -(0.02 +/- 0.03). The limits of detection and quantification were 2.0 and 7.0 mu g L(-1), respectively. The accuracy of the method was evaluated by recovery studies using spiked water samples that were also analyzed by molecular absorption spectrophotometry after reduction of paraquat with sodium dithionite in an alkaline medium. No evidence of statistically significant differences between the two methods was observed at the 95% confidence level.

Applying sequential injection analysis (SIA) and response surface methodology for optimization of Fenton-based processes

This work presents the use of sequential injection analysis (SIA) and the response surface methodology as a tool for optimization of Fenton-based processes. Alizarin red S dye (C.I. 58005) was used as a model compound for the anthraquinones family. whose pigments have a large use in coatings industry. The following factors were considered: [H(2)O(2)]:[Alizarin] and [H(2)O(2)]:[FeSO(4)] ratios and pH. The SIA system was designed to add reagents to the reactor and to perform on-line sampling of the reaction medium, sending the samples to a flow-through spectrophotometer for monitoring the color reduction of the dye. The proposed system fed the statistical program with degradation data for fast construction of response surface plots. After optimization, 99.7% of the dye was degraded and the TOC content was reduced to 35% of the original value. Low reagents consumption and high sampling throughput were the remarkable features of the SIA system. (C) 2008 Published by Elsevier B.V.

Under flow impedimetric measurements using magnetic particles for label-free detection affinity target

The necessity to adapt sensors based on electrochemical techniques for high throughput analysis control increases the interest to develop new analytical systems able to perform measurements under buffer now. In this report we explored the possibility of employing a new system to make impedimetric measurements to detect the interaction between proteins and small molecules. The well-known biotin-streptavidin interaction was adopted to evaluate the proposed assembly. This system allows us to perform experiments under flow. Magnetic beads functionalized with streptavidin were used and first characterized using AFM and FTIR. Non-faradic impedance spectroscopy allowed the detection of the biotin-streptavidin interaction. Using our new system and under a flow of PBS buffer, 5 10-5 M of biotin was detected with a stable signal. (c) 2007 Elsevier B.V. All rights reserved.

Square wave adsorptive cathodic stripping voltarnmetry automated by sequential injection analysis - Potentialities and limitations exemplified by the determination of methyl parathion in water samples

This paper describes the development and evaluation of a sequential injection method to automate the determination of methyl parathion by square wave adsorptive cathodic stripping voltammetry exploiting the concept of monosegmented flow analysis to perform in-line sample conditioning and standard addition. Accumulation and stripping steps are made in the sample medium conditioned with 40 mmol L-1 Britton-Robinson buffer (pH 10) in 0.25 mol L-1 NaNO3. The homogenized mixture is injected at a flow rate of 10 mu Ls(-1) toward the flow cell, which is adapted to the capillary of a hanging drop mercury electrode. After a suitable deposition time, the flow is stopped and the potential is scanned from -0.3 to -1.0 V versus Ag/AgCl at frequency of 250 Hz and pulse height of 25 mV The linear dynamic range is observed for methyl parathion concentrations between 0.010 and 0.50 mgL(-1), with detection and quantification limits of 2 and 7 mu gL(-1), respectively. The sampling throughput is 25 h(-1) if the in line standard addition and sample conditioning protocols are followed, but this frequency can be increased up to 61 h(-1) if the sample is conditioned off-line and quantified using an external calibration curve. The method was applied for determination of methyl parathion in spiked water samples and the accuracy was evaluated either by comparison to high performance liquid chromatography with UV detection, or by the recovery percentages. Although no evidences of statistically significant differences were observed between the expected and obtained concentrations, because of the susceptibility of the method to interference by other pesticides (e.g., parathion, dichlorvos) and natural organic matter (e.g., fulvic and humic acids), isolation of the analyte may be required when more complex sample matrices are encountered. (C) 2007 Elsevier B.V. All rights reserved.

Development and Application of a Highly Selective Biomimetic Sensor for Detection of Captopril, an Important Ally in Hypertension Control

A biomimetic sensor is proposed as a promising new analytical method for determination of captopril in different classes of samples. The sensor was prepared by modifying a carbon paste electrode with iron (II) phthalocyanine bis(pyridine) [FePe(dipy)] complex. Amperometric measurements in a batch analytical mode were first carried out in order to optimize the sensor response. An applied potential lower than 0.2 V vs Ag vertical bar AgCl in 0.1 mol L(-1) of TRIS buffer at pH 8.0 provided the best response, with a linear range of 2.5 x 10(-5) to 1.7 x 10(-4) mol L(-1). A detailed investigation of the selectivity of the sensor, employing seventeen other drugs, was also performed. Recovery studies were carried out using biological and environment samples in order to evaluate the sensor`s potential for use with these sample classes. Finally, the performance of the biomimetic sensor was optimized in a flow injection (FIA) system using a wall jet electrochemical cell. Under optimized flow conditions, a broad linear response range, from 5.0 x 10(-4) to 2.5 x 10(-2) mol L(-1), was obtained for captopril, with a sensitivity of 210 +/- 1 mu A L mol(-1).

