Assessment of short-read shotgun sequencing and microbiome analysis of faecal samples to discriminate between equol producers and non-producers.

Among the isoflavones and isoflavone-derived metabolites, equol, which in the human gut is synthesised from daidzein by minority bacterial populations, shows the strongest estrogenic and antioxidant activity. The beneficial effects on human health of isoflavone consumption might be partially or indeed totally attributable to this equol. Although some of the bacterial strains involved in its formation have been identified, the interplay between the composition and functionality of the gut microbiota and equol producer phenotype has hardly been studied. In this study, after shotgun metagenomic sequencing, different pipelines for the taxonomic and functional annotation of sequencing data were used in the search for similarities and differences in the faecal metagenome of equol-producing (n=3) and non-producing (n=2) women, with special focus on equol-producing taxa and their equol-associated genes. The taxonomic profiles of the samples differed significantly depending on the analytical method followed, although the microbial diversity detected by each tool was very similar at the phylum, genus and species levels. Equol-producing taxa were detected in both equol producers and non-producers, but no correlation between the abundance of equol-producing taxa and the equol producing/non-producing phenotype was found. Indeed, functional metagenomic analysis was unable to identify the genes involved in equol production, even in samples from equol producers. By aligning equol operons with the collected metagenomics data, a small number of reads mapping to equol-associated sequences were recognised in samples from both equol producers and equol non-producers, but only two reads mapping onto equol reductase-encoding genes in a sample from an equol producer. In conclusion, the taxonomic analysis of metagenomic data might not be suitable for detecting and quantifying equol-producing microbes in human faeces. Functional analysis of the data might provide an alternative. However, to detect the genetic makeup of the minority gut populations, more extensive sequencing than that achieved in the present study might be required.

Lung parenchyma and structure visualisation in paediatric chest MRI: a comparison of different short and ultra-short echo time protocols.

AIM: To evaluate image quality acquired at lung imaging using magnetic resonance imaging (MRI) sequences using short and ultra-short (UTE) echo times (TEs) with different acquisition strategies (breath-hold, prospective, and retrospective gating) in paediatric patients and in healthy volunteers. MATERIALS AND METHODS: End-inspiratory and end-expiratory three-dimensional (3D) spoiled gradient (SPGR3D) and 3D zero echo-time (ZTE3D), and 3D UTE free-breathing (UTE3D), prospective projection navigated radial ZTE3D (ZTE3D vnav), and four-dimensional ZTE (ZTE4D) were performed using a 1.5 T MRI system. For quantitative assessment, the contrast-to-noise ratio (CNR) and signal-to-noise ratio (SNR) values were calculated. To evaluate image quality, qualitative scoring was undertaken on all sequences to evaluate depiction of intrapulmonary vessels, fissures, bronchi, imaging noise, artefacts, and overall acceptability. RESULTS: Eight cystic fibrosis (CF) patients (median age 14 years, range 13-17 years), seven children with history of prematurity with or without bronchopulmonary dysplasia (BPD; median 10 years, range 10-11 years), and 10 healthy volunteers (median 32 years, range 20-52 years) were included in the study. ZTE3D vnav provided the most reliable output in terms of image quality, although scan time was highly dependent on navigator triggering efficiency and respiratory pattern. CONCLUSIONS: Best image quality was achieved with prospective ZTE3D and UTE3D readouts both in children and volunteers. The current implementation of retrospective ZTE3D readout (ZTE4D) did not provide diagnostic image quality but rather introduced artefacts over the entire imaging volume mimicking lung pathology.

Deep learning for improving ZTE MRI images in free breathing.

