A custom-built insulin resistance gene chip

Objectives/Aim—Microarray (gene chip) technology offers a powerful new tool for analyzing the expression of large numbers of genes in many experimental samples. The aim of this study was to design, construct, and use a gene chip to measure the expression levels of key genes in metabolic pathways related to insulin resistance.
Methods—We selected genes that were implicated in the development of insulin resistance, including genes involved in insulin signaling; glucose uptake, oxidation, and storage; fat uptake, oxidation, and storage; cytoskeletal components; and transcription factors. The key regulatory genes in the pathways were identified, along with other recently identified candidate genes such as calpain-10. A total of 242 selected genes (including 32 internal control elements) were sequence-verified, purified, and arrayed on aldehyde-coated slides.
Results—Where more than 1 clone containing the gene of interest was available, we chose those containing the genes in the 5' orientation and an insert size of around 1.5 kb. Of the 262 clones purchased, 56 (21%) were found to contain sequences other than those expected. In addition, 2 (1%) did not grow under standard conditions and were assumed to be nonviable. In these cases, alternate clones containing the gene of interest were chosen as described above. The current version of the Insulin Resistance Gene Chip contains 210 genes of interest, plus 48 control elements. A full list of the genes is available at http://www.hbs.deakin.edu.au/mru/research/gene_chip_tech/genechip_three.htm/.
Conclusions—The human Insulin Resistance Gene Chip that we have constructed will be a very useful tool for investigating variation in the expression of genes relevant to insulin resistance under various experimental conditions. Initially, the gene chip will be used in studies such as exercise interventions, fasting, euglycemic-hyperinsulinemic clamps, and administration of antidiabetic agents

Development of an automated DNA purification module using a micro-fabricated pillar chip

We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 × 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 µm, respectively, which provides a relatively large surface area (ca. 3 cm²) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.

Optical micro analysis in lab-on-a-chips

Lab-on-a-chips are microfluidic devices which include biological laboratory functions on a single chip. They offer great advantages in terms of speed of analysis, precision, low sample and reagent consumption and automation of analysis. An efficient detection method in lab-on-a-chips is essential for the detection and quantification of chemical and biological parameters under examination. This review paper focuses on the recent research on optical detection techniques for LOC applications. Furthermore, several emerging detection paradigms which are still being matured are covered in this paper. Also, an analysis of the performance of different optical detection methods is given.

Dynamic analysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immobilization technology

Quantification of programmed and accidental cell death provides useful end-points for the anticancer drug efficacy assessment. Cell death is, however, a stochastic process. Therefore, the opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. In this work, we describe the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilization platform for the realtime analysis of cancer drug-induced cytotoxicity. Microelectrode arrays were designed to generate weak electro-thermal vortices that support efficient drug mixing and rapid cell immobilization at the delta-shape regions of strong electric field formed between the opposite microelectrodes. We applied this technology to the dynamic analysis of hematopoietic tumor cells that represent a particular challenge for real-time imaging due to their dislodgement during image acquisition. The present study was designed to provide a comprehensive mechanistic rationale for accelerated cell-based assays on DEP chips using real-time labeling with cell permeability markers. In this context, we provide data on the complex behavior of viable vs dying cells in the DEP fields and probe the effects of DEP fields upon cell responses to anticancer drugs and overall bioassay performance. Results indicate that simple DEP cell immobilization technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells. This ability is of particular importance in studying the outcome of patient derived cancer cells, when exposed to therapeutic drugs, as these cells are often rare and difficult to collect, purify and immobilize.

Global and threshold-free transcriptional regulatory networks reconstruction through integrating ChIP-Chip and expression data

Inferring transcriptional regulatory networks from high-throughput biological data is a major challenge to bioinformatics today. To address this challenge, we developed TReNGO (Transcriptional Regulatory Networks reconstruction based on Global Optimization), a global and threshold-free algorithm with simulated annealing for inferring regulatory networks by the integration of ChIP-chip and expression data. Superior to existing methods, TReNGO was expected to find the optimal structure of transcriptional regulatory networks without any arbitrary thresholds or predetermined number of transcriptional modules (TMs). TReNGO was applied to both synthetic data and real yeast data in the rapamycin response. In these applications, we demonstrated an improved functional coherence of TMs and TF (transcription factor)- target predictions by TReNGO when compared to GRAM, COGRIM or to analyzing ChIP-chip data alone. We also demonstrated the ability of TReNGO to discover unexpected biological processes that TFs may be involved in and to also identify interesting novel combinations of TFs.

