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PubMed2000

Optimization of cDNA representational difference analysis for the identification of differentially expressed mRNAs.

Representational difference analysis (RDA) is a powerful and sensitive tool for identification of differentially expressed genes (M. Hubank and D. G. Schatz, 1999, Methods Enzymol. 303, 325-349; 1994, Nucleic Acids Res. 22, 5640-5648) that will identify both up- and downregulated genes differentially expressed between two cDNA populations. This manuscript provides a thorough description of an optimized RDA method. This procedure while still based on the traditional RDA originally developed by Lisitsyn and co-workers(N. A. Lisitsyn, 1995, Trends Genet. 11, 303-307; N. A. Lisitsyn, F. S. Leach, B. Vogelstein, and M. H. Wigler, 1994, Cold Spring Harbor Symp. Quant. BioL 59, 585-587; N. Lisitsyn, N. Lisitsyn, and M. Wigler, 1993, 259, 946-951) and modified by Hubank and Schatz for RNA (1994, Nucleic Acids Res. 22, 5640-5648) is improved and requires less starting material than many existing methods. Several key modifications are included (1). Size-exclusion gel-filtration microspin columns are used throughout the procedure to remove the primers and low molecular weight cDNAs. This results in reducing the number of ethanol precipitations required and in improving the yield of desirable amplification products (2). Elimination of the mung bean nuclease treatment in favor of a simple dilution of PCR serves as a means of markedly reducing the single-stranded cDNAs that can interfere with the amplification of differentially expressed products (3). The use of up to six unique noninteracting primers ensures that no anomalous amplification occurs due to carryover of primers or incomplete digestion from the ends of the cDNAs (4). A set of cDNA standards was developed and various concentrations were used to better characterize the ability of representational difference analysis to identify rare messages in a complex cDNA population (5). Integral to this manuscript, a detailed laboratory protocol is available from the authors (craig.byus@ucr.edu) and provides a step-by-step description of the modified procedure.

Base Sequence↗

PubMed1994

Predominance of T cell receptor V delta 3 in small bowel biopsies from coeliac disease patients.

Increased numbers of T cells bearing the gamma delta antigen receptor (gamma delta T cells) have been reported in small bowel biopsies of patients with latent, active or treated coeliac disease. We have studied jejunal biopsies from seven children with coeliac disease and 10 children with normal gut histology to characterize gamma delta T cell receptor (TCR) variable region gene subfamily expression in resident gamma delta T cells and compared the results with the findings in peripheral blood mononuclear cells (PBMC) obtained on the same day as the gut biopsy. Molecular analysis of RNA extracted from PBMC and biopsies was performed by reverse transcription and amplification with the polymerase chain reaction using primers specific for six TCR V delta families and four TCR V gamma families. We report, first, that a significantly increased number of gamma delta T cells expressing the TCR V delta 3 subfamily (P = 0.008) was observed in jejunal biopsies from children with coeliac disease, and second, that gamma delta T cell V region subfamily populations in gut differed from those seen in PBMC for both control and coeliac patients. Significantly reduced numbers of TCR V delta 2, V delta 3, V delta 5 (P < 0.01) and V gamma 2, V gamma 4 (P < 0.01) T cells were found in gut compared with PBMC. The difference in gamma delta T cell repertoire observed between gut and blood may reflect differences in the nature of the antigens usually encountered in these two compartments. The over-representation of TCR V delta 3 in patients with coeliac disease suggests a specific role for these cells in the induction or maintenance of the jejunal abnormality associated with this disease.

Base Sequence↗

PubMed2025

Selecting candidate Neisseria gonorrhoeae strains for oropharyngeal gonorrhoea human challenge: a genomics-based analysis of clinical isolates.

