CiteULike: Tag technique

Deciphering Protein–Protein Interactions. Part I. Experimental Techniques and Databases

Benjamin Shoemaker — 2007-03-31T13:18:05-00:00

PLoS Computational Biology, Vol. 3, No. 3. (1 March 2007), e42.

Competitive Strategy: Techniques for Analyzing Industries and Competitors

Michael Porter — 2007-05-15T11:01:11-00:00

(01 June 1998)

Now nearing its 60th printing in English and translated into nineteen languages, Michael E. Porter's Competitive Strategy has transformed the theory, practice, and teaching of business strategy throughout the world. Electrifying in its simplicity -- like all great breakthroughs -- Porter's analysis of industries captures the complexity of industry competition in five underlying forces. Porter introduces one of the most powerful competitive tools yet developed: his three generic strategies -- lowest cost, differentiation, and focus -- which bring structure to the task of strategic positioning. He shows how competitive advantage can be defined in terms of relative cost and relative prices, thus linking it directly to profitability, and presents a whole new perspective on how profit is created and divided. In the almost two decades since publication, Porter's framework for predicting competitor behavior has transformed the way in which companies look at their rivals and has given rise to the new discipline of competitor assessment.More than a million managers in both large and small companies, investment analysts, consultants, students, and scholars throughout the world have internalized Porter's ideas and applied them to assess industries, understand competitors,, and choose competitive positions. The ideas in the book address the underlying fundamentals of competition in a way that is independent of the specifics of the ways companies go about competing.Competitive Strategy has filled a void in management thinking. It provides an enduring foundation and grounding point on which all subsequent work can be built. By bringing a disciplined structure to the question of how firms achieve superior profitability, Porter's rich frameworks and deep insights comprise a sophisticated view of competition unsurpassed in the last quarter-century.

A two-phase sampling technique for information extraction from hidden web databases

YL Hedley — 2004-12-02T14:35:58-00:00

(2004), pp. 1-8.

Convexity Rule for Shape Decomposition Based on Contour Evolution Hierarchy

Longin Latecki — 2007-02-22T13:00:36-00:00

We concentrate here on decomposition of 2D objects into meaningful parts of visual form, shortly visual parts. It is a simple observation that convex parts of objects determine visual parts. However, the problem is that many significant visual parts are not convex, since a visual part may have concavities. We solve this problem by identifying convex parts at different stages of a proposed contour evolution method in which significant visual parts will become convex object parts at higher...

Shape representation by multiscale contour approximation

A Bengtsson — 2007-02-22T12:51:40-00:00

Pattern Analysis and Machine Intelligence, IEEE Transactions on, Vol. 13, No. 1. (1991), pp. 85-93.

An approach is presented for deriving qualitative descriptions of contours containing structures at different (unknown) scales. The descriptions are in terms of straight arcs, curved arcs with sign of curvature, corners, and points delimiting the arcs: inflexion points and transitions from straight to curved. Furthermore, the tangents at these points are derived. The approach is based on the construction of a hierarchic family of polygons, having the scale-space property of causality; structure can only disappear as scale goes from fine to coarse. Using the principle that structures that are stable over scale represent significant properties, the features of the descriptive representations are then derived

Multiscale Contour Segmentation and Approximation: An Algorithm Based on the Geometry of Regular Inscribed Polygons

Robert Bergevin — 2007-02-22T12:43:29-00:00

Computer Vision and Image Understanding, Vol. 71, No. 1. (July 1998), pp. 55-73.

This paper describes an algorithm to segment and approximate a contour into circular arcs and straight line segments. This algorithm finds an adequate set of circular arcs and straight line segments to describe the contour shape. The resulting description respects a large number of shape representation criteria much better than any previous method. These criteria support efficient, general-purpose, unique, shape preserving, local, robust, stable, invariant, multiscale, coherent, hierarchical, and multipart shape representations. The adequacy of the algorithm is a direct consequence of the formal computational theory on which it is based. The resulting description may depend on sensor accuracy, sampling step size, minimum and maximum contour curvatures, and scale. The algorithm is thus parameterized using a small number of thresholds related to these data set and task properties. Predictions regarding the ground-truth performance of the algorithm can be made given some knowledge of the above properties. Experimental comparative results are presented for contour data of varying complexity.