Review of low actuation voltage RF MEMS electrostatic switches based on metallic and carbon alloys

Radio frequency micro electro mechanical systems (RF MEMS) have enabled a new generation of devices that bring many advantages due to their very high performances. There are many incentives for the integration of the RF MEMS switches and electronic devices on the same chip. However, the high actuation voltage of RF MEMS switches compared to electronic devices poses a major problem. By reducing the actuation voltage of the RF MEMS switch, it is possible to integrate it into current electronic devices. Lowering the actuation voltage will have an impact on RF parameters of the RF MEMS switches. This investigation focuses on recent progress in reducing the actuation voltage with an emphasis on a modular approach that gives acceptable design parameters. A number of rules that should be considered in design and fabrication of low actuation RF MEMS switches are suggested.

Development of microfluidic-based analytical methodology for studying the effects of chemotherapy agents on cancer tissue

Whilst a multitude of techniques have been employed to study the biology of tumour tissue and its response to chemotherapeutic reagents, most current methodologies do not capture the sophistication of the in vivo environment. Microfluidics however offers the ability to maintain and interrogate primary tissue samples in an environment with biomimetic flow characteristics. In this study head and neck squamous cell carcinoma (HNSCC) tumour biopsies have been used to investigate the performance of a microfluidic device for generating clinically-useful information. The response of fresh and cryogenically-frozen primary HNSCC or metastatic lymph node samples to chemotherapy drugs (cisplatin, 5-flurouracil or docetaxel), alone and in combination, were monitored for both proliferation (water-soluble tetrazolium salt metabolism) and cell death biomarker release (lactate dehydrogenase, LDH) “off-chip”. The frozen tissue showed no significant difference in terms of either proliferation or LDH release in comparison with the matched fresh samples. Administration of all drugs caused cell death, in a dose-response manner, with the combination showing the greatest amount of cytotoxicity particularly at days 8 and 9; correlating well with published clinical data. The system described here offers an innovative method for studying the tumour microenvironment in vitro and, through incorporation of relevant analytical modules, provides the basis of a pre-clinical device that can be used to define personalised treatment regimens.

Direct processing of clinically relevant large volume samples for the detection of sexually transmitted infectious agents from urine on a microfluidic device

Urine is a preferred specimen for nucleic acid-based detection of sexually transmitted infections (STIs) but represents a challenge for microfluidic devices due to low analyte concentrations. We present an extraction methodology enabling rapid on-chip nucleic acid purification directly from clinically relevant sample volumes up to 1 ml and subsequent PCR amplification detection.

Paediatric leukaemia DNA methylation profiling using MBD enrichment and SOLiD sequencing on archival bone marrow smears

BACKGROUND: Acute Lymphoblastic Leukaemia (ALL) is the most common cancer in children. Over the past four decades, research has advanced the treatment of this cancer from a less than 60% chance of survival to over 85% today. The causal molecular mechanisms remain unclear. Here, we performed sequencing-based genomic DNA methylation profiling of eight paediatric ALL patients using archived bone marrow smear microscope slides. FINDINGS: SOLiD™ sequencing data was collected from Methyl-Binding Domain (MBD) enriched fractions of genomic DNA. The primary tumour and remission bone marrow sample was analysed from eight patients. Four patients relapsed and the relapsed tumour was analysed. Input and MBD-enriched DNA from each sample was sequenced, aligned to the hg19 reference genome and analysed for enrichment peaks using MACS (Model-based Analysis for ChIP-Seq) and HOMER (Hypergeometric Optimization of Motif EnRichment). In total, 3.67 gigabases (Gb) were sequenced, 2.74 Gb were aligned to the reference genome (average 74.66% alignment efficiency). This dataset enables the interrogation of differential DNA methylation associated with paediatric ALL. Preliminary results reveal concordant regions of enrichment indicative of a DNA methylation signature. CONCLUSION: Our dataset represents one of the first SOLiD™MBD-Seq studies performed on paediatric ALL and is the first to utilise archival bone marrow smears. Differential DNA methylation between cancer and equivalent disease-free tissue can be identified and correlated with existing and published genomic studies. Given the rarity of paediatric haematopoietic malignancies, relative to adult counterparts, our demonstration of the utility of archived bone marrow smear samples to high-throughput methylation sequencing approaches offers tremendous potential to explore the role of DNA methylation in the aetiology of cancer.

Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells

The role of circulating tumor cells (CTCs) in disease diagnosis, prognosis, monitoring of the therapeutic efficacy, and clinical decision making is immense and has attracted tremendous focus in the last decade. We designed and fabricated simple, flat channel microfluidic devices polydimethylsiloxane (PDMS based) functionalized with locked nucleic acid (LNA) modified aptamers (targeting epithelial cell adhesion molecule (EpCAM) and nucleolin expression) for quick and efficient capture of CTCs and cancer cells. With optimized flow rates (10 μl/min), it was revealed that the aptamer modified devices offered reusability for up to six times while retaining optimal capture efficiency (>90%) and specificity. High capture sensitivity (92%) and specificity (100%) was observed in whole blood samples spiked with Caco-2 cells (10-100 cells/ml). Analysis of blood samples obtained from 25 head and neck cancer patients on the EpCAM LNA aptamer functionalized chip revealed that an average count of 5 ± 3 CTCs/ml of blood were captured from 22/25 samples (88%). EpCAM intracellular domain (EpICD) immunohistochemistry on 9 oral squamous cell carcinomas showed the EpICD positivity in the tumor cells, confirming the EpCAM expression in CTCs from head and neck cancers. These microfluidic devices also maintained viability for in vitro culture and characterization. Use of LNA modified aptamers provided added benefits in terms of cost effectiveness due to increased reusability and sustainability of the devices. Our results present a robust, quick, and efficient CTC capture platform with the use of simple PDMS based devices that are easy to fabricate at low cost and have an immense potential in cancer diagnosis, prognosis, and therapeutic planning.

A preliminary study on machinability of super austenitic stainless steel

Stainless steel is the most widely used alloys of steel. The reputed variety of stainless steel having customised material properties as per the design requirements is Duplex Stainless Steel and Austenitic Stainless Steel. The Austenite Stainless Steel alloy has been developed further to be Super Austenitic Stainless Steel (SASS) by increasing the percentage of the alloying elements to form the half or more than the half of the material composition. SASS (Grade-AL-6XN) is an alloy steel containing high percentages of nickel (24%), molybdenum (6%) and chromium (21%). The chemical elements offer high degrees of corrosion resistance, toughness and stability in a large range of hostile environments like petroleum, marine and food processing industries. SASS is often used as a commercially viable substitute to high cost non-ferrous or non-metallic metals. The ability to machine steel effectively and efficiently is of utmost importance in the current competitive market. This paper is an attempt to evaluate the machinability of SASS which has been a classified material so far with very limited research conducted on it. Understanding the machinability of this alloy would assist in the effective forming of this material by metal cutting. The novelty of research associated with this is paper is reasonable taking into consideration the unknowns involved in machining SASS. The experimental design consists of conducting eight milling trials at combination of two different feed rates, 0.1 and 0.15 mm/tooth; cutting speeds, 100 and 150 m/min; Depth of Cut (DoC), 2 and 3 mm and coolant on for all the trials. The cutting tool has two inserts and therefore has two cutting edges. The trial sample is mounted on a dynamometer (type 9257B) to measure the cutting forces during the trials. The cutting force data obtained is later analyzed using DynaWare supplied by Kistler. The machined sample is subjected to surface roughness (Ra) measurement using a 3D optical surface profilometer (Alicona Infinite Focus). A comprehensive metallography process consisting of mounting, polishing and etching was conducted on a before and after machined sample in order to make a comparative analysis of the microstructural changes due to machining. The microstructural images were capture using a digital microscope. The microhardness test were conducted on a Vickers scale (Hv) using a Vickers microhardness tester. Initial bulk hardness testing conducted on the material show that the alloy is having a hardness of 83.4 HRb. This study expects an increase in hardness mostly due to work hardening may be due to phase transformation. The results obtained from the cutting trials are analyzed in order to judge the machinability of the material. Some of the criteria used for machinability evaluation are cutting force analysis, surface texture analysis, metallographic analysis and microhardness analysis. The methodology followed in each aspect of the investigation is similar to and inspired by similar research conducted on other materials. However, the novelty of this research is the investigation of various aspects of machinability and drawing comparisons between each other while attempting to justify each result obtained to the microstructural changes observed which influence the behaviour of the alloy. Due to the limited scope of the paper, machinability criteria such as chip morphology, Metal Removal Rate (MRR) and tool wear are not included in this paper. All aspects are then compared and the optimum machining parameters are justified with a scope for future investigations

