Pharmacological studies of Cav3.1 T-type calcium channels using automated patch-clamp techniques

T-type calcium channels are involved in a variety of physiological and pathophysiological processes, and thus could be therapeutic targets. However, there is no T-type channel selective blocker for use in clinical practice, demanding a need for the development of novel drugs where a higher-throughput screening system is required. Here we present pharmacological studies on Ca(v)3.1 T-type channels using automated patch-clamp. The IC(50) values obtained from automated patch-clamp and conventional one showed a good correlation (correlation coefficient of 0.82), suggesting that the automated patch-clamp is an efficient and reliable method for ranking the drug potencies for T-type channels.     

Production preparation and numerical control in PCB assembly

In this article, we analyze numerical control problems arising in a component insertion line for printed circuit board mounting. Such a line consists of a number of fully automated placement machines, connected by an automated, carrierless conveyor system. At each machine, the placement device consists of an arm equipped with a number of placement heads. Each head may serve certain component types; there is some freedom in the choice of equipment for each head. Components are supplied to each machine by tape feeders (each containing components of only one type) which are placed at certain feeder positions along the machine. Before actually operating such a line, a careful production preparation phase has to be completed, specifying for each printed circuit board type the exact way in which such a board should be mounted. Problems arising in this production preparation phase include: the choice of heads to be mounted on the placement arm of each machine, the choice of components to be placed at each machine (workload balancing), an assignment of feeders to feeder positions on each machine, clustering of components to be placed in one pick-and-place move, and the sequencing of component insertions at one machine. Formulations and solutions for all these problems are given and numerical results for some industrial examples are given. The numerical control system developed has been tested and implemented and has resulted in software, which is in use at several sites of Philips' Electronics.     

A Novel Image Recuperation Approach for Diagnosing and Ranking Retinopathy Disease Level Using Diabetic Fundus Image

Retinal fundus images are widely used in diagnosing and providing treatment for several eye diseases. Prior works using retinal fundus images detected the presence of exudation with the aid of publicly available dataset using extensive segmentation process. Though it was proved to be computationally efficient, it failed to create a diabetic retinopathy feature selection system for transparently diagnosing the disease state. Also the diagnosis of diseases did not employ machine learning methods to categorize candidate fundus images into true positive and true negative ratio. Several candidate fundus images did not include more detailed feature selection technique for diabetic retinopathy. To apply machine learning methods and classify the candidate fundus images on the basis of sliding window a method called, Diabetic Fundus Image Recuperation (DFIR) is designed in this paper. The initial phase of DFIR method select the feature of optic cup in digital retinal fundus images based on Sliding Window Approach. With this, the disease state for diabetic retinopathy is assessed. The feature selection in DFIR method uses collection of sliding windows to obtain the features based on the histogram value. The histogram based feature selection with the aid of Group Sparsity Non-overlapping function provides more detailed information of features. Using Support Vector Model in the second phase, the DFIR method based on Spiral Basis Function effectively ranks the diabetic retinopathy diseases. The ranking of disease level for each candidate set provides a much promising result for developing practically automated diabetic retinopathy diagnosis system. Experimental work on digital fundus images using the DFIR method performs research on the factors such as sensitivity, specificity rate, ranking efficiency and feature selection time.     

Monitoring of recombinant protein production using bioluminescence in a semiautomated fermentation process

On-line optimization of fermentation processes can be greatly aided by the availability of information on the physiological state of the cell. The goal of our "BioLux" research project was to design a recombinant cell capable of intracellular monitoring of product synthesis and to use it as part of an automated fermentation system. A recombinant plasmid was constructed containing an inducible promoter that controls the gene coding for a model protein and the genes necessary for bioluminescence. The cells were cultured in microfermenters equipped with an on-line turbidity sensor and a specially designed on-line light sensor capable of continuous measurement of bioluminescence. Initial studies were done under simple culture conditions, and a linear correlation between luminescence and protein production was obtained. Such specially designed recombinant bioluminescent cells can potentially be applied for model-based inference of intracellular product formation, as well as for optimization and control of recombinant fermentation processes.     

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike ¹. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex ². An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative ^{3, 4}: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening ⁵. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons ⁶; recently, we have also reported progress towards interrogating mammalian neurons ⁷. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre ⁸, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion. 相似文献   

Computational model for monitoring cholesterol metabolism

A non-deterministic finite automaton is designed to observe the cholesterol metabolism with the states of acceptance and rejection. The acceptance state of the automaton depicts the normal level of metabolism and production of good cholesterol as an end product. The rejection state of this machine shows the inhibition of enzymatic activity in cholesterol synthesis and removal of free fatty acids. The deficiency in human cholesterol metabolism pathway results in abnormal accumulation of cholesterol in plasma, arterial tissues leading to diseases such as hypercholesterolemia, atherosclerosis respectively and formation of gallstones. The designed machine can be used to monitor the cholesterol metabolism at molecular level through regulation of enzymes involved in the biosynthesis and metabolism of cholesterol for the treatment of diseases incident due to the respective metabolic disorder. In addition, an algorithm for this machine has been developed to compare the programmed string with the given string. This study demonstrates the construction of a machine that is used for the development of molecular targeted therapy for the disorders in cholesterol metabolism.     

