Analysis of locomotor activity rhythms in Drosophila

The genetic, molecular and anatomical dissection of the circadian clock in Drosophila and other higher organisms relies on the quantification of rhythmic phenotypes. Here, we introduce the methods currently in use in our laboratories for the analysis of fly locomotor activity rhythms. This phenotype provides a relatively simple, automated, efficient, reliable and robust output for the circadian clock. Thus it is not surprising that it is the preferred readout for measuring rhythmicity under a variety of conditions for most fly clock laboratories. The procedure requires at least 10 days of data collection and several days for analysis. In this protocol we advise on fly maintenance and on experimental design when studying the genetics of behavioral traits. We describe the setup for studying locomotor activity rhythms in the fruit fly and we introduce the statistical methods in use in our laboratories for the analysis of periodic data.     

Letter to the Editors: in Defense of Visual Inspection

Cornelissen and Haus (1) are certainly correct in their assertion that computerized data collection and analysis are useful and important tools in chronobiological research. We also agree that circadian rhythm data should be subjected to multiple complementary analyses whenever possible. In fact, both of our laboratories currently employ on-line collection of behavioural activity data, as well as several different computer-assisted statistical procedures, including analyses similar to those referred to by Cornelissen and Haus in their commentary.     

A centralized model for creating shared,standardized, microsatellite data that simplifies inter-laboratory collaboration

We demonstrate an efficient model for standardizing microsatellite DNA data among laboratories studying Oncorhynchus mykiss. Eight laboratories standardized 13 microsatellite loci following allele nomenclature of a central laboratory (average inter-laboratory genotyping concordance >98%). Following this central model, we have currently standardized 298 alleles from throughout the species native range. Although we focus here on O. mykiss, our experiences and recommendation apply equally to other broadly distributed species that may benefit from multi-laboratory collaborative data collection.     

An inter-laboratory comparison demonstrates that [1H]-NMR metabolite fingerprinting is a robust technique for collaborative plant metabolomic data collection

In any metabolomics experiment, robustness and reproducibility of data collection is of vital importance. These become more important in collaborative studies where data is to be collected on multiple instruments. With minimisation of variance in sample preparation and instrument performance it is possible to elucidate even subtle differences in metabolite fingerprints due to genotype or biological treatment. In this paper we report on an inter laboratory comparison of plant derived samples by [¹H]-NMR spectroscopy across five different sites and within those sites utilising instruments with different probes and magnetic field strengths of 9.4 T (400 MHz), 11.7 T (500 MHz) and 14.1 T (600 MHz). Whilst the focus of the study is on consistent data collection across laboratories, aspects of sample stability and the requirement for sample rotation within the NMR magnet are also discussed. Comparability of the datasets from participating laboratories was exceptionally good and the data were amenable to comparative analysis by multivariate statistics. Field strength differences can be adjusted for in the data pre-processing and multivariate analysis demonstrating that [¹H]-NMR fingerprinting is the ideal technique for large scale plant metabolomics data collection requiring the participation of multiple laboratories.     

MilQuant: a free, generic software tool for isobaric tagging-based quantitation

Isobaric tagging techniques such as iTRAQ and TMT are widely used in quantitative proteomics and especially useful for samples that demand in vitro labeling. Due to diversity in choices of MS acquisition approaches, identification algorithms, and relative abundance deduction strategies, researchers are faced with a plethora of possibilities when it comes to data analysis. However, the lack of generic and flexible software tool often makes it cumbersome for researchers to perform the analysis entirely as desired. In this paper, we present MilQuant, mzXML-based isobaric labeling quantitator, a pipeline of freely available programs that supports native acquisition files produced by all mass spectrometer types and collection approaches currently used in isobaric tagging based MS data collection. Moreover, aside from effective normalization and abundance ratio deduction algorithms, MilQuant exports various intermediate results along each step of the pipeline, making it easy for researchers to customize the analysis. The functionality of MilQuant was demonstrated by four distinct datasets from different laboratories. The compatibility and extendibility of MilQuant makes it a generic and flexible tool that can serve as a full solution to data analysis of isobaric tagging-based quantitation.     

Macromolecular cryocrystallography--methods for cooling and mounting protein crystals at cryogenic temperatures

Cryocrystallography is routinely used in macromolecular crystallography laboratories. The main advantage of X-ray diffraction data collection near 100K is that crystals display much less radiation damage than seen at room temperature. Techniques and tools are described to facilitate cryoprotecting and flash-cooling crystals for data collection.     

