The Open AUC Project

Progress in analytical ultracentrifugation (AUC) has been hindered by obstructions to hardware innovation and by software incompatibility. In this paper, we announce and outline the Open AUC Project. The goals of the Open AUC Project are to stimulate AUC innovation by improving instrumentation, detectors, acquisition and analysis software, and collaborative tools. These improvements are needed for the next generation of AUC-based research. The Open AUC Project combines on-going work from several different groups. A new base instrument is described, one that is designed from the ground up to be an analytical ultracentrifuge. This machine offers an open architecture, hardware standards, and application programming interfaces for detector developers. All software will use the GNU Public License to assure that intellectual property is available in open source format. The Open AUC strategy facilitates collaborations, encourages sharing, and eliminates the chronic impediments that have plagued AUC innovation for the last 20 years. This ultracentrifuge will be equipped with multiple and interchangeable optical tracks so that state-of-the-art electronics and improved detectors will be available for a variety of optical systems. The instrument will be complemented by a new rotor, enhanced data acquisition and analysis software, as well as collaboration software. Described here are the instrument, the modular software components, and a standardized database that will encourage and ease integration of data analysis and interpretation software.     

Parakeet: a digital twin software pipeline to assess the impact of experimental parameters on tomographic reconstructions for cryo-electron tomography

In cryo-electron tomography (cryo-ET) of biological samples, the quality of tomographic reconstructions can vary depending on the transmission electron microscope (TEM) instrument and data acquisition parameters. In this paper, we present Parakeet, a ‘digital twin’ software pipeline for the assessment of the impact of various TEM experiment parameters on the quality of three-dimensional tomographic reconstructions. The Parakeet digital twin is a digital model that can be used to optimize the performance and utilization of a physical instrument to enable in silico optimization of sample geometries, data acquisition schemes and instrument parameters. The digital twin performs virtual sample generation, TEM image simulation, and tilt series reconstruction and analysis within a convenient software framework. As well as being able to produce physically realistic simulated cryo-ET datasets to aid the development of tomographic reconstruction and subtomogram averaging programs, Parakeet aims to enable convenient assessment of the effects of different microscope parameters and data acquisition parameters on reconstruction quality. To illustrate the use of the software, we present the example of a quantitative analysis of missing wedge artefacts on simulated planar and cylindrical biological samples and discuss how data collection parameters can be modified for cylindrical samples where a full 180° tilt range might be measured.     

Bioinformatics tools for cancer metabolomics

It is well known that significant metabolic change take place as cells are transformed from normal to malignant. This review focuses on the use of different bioinformatics tools in cancer metabolomics studies. The article begins by describing different metabolomics technologies and data generation techniques. Overview of the data pre-processing techniques is provided and multivariate data analysis techniques are discussed and illustrated with case studies, including principal component analysis, clustering techniques, self-organizing maps, partial least squares, and discriminant function analysis. Also included is a discussion of available software packages.     

PC‐ORD version 5: A user‐friendly toolbox for ecologists

Recently, version 5 of PC‐ORD, one of the major commercial software packages for multivariate ecological community data analyses, was released. The new version offers a whole range of techniques and methods for analyses of ecological data. It includes modules for different types of ordination and classification, as well as other exploratory techniques such as species‐area curve analysis and indicator species analysis. Data are stored in spreadsheets and can be easily manipulated in various ways. In essence, version 5 of PC‐ORD offers the user a full toolbox for exploration and analysis of ecological data, packed in a user‐friendly environment.     

Sequence analysis of genes and genomes

A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.     

Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

Biophysical techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2′-CMP + RNASE and BH3I-1 + hBCL_XL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.     

Performance comparison and evaluation of software tools for microRNA deep-sequencing data analysis

With the development of next-generation sequencing (NGS) techniques, many software tools have emerged for the discovery of novel microRNAs (miRNAs) and for analyzing the miRNAs expression profiles. An overall evaluation of these diverse software tools is lacking. In this study, we evaluated eight software tools based on their common feature and key algorithms. Three deep-sequencing data sets were collected from different species and used to assess the computational time, sensitivity and accuracy of detecting known miRNAs as well as their capacity for predicting novel miRNAs. Our results provide useful information for researchers to facilitate their selection of the optimal software tools for miRNA analysis depending on their specific requirements, i.e. novel miRNAs discovery or miRNA expression profile analysis of sequencing data sets.     

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals’ positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.     