INTRODUCTION: Despite a growing interest in lung MRI, its broader use in a clinical setting remains challenging. Several factors limit the image quality of lung MRI, such as the extremely short T2 and T2* relaxation times of the lung parenchyma and cardiac and breathing motion. Zero Echo Time (ZTE) sequences are sensitive to short T2 and T2* species paving the way to improved "CT-like" MR images. To overcome this limitation, a retrospective respiratory gated version of ZTE (ZTE4D) which can obtain images in 16 different respiratory phases during free breathing was developed. Initial performance of ZTE4D have shown motion artifacts. To improve image quality, deep learning with fully convolutional neural networks (FCNNs) has been proposed. CNNs has been widely used for MR imaging, but it has not been used for improving free-breathing lung imaging yet. Our proposed pipeline facilitates the clinical work with patients showing difficulties/uncapable to perform breath-holding, or when the different gating techniques are not efficient due to the irregular respiratory pace. MATERIALS AND METHODS: After signed informed consent and IRB approval, ZTE4D free breathing and breath-hold ZTE3D images were obtained from 10 healthy volunteers on a 1.5 T MRI scanner (GE Healthcare Signa Artist, Waukesha, WI). ZTE4D acquisition captured all 16 phases of the respiratory cycle. For the ZTE breath-hold, the subjects were instructed to hold their breath in 5 different inflation levels ranging from full expiration to full inspiration. The training dataset consisting of ZTE-BH images of 10 volunteers was split into 8 volunteers for training, 1 for validation and 1 for testing. In total 800 ZTE breath-hold images were constructed by adding Gaussian noise and performing image transformations (translations, rotations) to imitate the effect of motion in the respiratory cycle, and blurring from varying diaphragm positions, as it appears for ZTE4D. These sets were used to train a FCNN model to remove the artificially added noise and transformations from the ZTE breath-hold images and reproduce the original quality of the images. Mean squared error (MSE) was used as loss function. The remaining 2 healthy volunteer's ZTE4D images were used to test the model and qualitatively assess the predicted images. RESULTS: Our model obtained a MSE of 0.09% on the training set and 0.135% on the validation set. When tested on unseen data the predicted images from our model improved the contrast of the pulmonary parenchyma against air filled regions (airways or air trapping). The SNR of the lung parenchyma was quantitatively improved by a factor of 1.98 and the CNR lung- blood, which is indicating the visibility of the intrapulmonary vessels, was improved by 4.2%. Our network was able to reduce ghosting artifacts, such as diaphragm movement and blurring, and enhancing image quality. DISCUSSION: Free-breathing 3D and 4D lung imaging with MRI is feasible, however its quality is not yet acceptable for clinical use. This can be improved with deep learning techniques. Our FCNN improves the visual image quality and reduces artifacts of free-breathing ZTE4D. Our main goal was rather to remove ghosting artifacts from the ZTE4D images, to improve diagnostic quality of the images. As main results of the network, diaphragm contour increased with sharper edges by visual inspection and less blurring of the anatomical structures and lung parenchyma. CONCLUSION: With FCNNs, image quality of free breathing ZTE4D lung MRI can be improved and enable better visualization of the lung parenchyma in different respiratory phases.

Accuracy and repeatability of joint sparsity multi-component estimation in MR Fingerprinting.

MR fingerprinting (MRF) is a promising method for quantitative characterization of tissues. Often, voxel-wise measurements are made, assuming a single tissue-type per voxel. Alternatively, the Sparsity Promoting Iterative Joint Non-negative least squares Multi-Component MRF method (SPIJN-MRF) facilitates tissue parameter estimation for identified components as well as partial volume segmentations. The aim of this paper was to evaluate the accuracy and repeatability of the SPIJN-MRF parameter estimations and partial volume segmentations. This was done (1) through numerical simulations based on the BrainWeb phantoms and (2) using in vivo acquired MRF data from 5 subjects that were scanned on the same week-day for 8 consecutive weeks. The partial volume segmentations of the SPIJN-MRF method were compared to those obtained by two conventional methods: SPM12 and FSL. SPIJN-MRF showed higher accuracy in simulations in comparison to FSL- and SPM12-based segmentations: Fuzzy Tanimoto Coefficients (FTC) comparing these segmentations and Brainweb references were higher than 0.95 for SPIJN-MRF in all the tissues and between 0.6 and 0.7 for SPM12 and FSL in white and gray matter and between 0.5 and 0.6 in CSF. For the in vivo MRF data, the estimated relaxation times were in line with literature and minimal variation was observed. Furthermore, the coefficient of variation (CoV) for estimated tissue volumes with SPIJN-MRF were 10.5% for the myelin water, 6.0% for the white matter, 5.6% for the gray matter, 4.6% for the CSF and 1.1% for the total brain volume. CoVs for CSF and total brain volume measured on the scanned data for SPIJN-MRF were in line with those obtained with SPM12 and FSL. The CoVs for white and gray matter volumes were distinctively higher for SPIJN-MRF than those measured with SPM12 and FSL. In conclusion, the use of SPIJN-MRF provides accurate and precise tissue relaxation parameter estimations taking into account intrinsic partial volume effects. It facilitates obtaining tissue fraction maps of prevalent tissues including myelin water which can be relevant for evaluating diseases affecting the white matter.

