Method for the generation and cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix

During puberty, pregnancy, lactation and postlactation, breast tissue undergoes extensive remodelling and the disruption of these events can lead to cancer. In vitro studies of mammary tissue and its malignant transformation regularly employ mammary epithelial cells cultivated on matrigel or floating collagen rafts. In these cultures, mammary epithelial cells assemble into three-dimensional structures resembling in vivo acini. We present a novel technique for generating functional mammary constructs without the use of matrix substitutes.

The cross-entropy method for network reliability estimation

Consider a network of unreliable links, modelling for example a communication network. Estimating the reliability of the network-expressed as the probability that certain nodes in the network are connected-is a computationally difficult task. In this paper we study how the Cross-Entropy method can be used to obtain more efficient network reliability estimation procedures. Three techniques of estimation are considered: Crude Monte Carlo and the more sophisticated Permutation Monte Carlo and Merge Process. We show that the Cross-Entropy method yields a speed-up over all three techniques.

On the estimation and comparison of short-rate models using the generalised method of moments

Subsequent to the influential paper of [Chan, K.C., Karolyi, G.A., Longstaff, F.A., Sanders, A.B., 1992. An empirical comparison of alternative models of the short-term interest rate. Journal of Finance 47, 1209-1227], the generalised method of moments (GMM) has been a popular technique for estimation and inference relating to continuous-time models of the short-term interest rate. GMM has been widely employed to estimate model parameters and to assess the goodness-of-fit of competing short-rate specifications. The current paper conducts a series of simulation experiments to document the bias and precision of GMM estimates of short-rate parameters, as well as the size and power of [Hansen, L.P., 1982. Large sample properties of generalised method of moments estimators. Econometrica 50, 1029-1054], J-test of over-identifying restrictions. While the J-test appears to have appropriate size and good power in sample sizes commonly encountered in the short-rate literature, GMM estimates of the speed of mean reversion are shown to be severely biased. Consequently, it is dangerous to draw strong conclusions about the strength of mean reversion using GMM. In contrast, the parameter capturing the levels effect, which is important in differentiating between competing short-rate specifications, is estimated with little bias. (c) 2006 Elsevier B.V. All rights reserved.

Development of an oligonucleotide-based SNP detection method on lateral flow strips using hexapet tages

Detection of point mutations or single nucleotide polymorphisms (SNPs) is important in relation to disease susceptibility or detection in pathogens of mutations determining drug resistance or host range. There is an emergent need for rapid detection methods amenable to point-of-care applications. The purpose of this study was to reduce to practice a novel method for SNP detection and to demonstrate that this technology can be used downstream of nucleic acid amplification. The authors used a model system to develop an oligonucleotide-based SNP detection system on nitrocellulose lateral flow strips. To optimize the assay they used cloned sequences of the herpes simplex virus-1 (HSV-1) DNA polymerase gene into which they introduced a point mutation. The assay system uses chimeric polymerase chain reaction (PCR) primers that incorporate hexameric repeat tags ("hexapet tags"). The chimeric sequences allow capture of amplified products to predefined positions on a lateral flow strip. These "hexapet" sequences have minimal cross-reactivity and allow specific hybridization-based capture of the PCR products at room temperature onto lateral flow strips that have been striped with complementary hexapet tags. The allele-specific amplification was carried out with both mutant and wild-type primer sets present in the PCR mix ("competitive" format). The resulting PCR products carried a hexapet tag that corresponded with either a wild-type or mutant sequence. The lateral flow strips are dropped into the PCR reaction tube, and mutant sequence and wild-type sequences diffuse along the strip and are captured at the corresponding position on the strip. A red line indicative of a positive reaction is visible after 1 minute. Unlike other systems that require separate reactions and strips for each target sequence, this system allows multiplex PCR reactions and multiplex detection on a single strip or other suitable substrates. Unambiguous visual discrimination of a point mutation under room temperature hybridization conditions was achieved with this model system in 10 minutes after PCR. The authors have developed a capture-based hybridization method for the detection and discrimination of HSV-1 DNA polymerase genes that contain a single nucleotide change. It has been demonstrated that the hexapet oligonucleotides can be adapted for hybridization on the lateral flow strip platform for discrimination of SNPs. This is the first step in demonstrating SNP detection on lateral flow using the hexapet oligonucleotide capture system. It is anticipated that this novel system can be widely used in point-of-care settings.

Composite auction method for suppressing unreasonable electricity price spikes in a competitive electricity market

The existence of undesirable electricity price spikes in a competitive electricity market requires an efficient auction mechanism. However, many of the existing auction mechanism have difficulties in suppressing such unreasonable price spikes effectively. A new auction mechanism is proposed to suppress effectively unreasonable price spikes in a competitive electricity market. It optimally combines system marginal price auction and pay as bid auction mechanisms. A threshold value is determined to activate the switching between the marginal price auction and the proposed composite auction. Basically when the system marginal price is higher than the threshold value, the composite auction for high price electricity market is activated. The winning electricity sellers will sell their electricity at the system marginal price or their own bid prices, depending on their rights of being paid at the system marginal price and their offers' impact on suppressing undesirable price spikes. Such economic stimuli discourage sellers from practising economic and physical withholdings. Multiple price caps are proposed to regulate strong market power. We also compare other auction mechanisms to highlight the characteristics of the proposed one. Numerical simulation using the proposed auction mechanism is given to illustrate the procedure of this new auction mechanism.

