Fingerprint analysis of eucommia bark by LC-DAD and LC-MS with the aid of chemometrics

Chromatographic fingerprints of 46 Eucommia Bark samples were obtained by liquid chromatography-diode array detector (LC-DAD). These samples were collected from eight provinces in China, with different geographical locations, and climates. Seven common LC peaks that could be used for fingerprinting this common popular traditional Chinese medicine were found, and six were identified as substituted resinols (4 compounds), geniposidic acid and chlorogenic acid by LC-MS. Principal components analysis (PCA) indicated that samples from the Sichuan, Hubei, Shanxi and Anhui—the SHSA provinces, clustered together. The other objects from the four provinces, Guizhou, Jiangxi, Gansu and Henan, were discriminated and widely scattered on the biplot in four province clusters. The SHSA provinces are geographically close together while the others are spread out. Thus, such results suggested that the composition of the Eucommia Bark samples was dependent on their geographic location and environment. In general, the basis for discrimination on the PCA biplot from the original 46 objects× 7 variables data matrix was the same as that for the SHSA subset (36 × 7 matrix). The seven marker compound loading vectors grouped into three sets: (1) three closely correlating substituted resinol compounds and chlorogenic acid; (2) the fourth resinol compound identified by the OCH3 substituent in the R4 position, and an unknown compound; and (3) the geniposidic acid, which was independent of the set 1 variables, and which negatively correlated with the set 2 ones above. These observations from the PCA biplot were supported by hierarchical cluster analysis, and indicated that Eucommia Bark preparations may be successfully compared with the use of the HPLC responses from the seven marker compounds and chemometric methods such as PCA and the complementary hierarchical cluster analysis (HCA).

Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

Development and validation of a ultra performance LC-ESI/MS method for analysis of metabolic phenotypes of healthy men in day and night urine samples

Ultra-performance LC coupled to quadrupole TOF/MS (UPLC-QTOF/MS) in positive and negative ESI was developed and validated to analyze metabolite profiles for urine from healthy men during the day and at night. Data analysis using principal components analysis (PCA) revealed differences between metabolic phenotypes of urine in healthy men during the day and at night. Positive ions with mass-to-charge ratio (m/z) 310.24 (5.35 min), 286.24 (4.74 min) and 310.24 (5.63 min) were elevated in the urine from healthy men at night compared to that during the day. Negative ions elevated in day urine samples of healthy men included m/z 167.02 (0.66 min), 263.12 (2.55 min) and 191.03 (0.73 min), whilst ions m/z 212.01 (4.77 min) were at a lower concentration in urine of healthy men during the day compared to that at night. The ions m/z 212.01 (4.77 min), 191.03 (0.73 min) and 310.24 (5.35 min) preliminarily correspond to indoxyl sulfate, citric acid and N-acetylneuraminic acid, providing further support for an involvement of phenotypic difference in urine of healthy men in day and night samples, which may be associated with notably different activities of gut microbiota, velocity of tricarboxylic acid cycle and activity of sialic acid biosynthesis in healthy men as regulated by circadian rhythm of the mammalian bioclock.

Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand β5

Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.

Design, implementation and multisite evaluation of a system suitability protocol for the quantitative assessment of instrument performance in liquid chromatography-multiple reaction monitoring-MS (LC-MRM-MS)

Multiple reaction monitoring (MRM) mass spectrometry coupled with stable isotope dilution (SID) and liquid chromatography (LC) is increasingly used in biological and clinical studies for precise and reproducible quantification of peptides and proteins in complex sample matrices. Robust LC-SID-MRM-MS-based assays that can be replicated across laboratories and ultimately in clinical laboratory settings require standardized protocols to demonstrate that the analysis platforms are performing adequately. We developed a system suitability protocol (SSP), which employs a predigested mixture of six proteins, to facilitate performance evaluation of LC-SID-MRM-MS instrument platforms, configured with nanoflow-LC systems interfaced to triple quadrupole mass spectrometers. The SSP was designed for use with low multiplex analyses as well as high multiplex approaches when software-driven scheduling of data acquisition is required. Performance was assessed by monitoring of a range of chromatographic and mass spectrometric metrics including peak width, chromatographic resolution, peak capacity, and the variability in peak area and analyte retention time (RT) stability. The SSP, which was evaluated in 11 laboratories on a total of 15 different instruments, enabled early diagnoses of LC and MS anomalies that indicated suboptimal LC-MRM-MS performance. The observed range in variation of each of the metrics scrutinized serves to define the criteria for optimized LC-SID-MRM-MS platforms for routine use, with pass/fail criteria for system suitability performance measures defined as peak area coefficient of variation <0.15, peak width coefficient of variation <0.15, standard deviation of RT <0.15 min (9 s), and the RT drift <0.5min (30 s). The deleterious effect of a marginally performing LC-SID-MRM-MS system on the limit of quantification (LOQ) in targeted quantitative assays illustrates the use and need for a SSP to establish robust and reliable system performance. Use of a SSP helps to ensure that analyte quantification measurements can be replicated with good precision within and across multiple laboratories and should facilitate more widespread use of MRM-MS technology by the basic biomedical and clinical laboratory research communities.

