A method for the analysis of sigmoid stimulus-response curves.

This paper describes a method, using an interactive computer programme, for fitting a curve to data which is expected to assume a sigmoid form. The programme is specifically designed to generate best estimates of the limits and maximum slope of stimulus-response curves for the carotid sinus baroreceptor reflex, but is applicable to curve-fitting tasks in other fields such as immunology, pharmacology, biochemistry and neurophysiology.     

A Fourier method for the analysis of exponential decay curves.

A method based on the Fourier convolution theorem is developed for the analysis of data composed of random noise, plus an unknown constant "base line," plus a sum of (or an integral over a continuous spectrum of) exponential decay functions. The Fourier method's usual serious practical limitation of needing high accuracy data over a very wide range is eliminated by the introduction of convergence parameters and a Gaussian taper window. A computer program is described for the analysis of discrete spectra, where the data involves only a sum of exponentials. The program is completely automatic in that the only necessary inputs are the raw data (not necessarily in equal intervals of time); no potentially biased initial guesses concerning either the number or the values of the components are needed. The outputs include the number of components, the amplitudes and time constants together with their estimated errors, and a spectral plot of the solution. The limiting resolving power of the method is studied by analyzing a wide range of simulated two-, three-, and four-component data. The results seem to indicate that the method is applicable over a considerably wider range of conditions than nonlinear least squares or the method of moments.     

A method for estimating penetrance from families sampled for linkage analysis

When a gene variant is discovered to segregate with a disease, it may be of interest to estimate the risk (or the age-specific risk) of the disease to carriers of the variant. The families that contributed to the discovery of the variant would typically contain multiple carriers, and so, especially if the variant is rare, might prove a valuable source of study subjects for estimation of the risk. These families, by virtue of having brought the gene in question to the attention of researchers, however, may not be representative of the relationship between carrier status and the risk of the disease in the population. Using these families for risk estimation could bias the observed association between the variant and the risk. The purpose here is to present an approach to adjusting for the potential bias while using the families from linkage analysis to estimate the risk.     

A new method for the analysis of sums of exponential decay curves

A method based on Fourier Transform is presented for the representation of data by an arbitrary sum of exponentials or Gaussian functions. The method has been successfully applied to the type of data sets which arise in pharmacokinetic studies. Two techniques for error ripple elinination are discussed.     

A versatile method for deciphering plant membrane proteomes

Proteomics is a very powerful approach to link the information contained in sequenced genomes, like that of Arabidopsis, to the functional knowledge provided by studies of plant cell compartments. This article summarizes the different steps of a versatile strategy that has been developed to decipher plant membrane proteomes. Initiated with envelope membranes from spinach chloroplasts, this strategy has been adapted to thylakoids, and further extended to a series of membranes from the model plant Arabidopsis: chloroplast envelope membranes, plasma membrane, and mitochondrial membranes. The first step is the preparation of highly purified membrane fractions from plant tissues. The second step in the strategy is the fractionation of membrane proteins on the basis of their physico-chemical properties. Chloroform/methanol extraction and washing of membranes with NaOH, NaCl or any other agent led to the simplification of the protein content of the fraction to be analysed. The next step is the genuine proteomic step, i.e. the separation of proteins by 1D-gel electrophoresis followed by in-gel proteolytic digestion of the polypeptides, analysis of the proteolytic peptides using mass spectrometry, and protein identification by searching through databases. The last step is the validation of the procedure by checking the subcellular location. The results obtained by using this strategy demonstrate that a combination of different proteomics approaches, together with bioinformatics, indeed provide a better understanding of the biochemical machinery of the different plant membranes at the molecular level.     