BACKGROUND: Neisseria gonorrhoeae is a human pathogen of major public health importance due to its increasing global prevalence and antimicrobial resistance (AMR). Evidence suggests that oropharyngeal infection plays a key role in N gonorrhoeae transmission and AMR; however, our understanding of oropharyngeal gonorrhoea pathogenesis is poor. A controlled human infection model (CHIM) for oropharyngeal gonorrhoea will improve understanding of infection and accelerate urgently needed novel gonorrhoea prevention and therapeutic strategies. As the first step in the development of this CHIM, we describe a systematic approach to CHIM strain selection that leverages genomics and clinical data. METHODS: In this genomics-based analysis, we applied a systematic N gonorrhoeae challenge strain selection strategy incorporating genomic and clinical data to a primary dataset of clinical isolates of N gonorrhoeae collected from adult patients in Victoria, Australia, between Jan 1 and Dec 31, 2017, and July 1, 2019, and June 30, 2021. This selection strategy used clinical, phenotypic, and genomic characteristics to define a set of eight criteria that aimed to ensure the contemporary global clinical relevance of the candidate strains; select strains that would be applicable for the assessment of current and future gonorrhoea vaccines; and maximise participant safety by reducing the risk of disseminated gonococcal infection and clinically significant AMR. We applied these criteria to our primary dataset to generate a panel of potential challenge strains. From this final dataset of potential challenge strains, we predetermined that we would select up to ten isolates to proceed to the next stage of detailed phenotypic characterisation for final N gonorrhoeae CHIM strain selection. FINDINGS: 5881 isolates comprised the primary dataset. After application of the selection criteria, most of the isolates (5795 [98·6%] of 5881) were excluded, mostly due to having clinically significant AMR and poor contemporary global clinical relevance. The remaining 86 N gonorrhoeae challenge strain candidates comprised five multilocus sequence types and six N gonorrhoeae multiantigen sequence types, many of which were represented by a single isolate. Of these 86 strains, five isolates were selected to maximise coverage of the phylogenetically distinct groups within the 86 candidate challenge strains and ensure representation of strains collected from various anatomical sites. INTERPRETATION: We transparently describe a novel, systematic, and rational genomics-based strategy for oropharyngeal gonorrhoea CHIM strain selection that improves the efficiency and transparency of CHIM strain selection and enables identification of contemporary and clinically relevant potential challenge strains. A final N gonorrhoeae challenge strain will be selected from the subset of five shortlisted candidates after detailed phenotypic assessment. FUNDING: Medical Research Future Fund, Australian National Health and Medical Research Council and Australian Government Research Training Program.

Humans↗

PubMed2002

An in vitro study to evaluate the accuracy of stereotactic localization using magnetic resonance imaging by means of the Leksell stereotactic system.

The advantages of using magnetic resonance imaging (MRI) as opposed to computed tomographic (CT) scans or ventriculography in stereotactic surgery include the increased tissue contrast of the lesion or target, direct non-reformatted multiplanar imaging and target coordinate determination as well as reduced imaging artefacts produced by the stereotactic frame. One disadvantage of MR stereotaxis, however, is the potential for anatomic inaccuracy due to equipment-induced inhomogeneities of the magnetic field. The authors present an experimental study on an in vitro model to examine the accuracy of target localization using the Leksell stereotactic frame and MR imaging. Ten formalin-fixed brains taken from patients who had died of non-neurological diseases were sealed in a properly modelled plaster-cast shell simulating the skull bone. These models were fixed in the Leksell stereotactic frame and high-field MR images were performed (Siemens Magnetom SP 1.5 Tesla, T1-weighted spin echo sequences, TR/TE 600/15 ms, slice thickness 2 mm, FOV 300 mm). Following electrocoagulation of different targets on both lentiforme nuclei, the localization and extension of the lesions were controlled by MRI. A gross-/histopathological verification was performed. This model allows a good representation of the anatomic structures without any artefacts. The postoperative MRI control and the pathological examination of the lesions matched well with the preoperatively defined targets. The correlation of coordinates and measurements obtained with the pathological studies were within a +/- 2 mm range in all cases.

Brain↗

PubMed2006

Concept of sample in OMICS technology.

Fundamental biological processes can now be studied by applying the full range of OMICS technologies (genomics, transcriptomics, proteomics, metabolomics, and beyond) to the same biological sample. Clearly, it would be desirable if the concept of sample were shared among these technologies, especially as up until the time a biological sample is prepared for use in a specific OMICS assay, its description is inherently technology independent. Sharing a common informatic representation would encourage data sharing (rather than data replication), thereby reducing redundant data capture and the potential for error. This would result in a significant degree of harmonization across different OMICS data standardization activities, a task that is critical if we are to integrate data from these different data sources. Here, we review the current concept of sample in OMICS technologies as it is being dealt with by different OMICS standardization initiatives and discuss the special role that the newly formed Genomic Standards Consortium (GSC) might have to play in this domain.

Animals↗

PubMed2000

Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype.

We have applied a sensitive global analysis of TCR heterogeneity to compare clonal dynamics of CD4(+) and CD8(+) T cells in acute infectious mononucleosis. Using this approach, we are able to identify a broad representation of the total virus-specific population without the bias of in vitro culture and then to track their phenotype and fate by their unique molecular footprint. We demonstrate a large number of Ag-driven clones using different TCRs in the acute phase, all CD8(+). The diverse large clones generated in the CD8 subset in response to this virus contrast with the complete lack of detectable clonal expansion in the CD4 compartment. Many of the same clones remain detectable in directly ex vivo CD8(+) T cells for at least a year after resolution of infectious mononucleosis, although the clone size is reduced. Thus, memory CD8 cells following EBV infection persist at relatively high circulating frequency and represent a subset of the large range of clonotypes comprising the acute effectors. Separation of samples into CD45RA (naive) and CD45RO (memory) fractions shows the accumulation of identical CDR3 region defined clonotypes in both CD45RO and CD45RA fractions and sequencing confirms that dominant long-lived monoclonal expansions can reside in the CD45RA pool.