Fractional order vs. exponential fitting in UTE MR imaging of the patellar tendon.

PURPOSE: Quantification of the T2∗ relaxation time constant is relevant in various magnetic resonance imaging applications. Mono- or bi-exponential models are typically used to determine these parameters. However, in case of complex, heterogeneous tissues these models could lead to inaccurate results. We compared a model, provided by the fractional-order extension of the Bloch equation with the conventional models. METHODS: Axial 3D ultra-short echo time (UTE) scans were acquired using a 3.0 T MRI and a 16-channel surface coil. After image registration, voxel-wise T2∗ was quantified with mono-exponential, bi-exponential and fractional-order fitting. We evaluated all three models repeatability and the bias of their derived parameters by fitting at various noise levels. To investigate the effect of the SNR for the different models, a Monte-Carlo experiment with 1000 repeats was performed for different noise levels for one subject. For a cross-sectional investigation, we used the mean fitted values of the ROIs in five volunteers. RESULTS: Comparing the mono-exponential and the fractional order T2∗ maps, the fractional order fitting method yielded enhanced contrast and an improved delineation of the different tissues. In the case of the bi-exponential method, the long T2∗ component map demonstrated the anatomy clearly with high contrast. Simulations showed a nonzero bias of the parameters for all three mathematical models. ROI based fitting showed that the T2∗ values were different depending on the applied method, and they differed most for the patellar tendon in all subjects. CONCLUSIONS: In high SNR cases, the fractional order and bi-exponential models are both performing well with low bias. However, in all observed cases, one of the bi-exponential components has high standard deviation in T2∗. The bi-exponential model is suitable for T2∗ mapping, but we recommend using the fractional order model for cases of low SNR.

Compressed Sensing 3D-GRASE for faster High-Resolution MRI.

PURPOSE: High-resolution three-dimensional (3D) structural MRI is useful for delineating complex or small structures of the body. However, it requires long acquisition times and high SAR, limiting its clinical use. The purpose of this work is to accelerate the acquisition of high-resolution images by combining compressed sensing and parallel imaging (CSPI) on a 3D-GRASE sequence and to compare it with a (CS)PI 3D-FSE sequence. Several sampling patterns were investigated to assess their influence on image quality. METHODS: The proposed k-space sampling patterns are based on two undersampled k-space grids, variable density (VD) Poisson-disc, and VD pseudo-random Gaussian, and five different trajectories described in the literature. Bloch simulations are performed to obtain the transform point spread function and evaluate the coherence of each sampling pattern. Image resolution was assessed by the full-width at half-maximum (FWHM). Prospective CSPI 3D-GRASE phantom and in vivo experiments in knee and brain are carried out to assess image quality, SNR, SAR, and acquisition time compared to PI 3D-GRASE, PI 3D-FSE, and CSPI 3D-FSE acquisitions. RESULTS: Sampling patterns with VD Poisson-disc obtain the lowest coherence for both PD-weighted and T2 -weighted acquisitions. VD pseudo-random Gaussian obtains lower FWHM, but higher sidelobes than VD Poisson-disc. CSPI 3D-GRASE reduces acquisition time (43% for PD-weighted and 40% for T2 -weighted) and SAR (∼45% for PD-weighted and T2 -weighted) compared to CSPI 3D-FSE. CONCLUSIONS: CSPI 3D-GRASE reduces acquisition time compared to a CSPI 3DFSE acquisition, preserving image quality. The design of the sampling pattern is crucial for image quality in CSPI 3D-GRASE image acquisitions.

A neuroradiologist's guide to arterial spin labeling MRI in clinical practice.