Fast and Simple Nuclear Magnetic Resonance Method To Measure Conjugated Linoleic Acid in Beef

Conjugated linoleic acids (CLAs) are a group of linoleic acid isomers that are naturally found in food products originating from ruminants (meat and dairy). These acids have received special attention in recent years due to their potential human health benefits. Research efforts have been proposed to increase the CLA content in beef to improve public health. However, because there are more than 30 million beef cattle used each year by the American food industry, it will be necessary to ensure their content in a large number of samples. Therefore, it is important to have an inexpensive and rapid analytical method to measure CLA content in food products. Because gas chromatography (GC), a current popular method for measuring CLAs, is slow, this paper describes a nuclear magnetic resonance spectroscopy ((1)H NMR) method that is potentially >10 times faster than the GC method. Analyses show a correlation coefficient of 0.97, indicating the capacity of NMR to quantify the CLA content in beef samples. Furthermore, the method proposed herein is simple and does not require sophisticated sample preparation.

Fast Acquisition of (13)C NMR Spectra using the Steady-state Free Precession Sequence

In this paper, we show that the steady-state free precession sequence can be used to acquire (13)C high-resolution nuclear magnetic resonance spectra and applied to qualitative analysis. The analysis of brucine sample using this sequence with 60 degrees flip angle and time interval between pulses equal to 300 ms (acquisition time, 299.7 ms; recycle delay, 300 ms) resulted in spectrum with twofold enhancement in signal-to-noise ratio, when compared to standard (13)C sequence. This gain was better when a much shorter time interval between pulses (100 ms) was applied. The result obtained was more than fivefold enhancement in signal-to-noise ratio, equivalent to more than 20-fold reduction in total data recording time. However, this short time interval between pulses produces a spectrum with severe phase and truncation anomalies. We demonstrated that these anomalies can be minimized by applying an appropriate apodization function and plotting the spectrum in the magnitude mode.

Load balancing solution and evaluation of F5 content switch equipment

The Thesis focused on hardware based Load balancing solution of web traffic through a load balancer F5 content switch. In this project, the implemented scenario for distributing HTTPtraffic load is based on different CPU usages (processing speed) of multiple member servers.Two widely used load balancing algorithms Round Robin (RR) and Ratio model (weighted Round Robin) are implemented through F5 load balancer. For evaluating the performance of F5 content switch, some experimental tests has been taken on implemented scenarios using RR and Ratio model load balancing algorithms. The performance is examined in terms of throughput (bits/sec) and Response time of member servers in a load balancing pool. From these experiments we have observed that Ratio Model load balancing algorithm is most suitable in the environment of load balancing servers with different CPU usages as it allows assigning the weight according to CPU usage both in static and dynamic load balancing of servers.

Model Configuration And Data Management In The Short-Term Water Information Forecasting Tools

The Short-term Water Information and Forecasting Tools (SWIFT) is a suite of tools for flood and short-term streamflow forecasting, consisting of a collection of hydrologic model components and utilities. Catchments are modeled using conceptual subareas and a node-link structure for channel routing. The tools comprise modules for calibration, model state updating, output error correction, ensemble runs and data assimilation. Given the combinatorial nature of the modelling experiments and the sub-daily time steps typically used for simulations, the volume of model configurations and time series data is substantial and its management is not trivial. SWIFT is currently used mostly for research purposes but has also been used operationally, with intersecting but significantly different requirements. Early versions of SWIFT used mostly ad-hoc text files handled via Fortran code, with limited use of netCDF for time series data. The configuration and data handling modules have since been redesigned. The model configuration now follows a design where the data model is decoupled from the on-disk persistence mechanism. For research purposes the preferred on-disk format is JSON, to leverage numerous software libraries in a variety of languages, while retaining the legacy option of custom tab-separated text formats when it is a preferred access arrangement for the researcher. By decoupling data model and data persistence, it is much easier to interchangeably use for instance relational databases to provide stricter provenance and audit trail capabilities in an operational flood forecasting context. For the time series data, given the volume and required throughput, text based formats are usually inadequate. A schema derived from CF conventions has been designed to efficiently handle time series for SWIFT.