Implementation of soft processor based SOC for JPEG compression on FPGA

With the advent of semiconductor process and EDA tools technology, IC designers can integrate more functions. However, to reduce the demand of time-to-market and tackle the increasing complexity of SoC, the need of fast prototyping and testing is growing. Taking advantage of deep submicron technology, modern FPGAs provide a fast and low-cost prototyping with large logic resources and high performance. So the hardware is mapped onto an emulation platform based on FPGA that mimics the behaviour of SOC. In this paper we use FPGA as a system on chip which is then used for image compression by 2-D DCT respectively and proposed SoC for image compression using soft core Microblaze. The JPEG standard defines compression techniques for image data. As a consequence, it allows to store and transfer image data with considerably reduced demand for storage space and bandwidth. From the four processes provided in the JPEG standard, only one, the baseline process is widely used. Proposed SoC for JPEG compression has been implemented on FPGA Spartan-6 SP605 evaluation board using Xilinx platform studio, because field programmable gate array have reconfigurable hardware architecture. Hence the JPEG image with high speed and reduced size can be obtained at low risk and low power consumption of about 0.699W. The proposed SoC for image compression is evaluated at 83.33MHz on Xilinx Spartan-6 FPGA.

Effect of particle characteristics on the two-body abrasive wear behaviour of a pearlitic steel

The specific wear rate and friction coefficient of a pearlitic microstructure subjected to different abrasive environments (i.e. SiC and alumina) were examined. A CSM high temperature pin-on-disc tribometer was used to simulate the two-body abrasive condition (i.e. the metallic surface abrading against the abrasive particles). The characteristics of the abrasive particles (i.e. particle size and density) revealed a significant impact on the amount of material loss. The specific wear rate of the pearlitic microstructure decreased with a reduction in the abrasive particle size, irrespective of the particle type. In addition, distinct particle deterioration mechanisms were observed during the abrasion process, which was largely determined by the abrasive particle size. Attrition, shelling and fracture were some of the dominant particle deterioration mechanisms occurring in both of the abrasive environments. SEM and EDX analysis on the wear debris displayed a unique metallic chip formation with respect to the particle type. Furthermore, the abrading efficiency (i.e. threshold level) of the abrasive particles was identified by means of interrupted abrasive wear tests. The dense packing nature of the alumina abrasive particles resulted in a significantly higher material removal rate than the SiC abrasive environment.

Investigation on the behavior of austenite and ferrite phases at stagnation region in the turning of duplex stainless steel alloys

This paper investigates the deformation mechanisms and plastic behavior of austenite and ferrite phases in duplex stainless steel alloys 2205 and 2507 under chip formation from a machine turning operation. SEM images and EBSD phase mapping of frozen chip root samples detected a build-up of ferrite bands in the stagnation region, and between 65 and 85 pct, more ferrite was identified in the stagnation region compared to austenite. SEM images detected micro-cracks developing in the ferrite phase, indicating ferritic build-up in the stagnation region as a potential triggering mechanism to the formation of built-up edge, as transgranular micro-cracks found in the stagnation region are similar to micro-cracks initiating built-up edge formation. Higher plasticity of austenite due to softening under high strain is seen responsible for the ferrite build-up. Flow lines indicate that austenite is plastically deforming at a greater rate into the chip, while ferrite shows to partition most of the strain during deformation. The loss of annealing twins and activation of multiple slip planes triggered at high strain may explain the highly plastic behavior shown by austenite.

Impacts of wear and geometry response of the cutting tool on machinability of super austenitic stainless steel

This paper presents a study of tool wear and geometry response whenmachinability tests were applied under milling operations onthe Super Austenitic Stainless Steel alloy AL-6XN. Eight milling trials were executed under two cutting speeds, two feed rates, andtwo depths of cuts. Cutting edge profile measurements were performed to reveal response of cutting edge geometry to the cuttingparameters and wear. A scanning electron microscope (SEM) was used to inspect the cutting edges. Results showed the presenceof various types of wear such as adhesion wear and abrasion wear on the tool rake and flank faces. Adhesion wear represents theformation of the built-up edge, crater wear, and chipping, whereas abrasion wear represents flank wear.Thecommonly formed wearwas crater wear. Therefore, the optimum tool life among the executed cutting trails was identified according to minimum lengthand depth of the crater wear.The profile measurements showed the formation of new geometries for the worn cutting edges due toadhesion and abrasion wear and the cutting parameters.The formation of the built-up edge was observed on the rake face of thecutting tool. The microstructure of the built-up edge was investigated using SEM. The built-up edge was found to have the austeniteshear lamellar structure which is identical to the formed shear lamellae of the produced chip.