Making the Most of Using Depth Reasoning to Label Line Drawings of Engineering Objects

P Varley — 2007-02-09T10:36:23-00:00

(2004)

Automatic creation of B-rep models of engineering objects from freehand sketches would benefit designers. A subgoal is to take a single line drawing (with hidden lines removed), and from it deduce an initial 3D geometric realisation of the visible part of the object. Junction and line labels, and provisional depth coordinates, are important components of this frontal geometry.

Locating perceptually salient points on planar curves

MA Fischler — 2007-02-22T13:03:59-00:00

Pattern Analysis and Machine Intelligence, IEEE Transactions on, Vol. 16, No. 2. (1994), pp. 113-129.

This paper describes the underlying ideas and algorithmic details of a computer program that performs at a human level of competence for a significant subset of the curve partitioning task. It extends and rounds out the technique and philosophical approach originally presented by Fischler and Bolles (1986). In particular, it provides a unified strategy for selecting and dealing with interactions between salient points, even when these points are salient at different scales of resolution. Experimental results are presented involving on the order of 1000 real and synthetically generated images

Towards an Ideal Rowing Technique for Performance: The Contributions from Biomechanics

Clara Soper — 2007-08-22T17:32:26-00:00

(2004), pp. 825-848.

At international standard, sculling (two oars) and rowing (one oar) are competed on-water over 2000m. Race time is the critical measure of performance and is determined from mean skiff velocity during a race. Although a high proportion of race training is completed on-water, rowing ergometers are commonly used for performance testing, technique coaching, crew selection or for training during poor weather. Rowing biomechanics research has aimed to identify characteristics of successful sculling and sweep rowing strokes; however, biomechanical predictors of 2000m rowing performance are indistinct in the literature. If specific biomechanical parameters distinguish between ability levels and successful or unsuccessful techniques, these attributes can be considered when modifying technique or predicting future rowing performance. The kinematics and kinetics of the sculling and rowing movements have been described on ergometers, on-water and for novice and elite male and female rowers, but there is limited research on the ideal technique or how a rower’s anthropometry or boat set-up could help improve/optimise their rowing performance. Currently viewing the technique and providing verbal feedback is the primary tool used by a coach to help improve a rower’s technique and performance. The greater use of customised telemetered sensors on the rowing skiff can assist the coach and biomechanist with judging when performance (skiff velocity) improves with some form of intervention.

Quantifying small numbers of antibodies with a 'near-universal' protein-DNA chimera

Ian Burbulis — 2007-11-30T17:39:32-00:00

Nature Methods, Vol. 4, No. 12. (04 November 2007), pp. 1011-1013.

Predictive haemodynamics in a one-dimensional human carotid artery bifurcation. Part I: Application to stent design.

VB Kolachalama — 2007-12-16T23:23:31-00:00

IEEE Trans Biomed Eng, Vol. 54, No. 5. (May 2007), pp. 802-812.

A diagnostic technique is proposed to identify patients with carotid stenosis who could most benefit from angioplasty followed by stent implantation. This methodology involves performing a parametric study to investigate the haemodynamic behavior due to alterations in the stenosis shapes in the internal carotid artery (ICA). A pulsatile 1-D Navier-Stokes solver incorporating fluid-wall interactions for a Newtonian fluid which predicts pressure and flow in the human carotid artery bifurcation is used for the numerical simulations. In order to assess the performance of each individual geometry, we introduce pressure variation factor as a metric to directly compare the global effect of variations in the geometry. It is shown that the probability of an overall catastrophic effect is higher when the stenosis is present in the upstream segment of the ICA. Furthermore, maximum pressure is used to quantify the local effects of geometry changes. The location of the peak and extent of stenosis are found not to influence maximum pressure. We also show how these metrics respond after stent deployment into the stenosed part of the ICA. In particular, it is found that localized pressure peaks do not depend on the length of a stent. Finally, we demonstrate how these metrics may be applied to cost-effectively predict the benefit of stenting.

National survey of the technique of intravitreal triamcinolone injection in the United Kingdom

DR Anijeet — 2006-01-27T13:18:43-00:00

Eye, Vol. aop, No. current.

Single-neuron labeling with inducible Cre-mediated knockout in transgenic mice.