Arterial spin labeling (ASL) is a non-invasive MRI technique to measure cerebral blood flow (CBF). This review provides a practical guide and overview of the clinical applications of ASL of the brain, as well its potential pitfalls. The technical and physiological background is also addressed. At present, main areas of interest are cerebrovascular disease, dementia and neuro-oncology. In cerebrovascular disease, ASL is of particular interest owing to its quantitative nature and its capability to determine cerebral arterial territories. In acute stroke, the source of the collateral blood supply in the penumbra may be visualised. In chronic cerebrovascular disease, the extent and severity of compromised cerebral perfusion can be visualised, which may be used to guide therapeutic or preventative intervention. ASL has potential for the detection and follow-up of arteriovenous malformations. In the workup of dementia patients, ASL is proposed as a diagnostic alternative to PET. It can easily be added to the routinely performed structural MRI examination. In patients with established Alzheimer's disease and frontotemporal dementia, hypoperfusion patterns are seen that are similar to hypometabolism patterns seen with PET. Studies on ASL in brain tumour imaging indicate a high correlation between areas of increased CBF as measured with ASL and increased cerebral blood volume as measured with dynamic susceptibility contrast-enhanced perfusion imaging. Major advantages of ASL for brain tumour imaging are the fact that CBF measurements are not influenced by breakdown of the blood-brain barrier, as well as its quantitative nature, facilitating multicentre and longitudinal studies.

In vivo attenuation and genetic evolution of a ST247-SCCmecI MRSA clone after 13 years of pathogenic bronchopulmonary colonization in a patient with cystic fibrosis: implications of the innate immune response.

Methicillin-resistant Staphylococcus aureus (MRSA) causes chronic pulmonary infections in patients with cystic fibrosis (CF). This study tracks the 13-year evolution (1996-2009) of a single MRSA clone in a male patient with CF, evaluating both the host immunogenic response and the microbial variations. Whole-genome sequencing was performed for the initial (CF-96) and evolved (CF-09) isolates. The immunogenicity of CF-96 and CF-09 was evaluated by incubation with innate immune cells from healthy volunteers. We also studied the patient's innate immune response profile, cytokine production, expression of triggering receptor expressed on myeloid cells-1 (TREM-1), and phagocytosis. A total of 30 MRSA ST247-SCCmecI-pvl(-) isolates were collected, which evidenced a genome size reduction from the CF-96 ancestor to the evolved CF-09 strain. Up to six changes in the spa-type were observed over the course of the 13-year evolution. Cytokine production, TREM-1 expression, and phagocytosis were significantly lower for the healthy volunteer monocytes exposed to CF-09, compared with those exposed to CF-96. Patient monocytes exhibited a reduced inflammatory response when challenged with CF-09. Genetic changes in MRSA, leading to reduced immunogenicity and entry into the refractory state, may contribute to the attenuation of virulence and efficient persistence of the bacteria in the CF lung.

Beauty and ugliness in the bodies and faces of others: an fMRI study of person esthetic judgement.

Whether beauty and ugliness represent two independent judgement categories or, instead, opposite extremes of a single dimension is a matter of debate. In the present 3T-functional Magnetic Resonance Imaging (fMRI) study, 20 participants were scanned while judging faces and nude bodies of people classified as extremely ugly, extremely beautiful, or indifferent. Certain areas, such as the caudate/nucleus accumbens (NAcc) and the anterior cingulate cortex (ACC), exhibited a linear relationship across esthetic judgments supporting ugliness as the lowest extreme of a beauty continuum. Other regions, such as basal occipital areas, displayed an inverse pattern, with the highest activations for ugly and the lowest for beautiful ones. Further, several areas were involved alike by both the very beautiful and the very ugly stimuli. Among these, the medial orbitofrontal cortex (mOFC), as well as the posterior and medial portions of the cingulate gyrus. This is interpreted as the activation of neural circuits related to self- vs. other-assessment. Beauty and ugliness in the brain, at least in relation to natural and biologically and socially relevant stimuli (faces and bodies), appear tightly related and non-independent. Finally, neutral stimuli elicited strong and wide activations of the somatosensory and somatomotor systems together with longer reaction times and higher error rates, probably reflecting the difficulty of the human brain to classify someone as indifferent.

Gradient induced artifacts in simultaneous EEG-fMRI: Effect of synchronization on spiral and EPI k-space trajectories.

The nature of the gradient induced electroencephalography (EEG) artifact is analyzed and compared for two functional magnetic resonance imaging (fMRI) pulse sequences with different k-space trajectories: echo planar imaging (EPI) and spiral. Furthermore, the performance of the average artifact subtraction algorithm (AAS) to remove the gradient artifact for both sequences is evaluated. The results show that the EEG gradient artifact for spiral sequences is one order of magnitude higher than for EPI sequences due to the chirping spectrum of the spiral sequence and the dB/dt of its crusher gradients. However, in the presence of accurate synchronization, the use of AAS yields the same artifact suppression efficiency for both pulse sequences below 80Hz. The quality of EEG signal after AAS is demonstrated for phantom and human data. EEG spectrogram and visual evoked potential (VEP) are compared outside the scanner and use both EPI and spiral pulse sequences. MR related artifact residues affect the spectra over 40Hz (less than 0.2 µV up to 120Hz) and modify the amplitude of P1, N2 and P300 in the VEP. These modifications in the EEG signal have to be taken into account when interpreting EEG data acquired in simultaneous EEG-fMRI experiments.