De novo design of foldable proteins with smooth folding funnel: automated negative design and experimental verification

De novo sequence design of foldable proteins provides a way of investigating principles of protein architecture. We performed fully automated sequence design for a target structure having a three-helix bundle topology and synthesized the designed sequences. Our design principle is different from the conventional approach, in that instead of optimizing interactions within the target structure, we design the global shape of the protein folding funnel. This includes automated implementation of negative design by explicitly requiring higher free energy of the denatured state. The designed sequences do not have significant similarity to those of any natural proteins. The NMR and CD spectroscopic data indicated that one designed sequence has a well-defined three-dimensional structure as well as alpha-helical content consistent with the target.     

Exploring inconsistencies in genome-wide protein function annotations: a machine learning approach

Background  

Incorrectly annotated sequence data are becoming more commonplace as databases increasingly rely on automated techniques for annotation. Hence, there is an urgent need for computational methods for checking consistency of such annotations against independent sources of evidence and detecting potential annotation errors. We show how a machine learning approach designed to automatically predict a protein's Gene Ontology (GO) functional class can be employed to identify potential gene annotation errors.     

Automated molecular microscopy: the new Leginon system

We report here on the current state of our efforts in automated molecular microscopy. Our primary automated data acquisition software system, Leginon, has been completely redesigned over the past two years. The new distributed system has been developed using the Python programming language and is compatible with both Linux and Windows operating systems. The new flexible architecture was designed to allow for the development of customized data collection protocols, several of which are described here. The system has been used to acquire data for approximately 150 experiments and we have demonstrated the capacity for high throughput data acquisition by acquiring images of more than 100,000 particles in a single session at the microscope.     

High-resolution image analysis

In many departments of cytology, cytogenetics, hematology, and pathology, research projects using high-resolution computerized microscopy are now being mounted for computation of morphometric measurements on various structural components, as well as for determination of cellular DNA content. The majority of these measurements are made in a partially automated, computer-assisted mode, wherein there is strong interaction between the user and the computerized microscope. At the same time, full automation has been accomplished for both sample preparation and sample examination for clinical determination of the white blood cell differential count. At the time of writing, approximately 1,000 robot differential counting microscopes are in the field, analyzing images of human white blood cells, red blood cells, and platelets at the overall rate of about 100,000 slides per day. This mammoth through-put represents a major accomplishment in the application of machine vision to automated microscopy for hematology. In other areas of automated high-resolution microscopy, such as cytology and cytogenetics, no commercial instruments are available (although a few metaphase-finding machines are available and other new machines have been announced during the past year). This is a disappointing product, considering the nearly half century of research effort in these areas. This paper provides examples of the state of the art in automation of cell analysis for blood smears, cervical smears, and chromosome preparations. Also treated are new developments in multi-resolution automated microscopy, where images are now being generated and analyzed by a single machine over a range of 64:1 magnification and from 10,000 X 20,000 to 500 X 500 in total picture elements (pixels). Examples of images of human lymph node and liver tissue are presented. Semi-automated systems are not treated, although there is mention of recent research in the automation of tissue analysis.     

Automated whole-cell patch-clamp electrophysiology of neurons in vivo

Whole-cell patch-clamp electrophysiology of neurons is a gold-standard technique for high-fidelity analysis of the biophysical mechanisms of neural computation and pathology, but it requires great skill to perform. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes. We demonstrate good yield, throughput and quality of automated intracellular recording in mouse cortex and hippocampus.     

Image analysis combined with quantitative cytochemistry. Results and instrumental developments for cancer diagnosis

This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%.(ABSTRACT TRUNCATED AT 250 WORDS)     

基于USB数据采集器的膜片钳系统软件设计

为了控制新的基于USB总线的数据采集器进行膜片钳实验,设计和实现了能应用于Windows 2000／XP操作系统的膜片钳系统软件。软件运用面向对象程序设计方法和统一建模语言,实现了封接测试．电压门控和配体门控等实验模式。实验结果表明,该系统软件能够精确控制数据采集器进行高速数据采集,满足细胞生理实验的要求。     

Reconfigurable modular automation systems for automotive power-train manufacture

This paper describes research towards the realization of reconfigurable modular automated machines and the associated engineering methods and tools necessary to support their lifecycle needs. UK-based research, in collaboration with the Ford Motor Company and several machine builders, has resulted in the development of full-scale prototype reconfigurable modular automation systems for both engine assembly and machining applications. The implementation of an assembly system is featured in this paper. An engineering environment and associated reconfigurable component-based control system architecture have been created aimed at supporting the lifecycle needs of a new generation of agile automated systems, i.e., providing reconfigurable, easily scalable automated machinery. This approach has the potential to fit within a wider collaborative automation strategy where manufacturing systems are implemented as a conglomerate of distributed, autonomous, and reusable units.     