Paul Young — 2008-05-09T08:59:37-00:00

Nature neuroscience (4 May 2008)

To facilitate a functional analysis of neuronal connectivity in a mammalian nervous system that is tightly packed with billions of cells, we developed a new technique that uses inducible genetic manipulations in fluorescently labeled single neurons in mice. Our technique, single-neuron labeling with inducible Cre-mediated knockout (SLICK), is achieved by coexpressing a drug-inducible form of Cre recombinase and a fluorescent protein in a small subsets of neurons, thus combining the powerful Cre recombinase system for conditional genetic manipulation with fluorescent labeling of single neurons for imaging. Here, we demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we applied SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals and demonstrate a Cre-loxP compatible system for dissecting gene functions in single identifiable neurons.

Tissue-specific and reversible RNA interference in transgenic mice

Ross Dickins — 2007-06-28T22:35:21-00:00

Nature Genetics, Vol. 39, No. 7. (17 June 2007), pp. 914-921.

A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system.

FA Edwards — 2007-08-13T13:59:07-00:00

Pflugers Arch, Vol. 414, No. 5. (September 1989), pp. 600-612.

(1) A preparation is described which allows patch clamp recordings to be made on mammalian central nervous system (CNS) neurones in situ. (2) A vibrating tissue slicer was used to cut thin slices in which individual neurones could be identified visually. Localized cleaning of cell somata with physiological saline freed the cell membrane, allowing the formation of a high resistance seal between the membrane and the patch pipette. (3) The various configurations of the patch clamp technique were used to demonstrate recording of membrane potential, whole cell currents and single channel currents from neurones and isolated patches. (4) The patch clamp technique was used to record from neurones filled with fluorescent dyes. Staining was achieved by filling cells during recording or by previous retrograde labelling. (5) Thin slice cleaning and patch clamp techniques were shown to be applicable to the spinal cord and almost any brain region and to various species. These techniques are also applicable to animals of a wide variety of postnatal ages, from newborn to adult.

Patch-clamp recording from mossy fiber terminals in hippocampal slices.

J Bischofberger — 2007-08-13T13:55:31-00:00

Nat Protoc, Vol. 1, No. 4. (2006), pp. 2075-2081.

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size. Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2-5 microm. The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting. Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice. Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.

Blue native PAGE.

I Wittig — 2007-04-09T21:18:15-00:00

Nat Protoc, Vol. 1, No. 1. (2006), pp. 418-428.

Blue native PAGE (BN-PAGE) can be used for one-step isolation of protein complexes from biological membranes and total cell and tissue homogenates. It can also be used to determine native protein masses and oligomeric states and to identify physiological protein-protein interactions. Native complexes are recovered from gels by electroelution or diffusion and are used for 2D crystallization and electron microscopy or analyzed by in-gel activity assays or by native electroblotting and immunodetection. In this protocol, we describe methodology to perform BN-PAGE followed by (i) native extraction or native electroblotting of separated proteins, or (ii) a second dimension of tricine-SDS-PAGE or modified BN-PAGE, or (iii) a second dimension of isoelectric focusing (IEF) followed by a third dimension of tricine-SDS-PAGE for the separation of subunits of complexes. These protocols for 2D and 3D PAGE can be completed in 2 and 3 days.

Darwinian Evolution on a Chip

Brian Paegel — 2008-04-08T18:05:48-00:00

PLoS Biology, Vol. 6, No. 4. (1 April 2008), e85.

Computer control of Darwinian evolution has been demonstrated by propagating a population of RNA enzymes in a microfluidic device. The RNA population was challenged to catalyze the ligation of an oligonucleotide substrate under conditions of progressively lower substrate concentrations. A microchip-based serial dilution circuit automated an exponential growth phase followed by a 10-fold dilution, which was repeated for 500 log-growth iterations. Evolution was observed in real time as the population adapted and achieved progressively faster growth rates over time. The final evolved enzyme contained a set of 11 mutations that conferred a 90-fold improvement in substrate utilization, coinciding with the applied selective pressure. This system reduces evolution to a microfluidic algorithm, allowing the experimenter to observe and manipulate adaptation.

Fast Burst Correlation of Financial Data

Michail Vlachos — 2007-11-16T11:37:02-00:00

Knowledge Discovery in Databases: PKDD 2005 (2005), pp. 368-379.

We examine the problem of monitoring and identification of correlated burst patterns in multi-stream time series databases. Our methodology is comprised of two steps: a burst detection part, followed by a burst indexing step. The burst detection scheme imposes a variable threshold on the examined data and takes advantage of the skewed distribution that is typically encountered in many applications. The indexing step utilizes a memory-based interval index for effectively identifying the overlapping burst regions. While the focus of this work is on financial data, the proposed methods and data-structures can find applications for anomaly or novelty detection in telecommunications and network traffic, as well as in medical data. Finally, we manifest the real-time response of our burst indexing technique, and demonstrate the usefulness of the approach for correlating surprising volume trading events at the NY stock exchange.