New insights into the archaeal diversity of a hypersaline microbial mat obtained by a metagenomic approach.

A metagenomic approach was carried out in order to study the genetic pool of a hypersaline microbial mat, paying more attention to the archaeal community and, specifically, to the putatively methanogenic members. The main aim of the work was to expand the knowledge of a likely ecologically important archaeal lineage, candidate division MSBL1, which is probably involved in methanogenesis at very high salinities. The results obtained in this study were in accordance with our previous report on the bacterial diversity encountered by using a number of molecular techniques, but remarkable differences were found in the archaeal diversity retrieval by each of the procedures used (metagenomics and 16S rRNA-based methods). The lack of synteny for most of the metagenomic fragments with known genomes, together with the low degree of similarity of the annotated open reading frames (ORFs) with the sequences in the databases, reflected the high degree of novelty in the mat community studied. Linking the sequenced clones with representatives of division MSBL1 was not possible because of the lack of additional information concerning this archaeal group in the public gene repositories. However, given the high abundance of representatives of this division in the 16S rRNA clone libraries and the low identity of the archaeal clones with known genomes, it was hypothesized that some of them could arise from MSBL1 genomes. In addition, other prokaryotic groups known to be relevant in organic matter mineralization at high salinities were detected.

A reconfigurable, wearable, wireless ECG system.

New emerging concepts as "wireless hospital", "mobile healthcare" or "wearable telemonitoring" require the development of bio-signal acquisition devices to be easily integrated into the clinical routine. In this work, we present a new system for Electrocardiogram (ECG) acquisition and its processing, with wireless transmission on demand (either the complete ECG or only one alarm message, just in case a pathological heart rate detected). Size and power consumption are optimized in order to provide mobility and comfort to the patient. We have designed a modular hardware system and an autonomous platform based on a Field-Programmable Gate Array (FPGA) for developing and debugging. The modular approach allows to redesign the system in an easy way. Its adaptation to a new biomedical signal would only need small changes on it. The hardware system is composed of three layers that can be plugged/unplugged: communication layer, processing layer and sensor layer. In addition, we also present a general purpose end-user application developed for mobile phones or Personal Digital Assistant devices (PDAs).

[Functional magnetic resonance imaging of the visual cortex: relation between stimulus intensity and bold response]. / Resonancia magnética funcional de la corteza visual: estudio de las relaciones entre la intensidad del estímulo y la respuesta BOLD.

INTRODUCTION: Visual system is a high interest topic in neuroscience research. The new neuroimaging techniques, such as functional magnetic resonance imaging (fMRI), allow us to quickly improve our knowledge on the visual system using non-invasive methods. This work examines the effect of small changes in the intensity of a visual stimulus over the BOLD response in the visual cortex. AIMS: To perform a detailed analysis of the visual cortex reaction to different intensities of a light source and to verify the ties between the intensity of the visual stimulus and the cortical response. SUBJECTS AND METHODS: Using fMRI (3 T), we registered BOLD response (area and intensity of the signal change) in 20 photophobic patients and 20 controls while viewing different stimulus intensities from a light source. RESULTS: We found a direct relation between stimulus intensity and occipital response. We show that cortical reactivity is higher in patients with photophobia than normal controls, specially for the lower and medium intensities. CONCLUSIONS: fMRI is a valid and robust technique to register consistent and reproducible responses in different groups of subjects. It is useful for the study of normal cortex functioning as well as for clinical use.

Prediction of human protein function from post-translational modifications and localization features.

We have developed an entirely sequence-based method that identifies and integrates relevant features that can be used to assign proteins of unknown function to functional classes, and enzyme categories for enzymes. We show that strategies for the elucidation of protein function may benefit from a number of functional attributes that are more directly related to the linear sequence of amino acids, and hence easier to predict, than protein structure. These attributes include features associated with post-translational modifications and protein sorting, but also much simpler aspects such as the length, isoelectric point and composition of the polypeptide chain.