Photosynthetically versatile thin shade leaves: A paradox of irradiance-response curves

Thick sun leaves have a larger construction cost per unit leaf area than thin shade leaves. To re-evaluate the adaptive roles of sun and shade leaves, we compared the photosynthetic benefits relative to the construction cost of the leaves. We drew photosynthetically active radiation (PAR)-response curves using the leaf-mass-based photosynthetic rate to reflect the cost. The dark respiration rates of the sun and shade leaves of mulberry (Morus bombycis Koidzumi) seedlings did not differ significantly. At irradiances below 250 μmol m⁻² s⁻¹, the shade leaves tended to have a significantly larger net photosynthetic rate (P _N) than the sun leaves. At irradiances above 250 μmol m⁻² s⁻¹, the P _N did not differ significantly. The curves indicate that plants with thin shade leaves have a larger daily CO₂ assimilation rate per construction cost than those with thick sun leaves, even in an open habitat. These results are consistently explained by a simple model of PAR extinction in a leaf. We must target factors other than the effective assimilation when we consider the adaptive roles of thick sun leaves.     

A versatile method for functionalizing surfaces with bioactive glycans

Microarrays and biosensors owe their functionality to our ability to display surface-bound biomolecules with retained biological function. Versatile, stable, and facile methods for the immobilization of bioactive compounds on surfaces have expanded the application of high-throughput "omics"-scale screening of molecular interactions by nonexpert laboratories. Herein, we demonstrate the potential of simplified chemistries to fabricate a glycan microarray, utilizing divinyl sulfone (DVS)-modified surfaces for the covalent immobilization of natural and chemically derived carbohydrates, as well as glycoproteins. The bioactivity of the captured glycans was quantitatively examined by surface plasmon resonance imaging (SPRi). Composition and spectroscopic evidence of carbohydrate species on the DVS-modified surface were obtained by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. The site-selective immobilization of glycans based on relative nucleophilicity (reducing sugar vs amine- and sulfhydryl-derived saccharides) and anomeric configuration was also examined. Our results demonstrate straightforward and reproducible conjugation of a variety of functional biomolecules onto a vinyl sulfone-modified biosensor surface. The simplicity of this method will have a significant impact on glycomics research, as it expands the ability of nonsynthetic laboratories to rapidly construct functional glycan microarrays and quantitative biosensors.     

A versatile digital computer program for non-linear regression analysis

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1. A general purpose digital computer program is described for application to biological experiments that require a non-linear regression analysis. The mathematical function, or model, to be fitted to a given set of experimental data is written as a section within the program. Given initial estimates for the parameters of the function, the program uses an iterative procedure to adjust the parameters until the sum of squares of residuals has converged to a minimum.     

Maximum likelihood method for the analysis of time-resolved fluorescence decay curves

The usefulness of fluorescence techniques for the study of macromolecular structure and dynamics depends on the accuracy and sensitivity of the methods used for data analysis. Many methods for data analysis have been proposed and used, but little attention has been paid to the maximum likelihood method, generally known as the most powerful statistical method for parameter estimation. In this paper we study the properties and behavior of maximum likelihood estimates by using simulated fluorescence intensity decay data. We show that the maximum likelihood method provides generally more accurate estimates of lifetimes and fractions than does the standard least-squares approach especially when the lifetime ratios between individual components are small. Three novelties to the field of fluorescence decay analysis are also introduced and studied in this paper: a) discretization of the convolution integral based on the generalized integral mean value theorem: b) the likelihood ratio test as a tool to determine the number of exponential decay components in a given decay profile; and c) separability and detectability indices which provide measures on how accurately, a particular decay component can be detected. Based on the experience gained from this and from our previous study of the Padé-Laplace method, we make some recommendations on how the complex problem of deconvolution and parameter estimation of multiexponential functions might be approached in an experimental setting. Offprint requests to: F. G. Prendergast     

A versatile non-radioactive technique for restriction analysis of recombinant DNA

Restriction analysis of recombinant DNA is most frequently performed according to Smith and Birnstiel by labeling 5'-termini with 32P, followed by partial digestion, separation, and autoradiographic detection of labeled fragments. We describe a rapid, non-radioactive technique for restriction analysis of recombinant DNA which combines Southern blotting of partial restriction digests and hybridization with a vector-specific probe labeled with the steroid-hapten Digoxigenin for immunological detection. This technique has several advantages compared to conventional methods. Labeling with 32P is not necessary and as the labeled DNA-fragment used as probe is vector-specific, it can be applied for numerous constructs using the particular cloning vector (e.g.pBR322). Furthermore, the probe can be stored for several months and can be reused many times.     