A Comparison of String Distance Metrics for Name-Matching Tasks

William Cohen — 2006-11-28T00:06:50-00:00

(2003)

Using an open-source, Java toolkit of name-matching methods, we experimentally compare string distance metrics on the task of matching entity names. We investigate a number of different metrics proposed by different communities, including edit-distance metrics, fast heuristic string comparators , token-based distance metrics, and hybrid methods. Overall, the best-performing method is a hybrid scheme combining a TFIDF weighting scheme, which is widely used in information retrieval, with ...

Clustering by Compression

R Cilibrasi — 2006-07-11T14:03:35-00:00

Information Theory, IEEE Transactions on, Vol. 51, No. 4. (2005), pp. 1523-1545.

We present a new method for clustering based on compression. The method does not use subject-specific features or background knowledge, and works as follows: First, we determine a parameter-free, universal, similarity distance, the normalized compression distance or NCD, computed from the lengths of compressed data files (singly and in pairwise concatenation). Second, we apply a hierarchical clustering method. The NCD is not restricted to a specific application area, and works across application area boundaries. A theoretical precursor, the normalized information distance, co-developed by one of the authors, is provably optimal. However, the optimality comes at the price of using the noncomputable notion of Kolmogorov complexity. We propose axioms to capture the real-world setting, and show that the NCD approximates optimality. To extract a hierarchy of clusters from the distance matrix, we determine a dendrogram (ternary tree) by a new quartet method and a fast heuristic to implement it. The method is implemented and available as public software, and is robust under choice of different compressors. To substantiate our claims of universality and robustness, we report evidence of successful application in areas as diverse as genomics, virology, languages, literature, music, handwritten digits, astronomy, and combinations of objects from completely different domains, using statistical, dictionary, and block sorting compressors. In genomics, we presented new evidence for major questions in Mammalian evolution, based on whole-mitochondrial genomic analysis: the Eutherian orders and the Marsupionta hypothesis against the Theria hypothesis.

Identifying similarities, periodicities and bursts for online search queries

Michail Vlachos — 2006-11-29T14:21:03-00:00

(2004), pp. 131-142.

Automatic Meaning Discovery Using Google

Rudi Cilibrasi — 2008-02-15T17:23:13-00:00

(2004)

We have found a method to automatically extract the meaning of words and phrases from the world-wide-web using Google page counts. The approach is novel in its unrestricted problem domain, simplicity of implementation, and manifestly ontological underpinnings. The world-wide-web is the largest database on earth, and the latent semantic context information entered by millions of independent users averages out to provide automatic meaning of useful quality. We demonstrate positive...

Language Trees and Zipping

Dario Benedetto — 2005-09-09T06:35:45-00:00

Physical Review Letters, Vol. 88, No. 4. (2002)

In this Letter we present a very general method for extracting information from a generic string of characters, e.g., a text, a DNA sequence, or a time series. Based on data-compression techniques, its key point is the computation of a suitable measure of the remoteness of two bodies of knowledge. We present the implementation of the method to linguistic motivated problems, featuring highly accurate results for language recognition, authorship attribution, and language classification.

Multi-document summarization using off the shelf compression software

Amardeep Grewal — 2007-09-19T16:59:55-00:00

(2003), pp. 17-24.

Methodology for validating software metrics

NF Schneidewind — 2006-11-30T10:20:36-00:00

Software Engineering, IEEE Transactions on, Vol. 18, No. 5. (1992), pp. 410-422.

A comprehensive metrics validation methodology is proposed that has six validity criteria, which support the quality functions assessment, control, and prediction, where quality functions are activities conducted by software organizations for the purpose of achieving project quality goals. Six criteria are defined and illustrated: association, consistency, discriminative power, tracking, predictability, and repeatability. The author shows that nonparametric statistical methods such as contingency tables play an important role in evaluating metrics against the validity criteria. Examples emphasizing the discriminative power validity criterion are presented. A metrics validation process is defined that integrates quality factors, metrics, and quality functions

Towards optimizing rowing technique.