Evolution of gene order conservation in prokaryotes.

BACKGROUND: As more complete genomes are sequenced, conservation of gene order between different organisms is emerging as an informative property of the genomes. Conservation of gene order has been used for predicting function and functional interactions of proteins, as well as for studying the evolutionary relationships between genomes. The reasons for the maintenance of gene order are still not well understood, as the organization of the prokaryote genome into operons and lateral gene transfer cannot possibly account for all the instances of conservation found. Comprehensive studies of gene order are one way of elucidating the nature of these maintaining forces. RESULTS: Gene order is extensively conserved between closely related species, but rapidly becomes less conserved among more distantly related organisms, probably in a cooperative fashion. This trend could be universal in prokaryotic genomes, as archaeal genomes are likely to behave similarly to bacterial genomes. Gene order conservation could therefore be used as a valid phylogenetic measure to study relationships between species. Even between very distant species, remnants of gene order conservation exist in the form of highly conserved clusters of genes. This suggests the existence of selective processes that maintain the organization of these regions. Because the clusters often span more than one operon, common regulation probably cannot be invoked as the cause of the maintenance of gene order. CONCLUSIONS: Gene order conservation is a genomic measure that can be useful for studying relationships between prokaryotes and the evolutionary forces shaping their genomes. Gene organization is extensively conserved in some genomic regions, and further studies are needed to elucidate the reason for this conservation.

Bringing gene order into bacterial shape.

A different arrangement of a cluster of genes involved in division and cell-wall synthesis separates bacilli from other bacteria in a phylogenetic analysis. We conclude that the relationships between these genes are not random and might reflect significant events in the evolution of the coupling between growth and division in bacteria.

Functional classes in the three domains of life.

The evolutionary divergence among the three major domains of life can now be addressed through the first set of complete genomes from representative species. These model species from the three domains of life, Haemophilus influenzae for Bacteria, Saccharomyces cerevisiae for Eukarya, and Methanococcus jannaschii for Archaea, provide the basis for a universal functional classification and analysis. We have chosen 13 functional classes and three superclasses (ENERGY, COMMUNICATION and INFORMATION) as global descriptors of protein function. Compositional comparison of the three complete genomes reveals that functional classes are ubiquitous yet diverse in the three domains of life. Proteins related with ENERGY processes are generally represented in all three domains, while those related with COMMUNICATION represent the most distinctive functional feature of each single domain. Finally, functions related with INFORMATION processing (translation, transcription, and replication) show a complex behaviour. In Archaea, proteins in this superclass are related with proteins in either Eukarya or Bacteria, as recognized previously. The distribution of functional classes in the three domains accurately reflects the principal characteristics of cellular life forms.

Automated genome sequence analysis and annotation.

MOTIVATION: Large-scale genome projects generate a rapidly increasing number of sequences, most of them biochemically uncharacterized. Research in bioinformatics contributes to the development of methods for the computational characterization of these sequences. However, the installation and application of these methods require experience and are time consuming. RESULTS: We present here an automatic system for preliminary functional annotation of protein sequences that has been applied to the analysis of sets of sequences from complete genomes, both to refine overall performance and to make new discoveries comparable to those made by human experts. The GeneQuiz system includes a Web-based browser that allows examination of the evidence leading to an automatic annotation and offers additional information, views of the results, and links to biological databases that complement the automatic analysis. System structure and operating principles concerning the use of multiple sequence databases, underlying sequence analysis tools, lexical analyses of database annotations and decision criteria for functional assignments are detailed. The system makes automatic quality assessments of results based on prior experience with the underlying sequence analysis tools; overall error rates in functional assignment are estimated at 2.5-5% for cases annotated with highest reliability ('clear' cases). Sources of over-interpretation of results are discussed with proposals for improvement. A conservative definition for reporting 'new findings' that takes account of database maturity is presented along with examples of possible kinds of discoveries (new function, family and superfamily) made by the system. System performance in relation to sequence database coverage, database dynamics and database search methods is analysed, demonstrating the inherent advantages of an integrated automatic approach using multiple databases and search methods applied in an objective and repeatable manner. AVAILABILITY: The GeneQuiz system is publicly available for analysis of protein sequences through a Web server at http://www.sander.ebi.ac. uk/gqsrv/submit