Video tracking and analysis of sleep in Drosophila melanogaster

In the past decade, Drosophila has emerged as an ideal model organism for studying the genetic components of sleep as well as its regulation and functions. In fruit flies, sleep can be conveniently estimated by measuring the locomotor activity of the flies using techniques and instruments adapted from the field of circadian behavior. However, proper analysis of sleep requires degrees of spatial and temporal resolution higher than is needed by circadian scientists, as well as different algorithms and software for data analysis. Here I describe how to perform sleep experiments in flies using techniques and software (pySolo and pySolo-Video) previously developed in my laboratory. I focus on computer-assisted video tracking to monitor fly activity. I explain how to plan a sleep analysis experiment that covers the basic aspects of sleep, how to prepare the necessary equipment and how to analyze the data. By using this protocol, a typical sleep analysis experiment can be completed in 5-7 d.     

Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged in the biopharmaceutical industry as a technique to detect small quantities of protein aggregates. However, the limits of detection and quantitation of these aggregates are not yet well understood. Although diverse factors (molecule, instrument, technique, and software dependent) preclude an all-encompassing measurement of these limits for the complete system, it is possible to use simulated data to determine the quantitation limits of the data analysis software aspect. The current study examines the performance of the SEDFIT/c(s) data analysis tool with simulated antibody monomer/dimer and monomer/aggregate systems. Under completely ideal conditions (zero noise, known meniscus, and shape factor homogeneity), the software limit of quantitation was 0.01% for the monomer/aggregate system and 0.03% for the less well-resolved monomer/dimer system. Under more realistic conditions (0.005 OD root mean square [RMS] noise, shape factor variability, and long solution column), the software limits of quantitation were 0.2 and 0.6% (0.002 and 0.006 OD) for the monomer/aggregate and monomer/dimer systems, respectively. Interestingly, diminished quantitation accuracy at very low levels of oligomer was not accompanied by deterioration of fit quality (as measured by root mean square deviation [RMSD] and residuals bitmap images).     

An automated PCR platform with homogeneous time-resolved fluorescence detection and dry chemistry assay kits

We have developed a novel instrument platform, GenomEra, for small-scale analysis of nucleic acids. The platform combines a rapid thermal cycler, an integrated time-resolved fluorescence measurement unit, and user-friendly software for the analysis of results. Disposable low-cost plastic reaction vessels are designed specifically for the instrument and contain all of the assay-specific reagents in dry form. The appropriate assay protocol is specified on barcodes printed under the vessels and is automatically initiated by the software. Detection is based on the use of sequence-specific probes labeled with intrinsically fluorescent europium or terbium chelates and complementary quencher probes, which enable sensitive, homogeneous closed-tube assays without the risk of carryover contamination. The detection limit of the instrument (background + 3 SD) is approximately 20 pmol/L for both chelates with a dynamic range of nearly four orders of magnitude. The functionality of the platform is demonstrated with a dual-label homogeneous polymerase chain reaction (PCR) assay for the detection of Salmonella using a Magda CA Salmonella assay kit. An internal amplification control is included in each reaction to eliminate false negative results caused by PCR inhibition. Qualitative assay results are automatically interpreted by the software and are available 45 min after sample addition.     

Using Sculptor and Situs for simultaneous assembly of atomic components into low-resolution shapes

We describe an integrated software system called Sculptor that combines visualization capabilities with molecular modeling algorithms for the analysis of multi-scale data sets. Sculptor features extensive special purpose visualization techniques that are based on modern GPU programming and are capable of representing complex molecular assemblies in real-time. The integration of graphics and modeling offers several advantages. The user interface not only eases the usually steep learning curve of pure algorithmic techniques, but it also permits instant analysis and post-processing of results, as well as the integration of results from external software. Here, we implemented an interactive peak-selection strategy that enables the user to explore a preliminary score landscape generated by the colors tool of Situs. The interactive placement of components, one at a time, is advantageous for low-resolution or ambiguously shaped maps, which are sometimes difficult to interpret by the fully automatic peak selection of colors. For the subsequent refinement of the preliminary models resulting from both interactive and automatic peak selection, we have implemented a novel simultaneous multi-body docking in Sculptor and Situs that softly enforces shape complementarities between components using the normalization of the cross-correlation coefficient. The proposed techniques are freely available in Situs version 2.6 and Sculptor version 2.0.     

VisBio: a computational tool for visualization of multidimensional biological image data

New laser scanning microscopy techniques enable biologists to acquire larger, more complex image datasets. Emerging imaging modalities such as multispectral, harmonic, and fluorescence lifetime can generate data with six or more dimensions; however, existing software is not well suited to the visualization or analysis of such data. To address these concerns, we have developed VisBio, an application and toolkit for visualization and analysis of multidimensional, biological image data of any dimensionality.     