B Sanderson — 2007-08-08T14:06:23-00:00

Medicine and science in sports and exercise, Vol. 18, No. 4. (August 1986), pp. 454-468.

An equation is developed (and solved) to describe the speed of a rowing boat as a function of the movement of the sculler's center of mass relative to the boat and the force applied. A method is presented to determine the degree to which fluctuations in boat speed through the rowing cycle affect the amount of power necessary to propel the boat at some mean speed. By changing technique, it is possible to modify these fluctuations in order to achieve the higher mean speed for a given amount of propulsive power. An approximate calculation of the ratio of the power put into the boat's motion to the power lost as water movement in the oar "puddle" suggests that increasing the blade area of the oar will result in improved efficiency. A similarity analysis is undertaken to see if large rowers have an advantage over small rowers in races. Dependence of drag coefficients on scale suggest they do; however, this advantage is very small and would be largely compensated for if boats were made optimally light (in which case the ratio of boat mass to body mass decreases as body mass decreases). Regulations of international rowing fix a minimum boat mass regardless of the rowers mass, thereby discriminating against smaller rowers. Equations are developed to show how stroke rate should scale with body mass for geometrically similar rowers. The ratio of power expended in internal motions to power expended propelling the boat is investigated.

Multi-finger and whole hand gestural interaction techniques for multi-user tabletop displays

Mike Wu — 2006-09-08T02:29:50-00:00

(2003), pp. 193-202.

Web-Based GeneChip Analysis System for Large-Scale Collaborative Projects.

Manhong Dai — 2007-07-12T14:19:15-00:00

Bioinformatics (22 June 2007)

SUMMARY: The Web-Based GeneChip Analysis System (WGAS) is developed to overcome limitations in analysis setup efficiency, data and procedure sharing, as well as security issues in existing commercial and public domain solutions. It also incorporates unique functions and resources for more accurate and flexible GeneChip analysis. AVAILABILITY: WGAS is freely available at:http://arrayanalysis.mbni.med.umich.edu/arrayanalysis.html.

QPRIMER: A Quick Web-based Application for Designing Conserved PCR Primers from Multigenome Alignments.

Namshin Kim — 2007-07-09T21:53:25-00:00

Bioinformatics (28 June 2007)

SUMMARY: We have developed a quick web-based application for designing conserved genomic PCR and RT-PCR primers from multigenome alignments targeting specific exons or introns. We used Pygr (The Python Graph Database Framework for Bioinformatics) to query intervals from multigenome alignments, which gives us less than a millisecond access to any intervals of any genome within multigenome alignments. PRIMER3 was used to extract optimal primers from a gene of interest. QPRIMER creates an electronic genomic PCR image from a set of conserved primers as well as summary pages for primer alignments and products. QPRIMER supports human, mouse, rat, chicken, dog, zebrafish, and fruit fly. AVAILABILITY: http://www.bioinformatics.ucla.edu/QPRIMER/

Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends.

Chunsun Zhang — 2007-06-20T06:56:14-00:00

Nucleic Acids Res (18 June 2007)

The possibility of performing fast and small-volume nucleic acid amplification and analysis on a single chip has attracted great interest. Devices based on this idea, referred to as micro total analysis, microfluidic analysis, or simply 'Lab on a chip' systems, have witnessed steady advances over the last several years. Here, we summarize recent research on chip substrates, surface treatments, PCR reaction volume and speed, architecture, approaches to eliminating cross-contamination and control and measurement of temperature and liquid flow. We also discuss product-detection methods, integration of functional components, biological samples used in PCR chips, potential applications and other practical issues related to implementation of lab-on-a-chip technologies.

Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells.

S Gerecht — 2007-07-12T13:58:25-00:00

Proc Natl Acad Sci U S A, Vol. 104, No. 27. (3 July 2007), pp. 11298-11303.