Improved perfusion conditions for patch-clamp recordings on human erythrocytes

Various configurations of the patch-clamp method are powerful tools for examining the transport of charged solutes across biological membranes. Originally developed for the study of relatively large cells which adhere to solid surfaces under in vitro culture, these methods have been increasingly applied to small cells or organelles in suspension. Under these conditions, a number of significant technical problems may arise as a result of the smaller geometry. Here, we examined these problems using human erythrocytes infected with the malaria parasite, Plasmodium falciparum, a system where experimental differences and the technical difficulty of erythrocyte patch-clamp have hindered universal agreement on the properties of the induced ion channels. We found that patch-clamp recordings on infected erythrocytes are especially susceptible to artifacts from mechanical perturbations due to solution flow around the cell. To minimize these artifacts, we designed a new perfusion chamber whose geometry allows controlled solution flow around the fragile erythrocyte. Not only were recordings acquired in this chamber significantly less susceptible to perfusion artifacts, but the chamber permitted rapid and reversible application of known inhibitors with negligible mechanical agitation. Electrophysiological recordings then faithfully reproduced several findings made with more traditional methods. The new perfusion chamber should also be useful for patch-clamp recordings on blood cells, protoplasts, and organelles.     

Cyclops: new modular software suite for cryo-EM

Cyclops is a new computer program designed as a graphical front-end that allows easy control and interaction with tasks and programs for 3D reconstruction of biological complexes using cryo-electron microscopy. Cyclops' current plug-ins are designed for automated particle picking and include two new algorithms, automated carbon masking and quaternion based rotation space sampling, which are also presented here. Additional plug-ins are in the pipeline. Cyclops allows straightforward organization and visualization of all data and tasks and allows both interactive and batch-wise processing. Furthermore, it was designed for straightforward implementation in grid architectures. As a front-end to a collection of programs it provides a common interface to these programs, thus enhancing the usability of the suite and the productivity of the user.     

Design of net-charged abc-type collagen heterotrimers

Net-negatively-charged heterospecific A:B:C collagen peptide heterotrimers were designed using an automated computational approach. The design algorithm considers both target stability and the energy gap between the target states and misfolded competing states. Structural characterization indicates the net-negative charge balance on the new designs enhances the specificity of the target state at the expense of its stability.     

A miniaturized planar patch-clamp system for transportable use

In the last decade, planar patch-clamp (PPC) has emerged as an innovative technology allowing parallel recordings of cellular electrophysiological activity on planar substrates. If PPC is widely adopted by the pharmaceutical sector, it remains poorly extended to other areas (i.e. environment and safety organizations) probably because of the large, expensive and non-easily transportable format of those commercial equipments. The present work describes for the first time a new compact and transportable planar patch-clamp system (named Toxint'patch or TIP, for Toxin detection with integrated patch-clamp) focusing on environmental matters and meant to be used in coastal laboratories, for direct on-site monitoring of the seawater and shellfish quality. The TIP system incorporates silicon chips tailored to monitor cellular ionic currents from cultured cells stably expressing a phycotoxin molecular target. The functionality of this novel briefcase-sized PPC system is described in terms of fluidic control, electronic performances with amplifying and filtering boards and of user interface for data acquisition and control implemented on a computer.     

Automated patch-clamp technique: increased throughput in functional characterization and in pharmacological screening of small-conductance Ca2+ release-activated Ca2+ channels

The suitability of an automated patch clamp for the characterization and pharmacological screening of calcium release-activated calcium (CRAC) channels endogenously expressed in RBL-2H3 cells was explored with the QPatch system. CRAC currents (I( CRAC)) are small, and thus precise recordings require high signal-to-noise ratios obtained by high seal resistances. Automated whole-cell establishment resulted in membrane resistances of 1728 +/- 226 MOmega (n = 44). CRAC channels were activated by a number of methods that raise intracellular calcium concentration, including EGTA, ionomycin, Ins(1,4,5)P(3), and thapsigargin. I(CRAC) whole-cell currents ranged from 30 to 120 pA with rise times of 40 to 150 s. An initial delay in current activation was observed in particular when I(CRAC) was activated by passive store depletion using EGTA. Apparent rundown of I(CRAC) was commonly observed, and the current could be reactivated by subsequent addition of thapsigargin. I(CRAC) was blocked by SKF-96365 and 2-APB with IC(50) values of 4.7 +/- 1.1 microM (n = 9) and 7.5 +/- 0.7 (n = 9) microM, respectively. The potencies of these blockers were similar to values reported for I(CRAC) in similar conventional patch-clamp experiments. The study demonstrates that CRAC channels can be rapidly and efficiently targeted with automated patch-clamp techniques for characterization of physiological and pharmacological properties.