Enhancing repository fungal data for biogeographic analyses

Open-access occurrence data are useful for studying spatial patterns of fungi, but often have quality issues. These include errors in taxonomy and geo-coordinates, and incomplete coverage across areas and taxonomic groups. We identify 15 quality issues that can lead to incorrect biogeographic inference, and develop a reproducible pipeline that flags and removes problematic entries. This pipeline tests accuracy of geographic records and names. Then, if information on non-native status is unavailable or unreliable, it detects non-native species via a predictive model. Finally, it identifies spatial and environmental outliers and removes them when biologically improbable. We test the pipeline by cleaning data for Australian fungi, with 251,642 records retained after cleaning the initial 1,034,601 records. Exploratory analysis showed that the cleaned data is useful for analyses such as biogeographic regionalisation, but recording gaps and lack of saturation in collection effort also caution that more surveys are needed to improve collection completeness.     

Expert systems for clinical pathology reporting

* Conventional automated interpretative reporting systems use standard or "canned" comments for patient reports. These are result-specific and do not generally refer to the patient context. * Laboratory information systems (LIS) are limited in their application of patient-specific content of reporting. * Patient-specific interpretation requires extensive cross-referencing to other information contained in the LIS such as previous test results, other related tests, and clinical notes, both current and previous. * Expert systems have the potential to improve reporting quality by enabling patient-specific reporting in clinical laboratories.     

Evaluation of five strategies for obtaining a core subset from a large genetic resource collection of durum wheat

The use of plant genetic resources contained in a large collection may be enhanced by specifying subsamples, called core samples. Five strategies for selecting a core sample from a collection of 3000 durum wheat accessions were applied and evaluated using four qualitative and eight quantitative spike characters. Each of the following strategies generated about 500 accessions for the core sample: random, random-systematic according to chronology of entry of the accessions into the collection, stratified by countryof-origin, stratified by log frequency by country-of-origin, and stratified by canonical variables. The first three strategies produced samples representative of the whole collection, but the remaining two produced the desired effect of increasing frequencies from less-represented countries-of-origin for several characters. The stratified canonical sample increased phenotypic variances. The quality of core samples is dependent upon good passport and evaluation data to partition the collection. The multivariate approach is extremely useful, but requires considerable data from the whole collection. Ecogeographic origin may be used in the absence of evaluation data on several characters to select useful core samples.     

Criteria for Native Food Plant Collection in Northeastern Brazil

Different criteria are used by human populations around the world in the selection of preferred natural resources. In this study, the following hypotheses were tested: 1) the preference for food native species is related to the number of uses for which the species are cited; 2) the categories of food and medicinal use equally influence the selection of a species as preferred; 3) the preference among native food species is related to its frequency and abundance in the areas of collection. Data collection on species preference was conducted in communities located near the Araripe National Forest (FLONA) in Ceará, Northeastern, Brazil. Both the hypothesis that the preference would relate to the number of uses attributed to species and the hypothesis that food and medicinal categories equally influence the selection of species as preferred ones were corroborated. It was concluded in this study that people optimize both use and collection of native resources using criteria such as the versatility of uses for species and the abundance of the resource in the preferred collection areas. It is believed that this trend may be an adaptive strategy adopted by local communities for the exploitation of native resources.     

A new technique for obtaining mitotic chromosome spreads from fishes in the field

This study presents an adaptation of current methodologies for preparing mitotic chromosomes from fishes, optimized for use in the field. The high-quality preparations obtained using this modified methodology is suitable for subsequent chromosomal analysis. Importantly, this method is particularly useful when specimen collection sites are far from research laboratories or when researchers are working with highly sensitive species that do not survive long outside of their natural habitats.     

Further Evaluation of Immunofluorescence Techniques for Detection of Shigellae in Fecal Specimens

The fluorescent antibody (FA) tests for group B and group D Shigella were reevaluated. Duplicate swab specimens from patients suspected of having shigellosis were cultured shortly after collection and after transport in a soft-agar holding medium. Smears for FA examination were made at the same time. Results obtained for the group D Shigella agree closely with those obtained in previous studies. The percentage of isolations of group B Shigella from transported specimens which were positive by FA was 59.6% as compared to 39.3 and 53.3% in previous studies. S. flexneri was isolated from 76.7% of the FA-positive specimens when they were examined shortly after collection. More isolates of Shigella were obtained when specimens were examined by both methods than when either method was used alone. The results indicated that FA tests for both group B and group D Shigella are practical and may be useful to laboratories engaged in Shigella isolation.     