Control of self-renewal and differentiation of human ES cells (hESCs) remains a challenge. This is largely due to the use of culture systems that involve poorly defined animal products and do not mimic the normal developmental milieu. Routine protocols involve the propagation of hESCs on mouse fibroblast or human feeder layers, enzymatic cell removal, and spontaneous differentiation in cultures of embryoid bodies, and each of these steps involves significant variability of culture conditions. We report that a completely synthetic hydrogel matrix can support (i) long-term self-renewal of hESCs in the presence of conditioned medium from mouse embryonic fibroblast feeder layers, and (ii) direct cell differentiation. Hyaluronic acid (HA) hydrogels were selected because of the role of HA in early development and feeder layer cultures of hESCs and the controllability of hydrogel architecture, mechanics, and degradation. When encapsulated in 3D HA hydrogels (but not within other hydrogels or in monolayer cultures on HA), hESCs maintained their undifferentiated state, preserved their normal karyotype, and maintained their full differentiation capacity as indicated by embryoid body formation. Differentiation could be induced within the same hydrogel by simply altering soluble factors. We therefore propose that HA hydrogels, with their developmentally relevant composition and tunable physical properties, provide a unique microenvironment for the self-renewal and differentiation of hESCs.

Modulating the Expression of Disease Genes with RNA-Based Therapy.

Matthew Wood — 2007-07-12T08:09:02-00:00

PLoS Genet, Vol. 3, No. 6. (29 June 2007)

Conventional gene therapy has focused largely on gene replacement in target cells. However, progress from basic research to the clinic has been slow for reasons relating principally to the challenges of heterologous DNA delivery and regulation in vivo. Alternative approaches targeting RNA have the potential to circumvent some of these difficulties, particularly as the active therapeutic molecules are usually short oligonucleotides and the target gene transcript is under endogenous regulation. RNA-based strategies offer a series of novel therapeutic applications, including altered processing of the target pre-mRNA transcript, reprogramming of genetic defects through mRNA repair, and the targeted silencing of allele- or isoform-specific gene transcripts. This review examines the potential of RNA therapeutics, focusing on antisense oligonucleotide modification of pre-mRNA splicing, methods for pre-mRNA trans-splicing, and the isoform- and allele-specific applications of RNA interference.

Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements

Marcel Coolen — 2007-09-19T10:20:50-00:00

Nucl. Acids Res. (13 September 2007), gkm662.

CpG methylation is a key component of the epigenome architecture that is associated with changes in gene expression without a change to the DNA sequence. Since the first reports on deregulation of DNA methylation, in diseases such as cancer, and the initiation of the Human Epigenome Project, an increasing need has arisen for a detailed, high-throughput and quantitative method of analysis to discover and validate normal and aberrant DNA methylation profiles in large sample cohorts. Here we present an improved protocol using base-specific fragmentation and MALDI-TOF mass spectrometry that enables a sensitive and high-throughput method of DNA methylation analysis, quantitative to 5% methylation for each informative CpG residue. We have determined the accuracy, variability and sensitivity of the protocol, implemented critical improvements in experimental design and interpretation of the data and developed a new formula to accurately measure CpG methylation. Key innovations now permit determination of differential and allele-specific methylation, such as in cancer and imprinting. The new protocol is ideally suitable for detailed DNA methylation analysis of multiple genomic regions and large sample cohorts that is critical for comprehensive profiling of normal and diseased human epigenomes. 10.1093/nar/gkm662

Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats.

Robert J Osborne — 2008-02-18T10:40:08-00:00

Nucleic Acids Res (7 February 2008)

We describe conditions for producing uninterrupted expanded CTG repeats consisting of up to 2000 repeats using 29 DNA polymerase. Previously, generation of such repeats was hindered by CTG repeat instability in plasmid vectors maintained in Escherichia coli and poor in vitro ligation of CTG repeat concatemers due to strand slippage. Instead, we used a combination of in vitro ligation and 29 DNA polymerase to amplify DNA. Correctly ligated products generating a dimerized repeat tract formed substrates for rolling circle amplification (RCA). In the presence of two non-complementary primers, hybridizing to either strand of DNA, ligations can be amplified to generate microgram quantities of repeat containing DNA. Additionally, expanded repeats generated by rolling circle amplification can be produced in vectors for expression of expanded CUG (CUG(exp)) RNA capable of sequestering MBNL1 protein in cell culture. Amplification of dimerized expanded repeats (ADER) opens new possibilities for studies of repeat instability and pathogenesis in myotonic dystrophy, a neurological disorder caused by an expanded CTG repeat.

microChIP--a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies.