Work at hand : an exploration of gesture in the context of work and everyday life to inform the design of gestural input devices

This research explores gestures used in the context of activities in the workplace and in everyday life in order to understand requirements and devise concepts for the design of gestural information applicances. A collaborative method of video interaction analysis devised to suit design explorations, the Video Card Game, was used to capture and analyse how gesture is used in the context of six different domains: the dentist's office; PDA and mobile phone use; the experimental biologist's laboratory; a city ferry service; a video cassette player repair shop; and a factory flowmeter assembly station. Findings are presented in the form of gestural themes, derived from the tradition of qualitative analysis but bearing some similarity to Alexandrian patterns. Implications for the design of gestural devices are discussed.

Power network in the loop : subsystem testing using a switching amplifier

“Hardware in the Loop” (HIL) testing is widely used in the automotive industry. The sophisticated electronic control units used for vehicle control are usually tested and evaluated using HIL-simulations. The HIL increases the degree of realistic testing of any system. Moreover, it helps in designing the structure and control of the system under test so that it works effectively in the situations that will be encountered in the system. Due to the size and the complexity of interaction within a power network, most research is based on pure simulation. To validate the performance of physical generator or protection system, most testing is constrained to very simple power network. This research, however, examines a method to test power system hardware within a complex virtual environment using the concept of the HIL. The HIL testing for electronic control units and power systems protection device can be easily performed at signal level. But performance of power systems equipments, such as distributed generation systems can not be evaluated at signal level using HIL testing. The HIL testing for power systems equipments is termed here as ‘Power Network in the Loop’ (PNIL). PNIL testing can only be performed at power level and requires a power amplifier that can amplify the simulation signal to the power level. A power network is divided in two parts. One part represents the Power Network Under Test (PNUT) and the other part represents the rest of the complex network. The complex network is simulated in real time simulator (RTS) while the PNUT is connected to the Voltage Source Converter (VSC) based power amplifier. Two way interaction between the simulator and amplifier is performed using analog to digital (A/D) and digital to analog (D/A) converters. The power amplifier amplifies the current or voltage signal of simulator to the power level and establishes the power level interaction between RTS and PNUT. In the first part of this thesis, design and control of a VSC based power amplifier that can amplify a broadband voltage signal is presented. A new Hybrid Discontinuous Control method is proposed for the amplifier. This amplifier can be used for several power systems applications. In the first part of the thesis, use of this amplifier in DSTATCOM and UPS applications are presented. In the later part of this thesis the solution of network in the loop testing with the help of this amplifier is reported. The experimental setup for PNIL testing is built in the laboratory of Queensland University of Technology and the feasibility of PNIL testing has been evaluated using the experimental studies. In the last section of this thesis a universal load with power regenerative capability is designed. This universal load is used to test the DG system using PNIL concepts. This thesis is composed of published/submitted papers that form the chapters in this dissertation. Each paper has been published or submitted during the period of candidature. Chapter 1 integrates all the papers to provide a coherent view of wide bandwidth switching amplifier and its used in different power systems applications specially for the solution of power systems testing using PNIL.

Reduction of Secondary Degeneration after Spinal Cord Injury by Acute Delivery of Proinflammatory Growth Factors

INTRODUCTION Inflammation is a protective attempt to facilitate the removal of damaged tissue and to initiate the healing response in other tissues. However, after spinal cord injury (SCI), this response is prolonged leading to secondary degeneration and glial scarring. Here, we investigate the potential of sustained delivery of pro-inflammatory factors vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) to increase early inflammatory events and promote inflammatory resolution. Method Animal ethics approval was obtained from the Queensland University of Technology. Adult Wistar-Kyoto rats (12-16 weeks old) were subjected to laminectomies and T10 hemisections. Animals were then randomised to treatment (implantation of osmotic pump (Alzet) loaded with 5ug VEGF & 5 ug PDGF) or control groups (lesion control or lesion plus pump delivering PBS). Rats were sacrificed at one month and the spinal cords were harvested and examined by immunohistology, using anti-neurofilament-200(NF200) and anti- ionized calcium binding adapter molecule 1 (Iba1). One way ANOVA was used for statistic analysis. Results At 1 month, active pump-treated cords showed a high level of axonal filament throughout the defects as compared to the control groups. The mean lesion size, as measured by NF200, was 0.47mm2 for the lesion control, 0.39mm2 for the vehicle control and 0.078mm2 for the active pump group. Significant differences were detected between the active pump group and the two control groups (AP vs LC p= 0.017 AG vs VC p= 0.004). Iba-1 staining also showed significant differences in the post-injury inflammatory response. Discussion We have shown that axons and activated microglia are co-located in the lesion of the treated cord. We hypothesise the delivery of VEGF/PDGF increases the local vessel permeability to inflammatory cells and activates these along with the resident microglia to threshold population, which ultimately resolved the prolonged inflammation. Here, we have shown that maintaining the inflammatory signals for at least 7 days improved the morphology of the injured cord. Conclusion This study has shown that boosting inflammation, by delivery VEGF/PDGF, in the early phase of SCI helps to reduce secondary degeneration and may promote inflammation resolution. This treatment may provide a platform for other neuro-regenrative therapies.