Sistema de produção sincronizado : uma aplicação em processos produtivos de propriedade contínuos segundo a teoria das restrições

Este trabalho de conclusão investiga o efeito da geração de estoques intermediários nos indicadores principais empregados na Teoria das Restrições (Ganho, Despesa Operacional e Inventário) em uma unidade industrial de processo produtivo de Propriedade contínuo, que emprega embalagens, matérias-primas obtidas em larga escala e cadeias logísticas de longo curso. Este tipo de indústria produz bens de consumo imediato, com pouca variabilidade, de modo “empurrado”. A principal conseqüência é a perda do sincronismo na cadeia logística, resultando em uma grande quantidade de estoques intermediários e custos crescentes, relacionados principalmente ao custo de manutenção destes estoques. Através dos cinco passos de focalização e das ferramentas lógicas da Teoria das Restrições, propõe-se uma alternativa gerencial, que inclui o algoritmo Tambor-Pulmão-Corda e insere a organização em um processo de melhoria contínua, cujos impactos são avaliados por simulação computacional. Através de técnicas estatísticas e software apropriados, constrói-se um modelo de simulação computacional baseado em dados reais de uma planta produtora de cimento. A partir deste modelo, diferentes cenários são testados, descobrindo-se a condição ótima. Chega-se a uma conclusão, considerando a mudança na política de geração de estoques intermediários e seus impactos na redução de custos e riscos.

TCP HolyWood

We introduce a new end-to-end, sender side Transport Control Protocol called TCP HolyWood or in short TCP-HW. In a simulated wired environment, TCP HolyWood outperforms in average throughput, three of the more important TCP protocols ever made, we are talking about TCP Reno, TCP Westwood, and TCP Vegas; and in average jitter to TCP Reno and TCP Vegas too. In addition, according to Jain’s index, our proposal is as fair as TCP Reno, the Standard.

Multilayer optimization in radio resource allocation for the packet transmission in wireless networks

In the last decade mobile wireless communications have witnessed an explosive growth in the user’s penetration rate and their widespread deployment around the globe. It is expected that this tendency will continue to increase with the convergence of fixed Internet wired networks with mobile ones and with the evolution to the full IP architecture paradigm. Therefore mobile wireless communications will be of paramount importance on the development of the information society of the near future. In particular a research topic of particular relevance in telecommunications nowadays is related to the design and implementation of mobile communication systems of 4th generation. 4G networks will be characterized by the support of multiple radio access technologies in a core network fully compliant with the Internet Protocol (all IP paradigm). Such networks will sustain the stringent quality of service (QoS) requirements and the expected high data rates from the type of multimedia applications to be available in the near future. The approach followed in the design and implementation of the mobile wireless networks of current generation (2G and 3G) has been the stratification of the architecture into a communication protocol model composed by a set of layers, in which each one encompasses some set of functionalities. In such protocol layered model, communications is only allowed between adjacent layers and through specific interface service points. This modular concept eases the implementation of new functionalities as the behaviour of each layer in the protocol stack is not affected by the others. However, the fact that lower layers in the protocol stack model do not utilize information available from upper layers, and vice versa, downgrades the performance achieved. This is particularly relevant if multiple antenna systems, in a MIMO (Multiple Input Multiple Output) configuration, are implemented. MIMO schemes introduce another degree of freedom for radio resource allocation: the space domain. Contrary to the time and frequency domains, radio resources mapped into the spatial domain cannot be assumed as completely orthogonal, due to the amount of interference resulting from users transmitting in the same frequency sub-channel and/or time slots but in different spatial beams. Therefore, the availability of information regarding the state of radio resources, from lower to upper layers, is of fundamental importance in the prosecution of the levels of QoS expected from those multimedia applications. In order to match applications requirements and the constraints of the mobile radio channel, in the last few years researches have proposed a new paradigm for the layered architecture for communications: the cross-layer design framework. In a general way, the cross-layer design paradigm refers to a protocol design in which the dependence between protocol layers is actively exploited, by breaking out the stringent rules which restrict the communication only between adjacent layers in the original reference model, and allowing direct interaction among different layers of the stack. An efficient management of the set of available radio resources demand for the implementation of efficient and low complexity packet schedulers which prioritize user’s transmissions according to inputs provided from lower as well as upper layers in the protocol stack, fully compliant with the cross-layer design paradigm. Specifically, efficiently designed packet schedulers for 4G networks should result in the maximization of the capacity available, through the consideration of the limitations imposed by the mobile radio channel and comply with the set of QoS requirements from the application layer. IEEE 802.16e standard, also named as Mobile WiMAX, seems to comply with the specifications of 4G mobile networks. The scalable architecture, low cost implementation and high data throughput, enable efficient data multiplexing and low data latency, which are attributes essential to enable broadband data services. Also, the connection oriented approach of Its medium access layer is fully compliant with the quality of service demands from such applications. Therefore, Mobile WiMAX seems to be a promising 4G mobile wireless networks candidate. In this thesis it is proposed the investigation, design and implementation of packet scheduling algorithms for the efficient management of the set of available radio resources, in time, frequency and spatial domains of the Mobile WiMAX networks. The proposed algorithms combine input metrics from physical layer and QoS requirements from upper layers, according to the crosslayer design paradigm. Proposed schedulers are evaluated by means of system level simulations, conducted in a system level simulation platform implementing the physical and medium access control layers of the IEEE802.16e standard.