John Arne Dahl — 2008-01-20T10:45:07-00:00

Nucleic Acids Res (17 January 2008)

Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein-DNA interactions. Drawbacks of current ChIP assays however are a requirement for large cell numbers, which limits applicability of ChIP to rare cell samples, and/or lengthy procedures with limited applications. There are to date no protocols for fast and parallel ChIPs of post-translationally modified histones from small cell numbers or biopsies, and importantly, no protocol allowing for investigations of transcription factor binding in small cell numbers. We report here the development of a micro (mu) ChIP assay suitable for up to nine parallel quantitative ChIPs of modified histones or RNA polymerase II from a single batch of 1000 cells. muChIP can also be downscaled to monitor the association of one protein with multiple genomic sites in as few as 100 cells. muChIP is applicable to small fresh tissue biopsies, and a cross-link-while-thawing procedure makes the assay suitable for frozen biopsies. Using muChIP, we characterize transcriptionally permissive and repressive histone H3 modifications on developmentally regulated promoters in human embryonal carcinoma cells and in osteosarcoma biopsies. muChIP creates possibilities for multiple parallel and rapid transcription factor binding and epigenetic analyses of rare cell and tissue samples.

MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.

Margaret S Ebert — 2007-08-20T10:53:19-00:00

Nat Methods (12 August 2007)

MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.

Switching genes off—all the way

Nicole Rusk — 2007-09-01T06:04:58-00:00

Nature Methods, Vol. 4, No. 9., pp. 684-685.

Enzyme kinetics far from the standard quasi-steady-state and equilibrium approximations

S Schnell — 2006-10-20T23:17:40-00:00

Mathematical and Computer Modelling, Vol. 35, No. 1-2. (January 2002), pp. 137-144.

Analytic approximations of the time-evolution of the single enzyme-substrate reaction are valid for all but a small region of parameter space in the positive initial enzyme-initial substrate concentration plane. We find velocity equations for the substrate decomposition and product formation with the aid of the total quasi-steady-state approximation and an aggregation technique for cases where neither the more normally employed standard nor reverse quasi-steady-state approximations are valid. Applications to determining reaction kinetic parameters are discussed.

A genetic approach to identifying mitochondrial proteins.

T Ozawa — 2008-04-14T12:49:55-00:00

Nature biotechnology, Vol. 21, No. 3. (March 2003), pp. 287-293.

The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.

Yeast cells lacking the mitochondrial gene encoding the ATP synthase subunit 6 exhibit a selective loss of complex IV and unusual mitochondrial morphology.

M Rak — 2007-11-21T14:02:34-00:00

J Biol Chem, Vol. 282, No. 15. (13 April 2007), pp. 10853-10864.

Atp6p is an essential subunit of the ATP synthase proton translocating domain, which is encoded by the mitochondrial DNA (mtDNA) in yeast. We have replaced the coding sequence of Atp6p gene with the non-respiratory genetic marker ARG8m. Due to the presence of ARG8m, accumulation of rho-/rho0 petites issued from large deletions in mtDNA could be restricted to 20-30% by growing the atp6 mutant in media lacking arginine. This moderate mtDNA instability created favorable conditions to investigate the consequences of a specific lack in Atp6p. Interestingly, in addition to the expected loss of ATP synthase activity, the cytochrome c oxidase respiratory enzyme steady-state level was found to be extremely low (<5%) in the atp6 mutant. We show that the cytochrome c oxidase-poor accumulation was caused by a failure in the synthesis of one of its mtDNA-encoded subunits, Cox1p, indicating that, in yeast mitochondria, Cox1p synthesis is a key target for cytochrome c oxidase abundance regulation in relation to the ATP synthase activity. We provide direct evidence showing that in the absence of Atp6p the remaining subunits of the ATP synthase can still assemble. Mitochondrial cristae were detected in the atp6 mutant, showing that neither Atp6p nor the ATP synthase activity is critical for their formation. However, the atp6 mutant exhibited unusual mitochondrial structure and distribution anomalies, presumably caused by a strong delay in inner membrane fusion.

Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria.

N Bonnefoy — 2007-11-21T13:57:55-00:00

Methods Cell Biol, Vol. 80 (2007), pp. 525-548.

Bringing metabolic networks to life: integration of kinetic, metabolic, and proteomic data

Wolfram Liebermeister — 2006-12-16T01:45:39-00:00

Theoretical Biology and Medical Modelling, Vol. 3 (15 December 2006), 42.

Droplet microfluidics.

SY Teh — 2008-04-11T15:09:22-00:00

Lab on a chip, Vol. 8, No. 2. (February 2008), pp. 198-220.

Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of "digital fluidic" operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors, resulting in decreased reaction times. This, coupled with the precise generation and repeatability of droplet operations, has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as microreactors ranging from the nano- to femtoliter range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. This review will focus on the various droplet operations, as well as the numerous applications of the system. Due to advantages unique to droplet-based systems, this technology has the potential to provide novel solutions to today's biomedical engineering challenges for advanced diagnostics and therapeutics.

Learning with Kernels: Support Vector Machines, Regularization, Optimization, and Beyond (Adaptive Computation and Machine Learning)

Bernhard Schölkopf — 2007-12-08T16:45:46-00:00

(15 December 2001)

In the 1990s, a new type of learning algorithm was developed, based on results from statistical learning theory: the Support Vector Machine (SVM). This gave rise to a new class of theoretically elegant learning machines that use a central concept of SVMs—-kernels--for a number of learning tasks. Kernel machines provide a modular framework that can be adapted to different tasks and domains by the choice of the kernel function and the base algorithm. They are replacing neural networks in a variety of fields, including engineering, information retrieval, and bioinformatics. Learning with Kernels provides an introduction to SVMs and related kernel methods. Although the book begins with the basics, it also includes the latest research. It provides all of the concepts necessary to enable a reader equipped with some basic mathematical knowledge to enter the world of machine learning using theoretically well-founded yet easy-to-use kernel algorithms and to understand and apply the powerful algorithms that have been developed over the last few years.

A genetic method to identify mitochondrial proteins in living Mammalian cells.

T Ozawa — 2008-02-21T13:49:42-00:00

Methods Mol Biol, Vol. 390 (2007), pp. 119-130.

Mitochondria play pivotal roles in the metabolism and physiology of eukaryotic cells. The composition of mitochondria varies in different cell types, and therefore it is crucial to know the set of proteins that constitutes the organelle in each cell type. The identification of mitochondrial proteins has been furthered by biochemical methods, but half of mitochondrial proteins still remain unidentified. We have developed a genetic method to identify mitochondrial proteins with reconstitution of split enhanced green fluorescent protein (EGFP) by protein splicing. cDNA are randomly fused to the N-terminal half of EGFP and are introduced into cells expressing the C-terminal EGFP in the mitochondrial matrix. If the cDNA encodes a protein that is targeted into mitochondria, full-length EGFP is reconstituted in the mitochondrial matrix by protein splicing. The fluorescent cells are collected by fluorescence-activated cell sorting and the cDNA are identified by DNA sequencing. This method provides a means to map proteins distributed within mitochondria of different mammalian cells.

Systems Biology Toolbox for MATLAB: a computational platform for research in systems biology

Henning Schmidt — 2006-02-12T21:32:27-00:00

Bioinformatics, Vol. 22, No. 4. (15 February 2006), pp. 514-515.

Protein identification using 2D-LC-MS/MS

Claire Delahunty — 2006-11-13T19:35:28-00:00

Methods, Vol. 35, No. 3. (March 2005), pp. 248-255.

Multidimensional liquid chromatography techniques have been coupled to tandem mass spectrometry to provide a robust method to identify proteins in complex mixtures. Data acquisition is interfaced directly with search algorithms for identification through cross-correlation with databases. This review describes the most recent advances in methodologies for protein identification by mass spectrometry and describes the limitations of the application of the technologies.

Allotopic expression of yeast mitochondrial maturase to study mitochondrial import of hydrophobic proteins.

MG Claros — 2007-11-17T16:00:00-00:00

Methods Enzymol, Vol. 264 (1996), pp. 389-403.

Dynamic modelling and analysis of biochemical networks: mechanism-based models and model-based experiments.

Natal A W van Riel — 2006-11-21T10:24:14-00:00

Brief Bioinform (14 November 2006)

Systems biology applies quantitative, mechanistic modelling to study genetic networks, signal transduction pathways and metabolic networks. Mathematical models of biochemical networks can look very different. An important reason is that the purpose and application of a model are essential for the selection of the best mathematical framework. Fundamental aspects of selecting an appropriate modelling framework and a strategy for model building are discussed. Concepts and methods from system and control theory provide a sound basis for the further development of improved and dedicated computational tools for systems biology. Identification of the network components and rate constants that are most critical to the output behaviour of the system is one of the major problems raised in systems biology. Current approaches and methods of parameter sensitivity analysis and parameter estimation are reviewed. It is shown how these methods can be applied in the design of model-based experiments which iteratively yield models that are decreasingly wrong and increasingly gain predictive power.