Investigation of shaft voltage in wind turbine systems with induction generators

This paper presents the analysis of shaft voltage in different configurations of a doubly fed induction generator (DFIG) and an induction generator (IG) with a back-to-back inverter in wind turbine applications. Detailed high frequency model of the proposed systems have been developed based on existing capacitive couplings in IG & DFIG structures and common mode voltage sources. In this research work, several arrangements of DFIG based wind energy conversion systems (WES) are investigated in case of shaft voltage calculation and its mitigation techniques. Placements of an LC line filter in different locations and its effects on shaft voltage elimination are studied via Mathematical analysis and simulations. A pulse width modulation (PWM) technique and a back-to-back inverter with a bidirectional buck converter have been presented to eliminate the shaft voltage in a DFIG wind turbine.

A bidirectional two-leg resonant converter for high voltage pulsed power application

A high voltage pulsed power supply is proposed in this paper based on oscillation between an inductor and a capacitor in an LC circuit. A two-leg resonant circuit, supplied through an inverter with an alternative voltage waveform, can generate output voltage up to four times an input voltage magnitude. Bipolar and unipolar modulations are used in a single phase inverter to analyse their effects on the proposed resonant converter. Simulations have been carried out to evaluate the proposed topology and control.

Virtual fences for controlling cows

We describe a moving virtual fence algorithm for herding cows. Each animal in the herd is given a smart collar consisting of a GPS, PDA, wireless networking and a sound amplifier. Using the GPS, the animal's location can be verified relative to the fence boundary. When approaching the perimeter, the animal is presented with a sound stimulus whose effect is to move away. We have developed the virtual fence control algorithm for moving a herd. We present simulation results and data from experiments with 8 cows equipped with smart collars.

Dynamic virtual fences for controlling cows

A virtual fence is created by applying an aversive stimulus to an animal when it approaches a predefined boundary. It is implemented by a small animal-borne computer system with a GPS receiver. This approach allows the implementation of virtual paddocks inside a normal physically-fenced paddock. Since the fence lines are virtual they can be moved by programming to meet the needs of animal or land management. This approach enables us to consider animals as agents with natural mobility that are controllable and to apply a vast body of theory in motion planning. In this paper we describe a herd-animal simulator and physical experiments conducted on a small herd of 10 animals using a Smart Collar. The Smart Collar consists of a GPS, PDA, wireless networking and a sound amplifier. We describe a motion planning algorithm that can move a virtual paddock subject to landscape constraints which is suitable for mustering cows. We present simulation results and data from experiments with 8 cows equipped with Smart Collars.

Testing of a distributed generation system with a virtual grid

The paper proposes a solution for testing of a physical distributed generation system (DGs) along with a computer simulated network. The computer simulated network is referred as the virtual grid in this paper. Integration of DG with the virtual grid provides broad area of testing of power supplying capability and dynamic performance of a DG. It is shown that a DG can supply a part of load power while keeping Point of Common Coupling (PCC) voltage magnitude constant. To represent the actual load, a universal load along with power regenerative capability is designed with the help of voltage source converter (VSC) that mimics the load characteristic. The overall performance of the proposed scheme is verified using computer simulation studies.

Active power flow control in a distribution system using discontinuous voltage controller

stract This paper proposes a hybrid discontinuous control methodology for a voltage source converter (VSC), which is used in an uninterrupted power supply (UPS) application. The UPS controls the voltage at the point of common coupling (PCC). An LC filter is connected at the output of the VSC to bypass switching harmonics. With the help of both filter inductor current and filter capacitor voltage control, the voltage across the filter capacitor is controlled. Based on the voltage error, the control is switched between current and voltage control modes. In this scheme, an extra diode state is used that makes the VSC output current discontinuous. This diode state reduces the switching losses. The UPS controls the active power it supplies to a three-phase, four-wire distribution system. This gives a full flexibility to the grid to buy power from the UPS system depending on its cost and load requirement at any given time. The scheme is validated through simulation using PSCAD.

A new family of Marx generator based on resonant converter

This paper presents a novel topology to generate high voltage with utilization of slow and fast power switches. New concepts used in this topology include numbers of diode-capacitor units in parallel with resonant circuits which are connected to a positive buck-boost converter. The resonant circuit reverses the voltage polarity of the capacitors. This configuration has capability of generating a flexible high voltage with certain number of capacitors. The advantage of this topology is to use slow switches, less number of diodes and capacitors compare to Marx generator. Simulations have been performed to verify the proposed topology.