A versatile method for cell-specific profiling of translated mRNAs in Drosophila

In Drosophila melanogaster few methods exist to perform rapid cell-type or tissue-specific expression profiling. A translating ribosome affinity purification (TRAP) method to profile actively translated mRNAs has been developed for use in a number of multicellular organisms although it has only been implemented to examine limited sets of cell- or tissue-types in these organisms. We have adapted the TRAP method for use in the versatile GAL4/UAS system of Drosophila allowing profiling of almost any tissue/cell-type with a single genetic cross. We created transgenic strains expressing a GFP-tagged ribosomal protein, RpL10A, under the control of the UAS promoter to perform cell-type specific translatome profiling. The GFP::RpL10A fusion protein incorporates efficiently into ribosomes and polysomes. Polysome affinity purification strongly enriches mRNAs from expected genes in the targeted tissues with sufficient sensitivity to analyze expression in small cell populations. This method can be used to determine the unique translatome profiles in different cell-types under varied physiological, pharmacological and pathological conditions.     

A method for simultaneous alignment of multiple protein structures

Here, we present MultiProt, a fully automated highly efficient technique to detect multiple structural alignments of protein structures. MultiProt finds the common geometrical cores between input molecules. To date, most methods for multiple alignment start from the pairwise alignment solutions. This may lead to a small overall alignment. In contrast, our method derives multiple alignments from simultaneous superpositions of input molecules. Further, our method does not require that all input molecules participate in the alignment. Actually, it efficiently detects high scoring partial multiple alignments for all possible number of molecules in the input. To demonstrate the power of MultiProt, we provide a number of case studies. First, we demonstrate known multiple alignments of protein structures to illustrate the performance of MultiProt. Next, we present various biological applications. These include: (1) a partial alignment of hinge-bent domains; (2) identification of functional groups of G-proteins; (3) analysis of binding sites; and (4) protein-protein interface alignment. Some applications preserve the sequence order of the residues in the alignment, whereas others are order-independent. It is their residue sequence order-independence that allows application of MultiProt to derive multiple alignments of binding sites and of protein-protein interfaces, making MultiProt an extremely useful structural tool.     

A versatile ligation-independent cloning method suitable for high-throughput expression screening applications

This article describes the construction of a set of versatile expression vectors based on the In-Fusion™ cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion™ has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His₆-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.     

A new and versatile method for determination of thiolamines of biological importance

A method for the separation and quantitation of several important biological thiolamines is described. The procedure employs a C₁₈ reversed-phase HPLC system to separate the dinitrophenyl derivatives of reduced and oxidized glutathione and cysteine and relies on an internal standard, N^ϵ-methyllysine, to minimize experimental error. The method was validated in three matrices (water, HepG2 cell lysates, and mouse liver homogenates) using several criteria. The detector response was linear for the dinitrophenyl derivatives of glutathione, glutathione disulfide, cysteine, and cystine in the concentrations ranging from 10 to 50 nmol/ml. Inter- and intra-day variation, percent recovery in the biological matrices, and limits of detection and quantitation were determined. For the most accurate determination, it is essential that standard curves be produced daily and in the same matrix as that being analyzed.     

A simple, versatile method for GFP-based super-resolution microscopy via nanobodies

We developed a method to use any GFP-tagged construct in single-molecule super-resolution microscopy. By targeting GFP with small, high-affinity antibodies coupled to organic dyes, we achieved nanometer spatial resolution and minimal linkage error when analyzing microtubules, living neurons and yeast cells. We show that in combination with libraries encoding GFP-tagged proteins, virtually any known protein can immediately be used in super-resolution microscopy and that simplified labeling schemes allow high-throughput super-resolution imaging.