A target-field method to design circular biplanar coils for asymmetric shim and gradient fields

The paper presents a method for designing circular, shielded biplanar coils that can generate any desired field. A particular feature of these coils is that the target field may be located asymmetrically within the coil. A transverse component of the magnetic field produced by the coil is made to match a prescribed target field over the surfaces of two concentric spheres (the diameter of spherical volume) that define the target field location. The paper shows winding patterns and fields for several gradient and shim coils. It examines the effect that the finite coil size has on the winding patterns, using a Fourier-transform calculation for comparison.

A time-harmonic target-field method for designing unshielded RF coils in MRI

Time-harmonic methods are required in the accurate design of RF coils as operating frequency increases. This paper presents such a method to find a current density solution on the coil that will induce some desired magnetic field upon an asymmetrically located target region within. This inverse method appropriately considers the geometry of the coil via a Fourier series expansion, and incorporates some new regularization penalty functions in the solution process. A new technique is introduced by which the complex, time-dependent current density solution is approximated by a static coil winding pattern. Several winding pattern solutions are given, with more complex winding patterns corresponding to more desirable induced magnetic fields.

Shortcomings of protein removal prior to high performance liquid chromatographic analysis - A case study using method development for BAY 11-7082

During the analytical method development for BAY 11-7082 ((E)-3-[4-methylphenylsulfonyl]-2-propenenitrile), using HPLC-MS-MS and HPLC-UV, we observed that the protein removal process (both ultrafiltration and precipitation method using organic solvents) prior to HPLC brought about a significant reduction in the concentration of this compound. The use of a structurally similar internal standard, BAY 11-7085 ((E)-3-[4-t-butylphenylsulfonyl]-2-propenenitrile), was not effective in compensating for the loss of analyte as the extent of reduction was different to that of the analyte. We present here a systematic investigation of this problem and a new validated method for the determination of BAY 11-7082. (c) 2006 Elsevier B.V. All rights reserved.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization

Chromogenic (CISH) and fluorescent ( FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes ( BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi 29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12; 15)(p12; q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

New method to determine PSD using supercritical adsorption: Applied to methane adsorption in activated carbon

Adsorption of supercritical fluids is increasingly carried out to determine the micropore size distribution. This is largely motivated by the advances in the use of supercritical adsorption in high energy applications, such as hydrogen and methane storage in porous media. Experimental data are reported as mass excess versus pressure, and when these data are matched against the theoretical mass excess, significant errors could occur if the void volume used in the calculation of the experimental mass excess is incorrectly determined [Malbrunot, P.; Vidal, D.; Vermesse, J.; Chahine, R.; Bose, T. K. Langmuir 1997, 13, 539]. 1 The incorrect value for the void volume leads to a wrong description of the maximum in the plot of mass excess versus pressure as well as the part of the isotherm over the pressure region where the isotherm is decreasing. Because of this uncertainty in the maximum and the decreasing part of the isotherm, we propose a new method in which the problems associated with this are completely avoided. Our method involves only the relationship between the amount that is introduced into the adsorption cell and the equilibrium pressure. This information of direct experimental data has two distinct advantages. The first is that the data is the raw data without any manipulation (i.e., involving further calculations), and the second one is that this relationship always monotonically increases with pressure. We will illustrate this new method with the adsorption data of methane in a commercial sample of activated carbon.

Dose determination of haloperidol, risperidone and olanzapine using an in vivo dopamine D-2-receptor occupancy method in the rat

The purpose of the present study was to determine antipsychotic doses that achieve 80% striatal doparnine D-2-receptor occupancy for haloperidol, risperidone and olanzapine in rats. Wistar rats were treated with normal saline vehicle (controls), haloperidol (0.25 and 0.5 mg/kg/ day), risperidone (3, 5 and 6 mg/kg/day) and olanzapine (5 and 10 mg/kg/day) for 7 days via osmotic minipumps. Striatal and cerebellar tissue were collected and in vivo dopamine D2-receptor occupancies were determined using H-3-raclopride. The doses required to achieve dopamine D-2-receptor occupancy of 80% in 11- and 24-week old rats were: haloperidol 0.25 mg/kg/day, risperidone 5 mg/kg/day and olanzapine 10 mg/kg/day. (c) 2006 Elsevier B.V All rights reserved.

A method for screening the temperature dependence of three-dimensional crystal formation

Temperature is an important parameter controlling protein crystal growth. A new temperature-screening system (Thermo-screen) is described consisting of a gradient thermocycler fitted with a special crystallization-plate adapter onto which a 192-well sitting-drop crystallization plate can be mounted (temperature range 277-372 K; maximum temperature gradient 20 K; interval precision 0.3 K). The system allows 16 different conditions to be monitored simultaneously over a range of 12 temperatures and is well suited to conduct wide (similar to 20 K) and fine (similar to 3 K) temperature-optimization screens. It can potentially aid in the determination of temperature phase diagrams and run more complex temperature-cycling experiments for seeding and crystal growth.

A new decoupling method for quadrature coils in magnetic resonance imaging

A powerful decoupling method is introduced to obtain decoupled signal voltages from quadrature coils in magnetic resonance imaging (MRI). The new method uses the knowledge of the position of the signal source in MRI, the active slice, to define a new mutual impedance which accurately quantifies the coupling voltages and enables them to be removed almost completely. Results show that by using the new decoupling method, the percentage errors in the decoupled voltages are of the order of 10(-7)% and isolations between two coils are more than 170 dB.