An automated coordinate recorder for biometry

Many measuring devices used in micropaleontology are unsatisfactory because observations must be recorded manually. Moreover, most use a linear scale that depends on the presence of predetermined morphological loci. An x, y recorder, largely adapted from instruments available in geological laboratories, is discussed. It obviates manual recording, frees data collection from the constraint of homologous loci and. in some applications, permits more shape information to be collected.     

Randomised controlled trial of computer-held medical records in hypertensive patients.

A total of 278 hypertensive patients in three clinics were randomly allocated to have their medical records held in a computer system (136) or on standard hospital notes (142). For the computer system the doctor completed a structured input form, and the information on symptoms, physical findings, and diagnoses was more complete than that in the standard notes. This resulted in certain symptoms and risk factors being recognised more often when the computer system was used. The hypertension clinics'' routines were not disrupted by the introduction of a computer-held system, and follow-up consultation times were not affected by the type of records kept, although the first consultation took eight minutes longer when computer documents were completed. The patients remained in the trial for one year and clinical management was assessed from blood pressure control, drop-out rates, and the frequency of performing investigations. These estimates of management showed no significant difference between the two groups, but the attempt to tailor the computer system to help management made the system acceptable to the doctors using it. The computer system continues to be used and is providing data for research into hypertension.     

Current trends in the epidemiological typing of clinically relevant microbes in Europe

A questionnaire-based survey was performed to accumulate data on methodologies used in microbiology laboratories involved in epidemiological typing. Genotyping by PFGE and MLST are currently clearly preferred over phenotyping. The overall wish is to increase the activities by over 20% and additional resources would be used to invest in real-time typing.     

Assessing bias in experiment design for large scale mass spectrometry-based quantitative proteomics

Mass spectrometry-based proteomics holds great promise as a discovery tool for biomarker candidates in the early detection of diseases. Recently much emphasis has been placed upon producing highly reliable data for quantitative profiling for which highly reproducible methodologies are indispensable. The main problems that affect experimental reproducibility stem from variations introduced by sample collection, preparation, and storage protocols and LC-MS settings and conditions. On the basis of a formally precise and quantitative definition of similarity between LC-MS experiments, we have developed Chaorder, a fully automatic software tool that can assess experimental reproducibility of sets of large scale LC-MS experiments. By visualizing the similarity relationships within a set of experiments, this tool can form the basis of systematic quality control and thus help assess the comparability of mass spectrometry data over time, across different laboratories, and between instruments. Applying Chaorder to data from multiple laboratories and a range of instruments, experimental protocols, and sample complexities revealed biases introduced by the sample processing steps, experimental protocols, and instrument choices. Moreover we show that reducing bias by correcting for just a few steps, for example randomizing the run order, does not provide much gain in statistical power for biomarker discovery.     

Allelic ladders and reference genotypes for a rigorous standardization of poplar microsatellite data

A correct identification of members of the poplar hybrid complex Populus × canadensis is essential in breeding programs and studies in introgressive gene flow. Molecular marker protocols have been developed for this purpose. However, due to missing standards, these techniques have so far not been suited to the transfer of results between different laboratories. We present here a powerful system of nuclear microsatellite DNA (nSSR) fingerprints, standardized by allelic ladders and reference genotypes. Seven nSSR loci provided fingerprints of 65 commercial poplar clones. Their alleles were used to construct allelic ladders. Thus, a first standardized register of poplar clones is now available. All procedures were optimized according to simplified DNA extraction protocols, multiplexed PCR and electrophoresis procedures. Corresponding data originating from two different electrophoretic platforms in different laboratories were congruent when the allelic ladder was used. Unambiguous differentiation of the clones was based on a very low probability of identity (PI) of 1.95 × 10⁻⁸. Our results revealed discrepancies between clone denotations and genetic fingerprints. This suggests that, potentially, members of the clone collection could have been mixed up, thus confirming the demand for rigorous standards. The protocol presented can be exploited in a manifold way, e.g. to enlarge the present clonal molecular data base, or to use it for purposes of certification and control. Furthermore, the allelic ladders are recommended for use in poplar population genetic studies across different laboratories. The allelic ladders and single sample reference genotypes can be obtained on demand.     

AFM 4.0: a toolbox for DNA microarray analysis

We have developed a series of programs, collectively packaged as Array File Maker 4.0 (AFM), that manipulate and manage DNA microarray data. AFM 4.0 is simple to use, applicable to any organism or microarray, and operates within the familiar confines of Microsoft Excel. Given a database of expression ratios, AFM 4.0 generates input files for clustering, helps prepare colored figures and Venn diagrams, and can uncover aneuploidy in yeast microarray data. AFM 4.0 should be especially useful to laboratories that do not have access to specialized commercial or in-house software.