In-depth analysis of tandem mass spectrometry data from disparate instrument types

Mass spectrometric analyses of protein digests produce large numbers of fragmentation spectra that are not identified by routine database searching strategies. Some of these spectra could be identified by development of improved search engines. However, many of these spectra represent fragmentation of peptide components bearing modifications that are not routinely considered in database searches. Here we present new software within Protein Prospector that allows comprehensive analysis of data sets by analyzing the data at increasing levels of depth. Analysis of published data sets is presented to illustrate that the software is not biased to any instrument types. The results show that these data sets contain many modified peptides. As well as searching for known modification types, Protein Prospector permits the detection and identification of unexpected or novel modifications by searching for any mass shift within a user-specified mass range to any chosen amino acid(s). Several modifications never previously reported in proteomics data were identified in these standard data sets using this mass modification searching approach.     

Flash fluorometer made from off-the-shelf photographic equipment to measure tissue levels of fluorescein

A device to measure fluorescein in tissue has been constructed from standard photographic equipment--an electronic strobe and a flashmeter both covered with interference filters. The instrument works well in the light and need not touch the area being measured, an advantage over existing fluorometers. The instrument has been used to measure the amount of dye in flaps in rats, pigs, and three humans. The results revealed that the amount of dye in a freshly made flap was rarely as much as in normal skin, and skin with less than 20 percent of the dye of control areas usually sloughed, although there were exceptions. In the future the instrument will be improved, and its readings will be compared to those obtained from radioactive microspheres, the present "gold standard" of techniques to measure vascularity. The instrument can be used to estimate the blood supply to any tissue and seems to be as reliable as the dermofluorometers already on the market.     

TOPPAS: a graphical workflow editor for the analysis of high-throughput proteomics data

Mass spectrometry coupled to high-performance liquid chromatography (HPLC-MS) is evolving more quickly than ever. A wide range of different instrument types and experimental setups are commonly used. Modern instruments acquire huge amounts of data, thus requiring tools for an efficient and automated data analysis. Most existing software for analyzing HPLC-MS data is monolithic and tailored toward a specific application. A more flexible alternative consists of pipeline-based tool kits allowing the construction of custom analysis workflows from small building blocks, e.g., the Trans Proteomics Pipeline (TPP) or The OpenMS Proteomics Pipeline (TOPP). One drawback, however, is the hurdle of setting up complex workflows using command line tools. We present TOPPAS, The OpenMS Proteomics Pipeline ASsistant, a graphical user interface (GUI) for rapid composition of HPLC-MS analysis workflows. Workflow construction reduces to simple drag-and-drop of analysis tools and adding connections in between. Integration of external tools into these workflows is possible as well. Once workflows have been developed, they can be deployed in other workflow management systems or batch processing systems in a fully automated fashion. The implementation is portable and has been tested under Windows, Mac OS X, and Linux. TOPPAS is open-source software and available free of charge at http://www.OpenMS.de/TOPPAS .     

A fluorescence cytophotometer operated under computer control for multi-parameter cell analysis

Summary A new type of fluorescence cytophotometer has been developed for multi-parameter cell analysis (Olympus BH2-QRFL). For multi-color fluorescence cytophotometry, this instrument is equipped with four sets of interchangeable filters, each consisting of an excitation filter, a dichroic mirror with a barrier filter, and a measuring filter. For permitting automatic operation of the filter sets, the cytophotometer is connected on line with a personal computer (HP 85F). A desired sequence of filter sets can be memorized in the software and multiple cellular constituents can be rapidly and consecutively determined on a single cell basis. All data are stored in the same computer and can be retrieved for further statistical analysis and display either in tabular form, or as histogram, correlation histograms, two-dimensional scatter plot, or two-dimensional frequency distribution histogram, on the CRT (cathode ray tube) with simultaneous hard copy. As an example of multiparameter cell analysis, combined protein and DNA measurements were performed on normal, border-line, and cancerous gynecological cytology specimens by using the ninhydrin-Schiff and Feulgen techniques.     

WinPep 2.11: novel software for PC-based analyses of amino acid sequences.

WinPep 2.11 is the latest version of a user-friendly, versatile software for the analysis of amino acid sequences. WinPep was developed for Windows 95/98 and WindowsNT. Because of the standard windows techniques (copy & paste) and an intuitive user interface, no lengthy training is required to work with this program. Main features include calculation of molecular weight, isoelectric point, and molar absorption coefficients, as well as simulation of sequence specific cleavage. In addition to data analysis, data presentation is greatly assisted by the program, e.g., by producing helical wheel displays and hydropathy plots. The implemented functions of WinPep and it's usage are described in this publication.