A sensitive microextraction by packed sorbent-based methodology combined with ultra-high pressure liquid chromatography as a powerful technique for analysis of biologically active flavonols in wines

A new approach based on microextraction by packed sorbent (MEPS) and reversed-phase high-throughput ultra high pressure liquid chromatography (UHPLC) method that uses a gradient elution and diode array detection to quantitate three biologically active flavonols in wines, myricetin, quercetin, and kaempferol, is described. In addition to performing routine experiments to establish the validity of the assay to internationally accepted criteria (selectivity, linearity, sensitivity, precision, accuracy), experiments are included to assess the effect of the important experimental parameters such as the type of sorbent material (C2, C8, C18, SIL, and C8/SCX), number of extraction cycles (extract-discard), elution volume, sample volume, and ethanol content, on the MEPS performance. The optimal conditions of MEPS extraction were obtained using C8 sorbent and small sample volumes (250 μL) in five extraction cycle and in a short time period (about 5 min for the entire sample preparation step). Under optimized conditions, excellent linearity View the MathML source(Rvalues2>0.9963), limits of detection of 0.006 μg mL−1 (quercetin) to 0.013 μg mL−1 (myricetin) and precision within 0.5–3.1% were observed for the target flavonols. The average recoveries of myricetin, quercetin and kaempferol for real samples were 83.0–97.7% with relative standard deviation (RSD, %) lower than 1.6%. The results obtained showed that the most abundant flavonol in the analyzed samples was myricetin (5.8 ± 3.7 μg mL−1). Quercetin (0.97 ± 0.41 μg mL−1) and kaempferol (0.66 ± 0.24 μg mL−1) were found in a lower concentration. The optimized MEPSC8 method was compared with a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis HLB) were used as reference. MEPSC8 approach offers an attractive alternative for analysis of flavonols in wines, providing a number of advantages including highest extraction efficiency (from 85.9 ± 0.9% to 92.1 ± 0.5%) in the shortest extraction time with low solvent consumption, fast sample throughput, more environmentally friendly and easy to perform.

A fast method using a new hydrophilic–lipophilic balanced sorbent in combination with ultra-high performance liquid chromatography for quantification of significant bioactive metabolites in wines

This manuscript describes the development and validation of an ultra-fast, efficient, and high throughput analytical method based on ultra-high performance liquid chromatography (UHPLC) equipped with a photodiode array (PDA) detection system, for the simultaneous analysis of fifteen bioactive metabolites: gallic acid, protocatechuic acid, (−)-catechin, gentisic acid, (−)-epicatechin, syringic acid, p-coumaric acid, ferulic acid, m-coumaric acid, rutin, trans-resveratrol, myricetin, quercetin, cinnamic acid and kaempferol, in wines. A 50-mm column packed with 1.7-μm particles operating at elevated pressure (UHPLC strategy) was selected to attain ultra-fast analysis and highly efficient separations. In order to reduce the complexity of wine extract and improve the recovery efficiency, a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a new macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis™ HLB), was performed prior to UHPLC–PDA analysis. The calibration curves of bioactive metabolites showed good linearity within the established range. Limits of detection (LOD) and quantification (LOQ) ranged from 0.006 μg mL−1 to 0.58 μg mL−1, and from 0.019 μg mL−1 to 1.94 μg mL−1, for gallic and gentisic acids, respectively. The average recoveries ± SD for the three levels of concentration tested (n = 9) in red and white wines were, respectively, 89 ± 3% and 90 ± 2%. The repeatability expressed as relative standard deviation (RSD) was below 10% for all the metabolites assayed. The validated method was then applied to red and white wines from different geographical origins (Azores, Canary and Madeira Islands). The most abundant component in the analysed red wines was (−)-epicatechin followed by (−)-catechin and rutin, whereas in white wines syringic and p-coumaric acids were found the major phenolic metabolites. The method was completely validated, providing a sensitive analysis for bioactive phenolic metabolites detection and showing satisfactory data for all the parameters tested. Moreover, was revealed as an ultra-fast approach allowing the separation of the fifteen bioactive metabolites investigated with high resolution power within 5 min.