Acute Disease↗

PubMed2004

Biologically valid linear factor models of gene expression.

MOTIVATION: The identification of physiological processes underlying and generating the expression pattern observed in microarray experiments is a major challenge. Principal component analysis (PCA) is a linear multivariate statistical method that is regularly employed for that purpose as it provides a reduced-dimensional representation for subsequent study of possible biological processes responding to the particular experimental conditions. Making explicit the data assumptions underlying PCA highlights their lack of biological validity thus making biological interpretation of the principal components problematic. A microarray data representation which enables clear biological interpretation is a desirable analysis tool. RESULTS: We address this issue by employing the probabilistic interpretation of PCA and proposing alternative linear factor models which are based on refined biological assumptions. A practical study on two well-understood microarray datasets highlights the weakness of PCA and the greater biological interpretability of the linear models we have developed.

Algorithms↗

PubMed1995

Neural net representations of empirical protein potentials.

Recently, there has been considerable interest in deriving and applying knowledge-based, empirical potential functions for proteins. These empirical potentials have been derived from the statistics of interacting, spatially neighboring residues, as may be obtained from databases of known protein crystal structures. In this paper we employ neural networks to redefine empirical potential functions from the point of view of discrimination functions. This approach generalizes previous work, in which simple frequency counting statistics are used on a database of known protein structures. This generalization allows us to avoid restriction to strictly pairwise interactions. Instead of frequency counting to fix adjustable parameters, one now optimizes an objective function involving a neural network parameterized probability distribution. We show how our method reduces to previous work in special situations, but also allows extensions to include orders of interaction beyond pairwise interaction. Given the close packing of proteins, steric interactions etc., the inclusion of higher order interactions is critical for developing an accurate potential. A key feature in the approach we advocate is the development of a representation to describe the spatial location of interacting residues that exist in a sphere of small fixed radius around each residue. This is a "shape representation" problem that has a natural solution for the interaction neighborhoods of protein residues. We demonstrate in a series of numerical experiments that the neural network approach improves discrimination over that obtained by previous methodologies limited to pair-wise interactions.

Amino Acid Sequence↗

PubMed1999

Significance of Z-value statistics of Smith-Waterman scores for protein alignments.

The Z-value is an attempt to estimate the statistical significance of a Smith-Waterman dynamic alignment score (SW-score) through the use of a Monte-Carlo process. It partly reduces the bias induced by the composition and length of the sequences. This paper is not a theoretical study on the distribution of SW-scores and Z-values. Rather, it presents a statistical analysis of Z-values on large datasets of protein sequences, leading to a law of probability that the experimental Z-values follow. First, we determine the relationships between the computed Z-value, an estimation of its variance and the number of randomizations in the Monte-Carlo process. Then, we illustrate that Z-values are less correlated to sequence lengths than SW-scores. Then we show that pairwise alignments, performed on 'quasi-real' sequences (i.e., randomly shuffled sequences of the same length and amino acid composition as the real ones) lead to Z-value distributions that statistically fit the extreme value distribution, more precisely the Gumbel distribution (global EVD, Extreme Value Distribution). However, for real protein sequences, we observe an over-representation of high Z-values. We determine first a cutoff value which separates these overestimated Z-values from those which follow the global EVD. We then show that the interesting part of the tail of distribution of Z-values can be approximated by another EVD (i.e., an EVD which differs from the global EVD) or by a Pareto law. This has been confirmed for all proteins analysed so far, whether extracted from individual genomes, or from the ensemble of five complete microbial genomes comprising altogether 16956 protein sequences.

Computing Methodologies↗

PubMed2004

Completely automated, highly error-tolerant macromolecular structure determination from multidimensional nuclear overhauser enhancement spectra and chemical shift assignments.

The major rate-limiting step in high-throughput NMR protein structure determination involves the calculation of a reliable initial fold, the elimination of incorrect nuclear Overhauser enhancement (NOE) assignments, and the resolution of NOE assignment ambiguities. We present a robust approach to automatically calculate structures with a backbone coordinate accuracy of 1.0-1.5 A from datasets in which as much as 80% of the long-range NOE information (i.e., between residues separated by more than five positions in the sequence) is incorrect. The current algorithm differs from previously published methods in that it has been expressly designed to ensure that the results from successive cycles are not biased by the global fold of structures generated in preceding cycles. Consequently, the method is highly error tolerant and is not easily funnelled down an incorrect path in either three-dimensional structure or NOE assignment space. The algorithm incorporates three main features: a linear energy function representation of the NOE restraints to allow maximization of the number of simultaneously satisfied restraints during the course of simulated annealing; a method for handling the presence of multiple possible assignments for each NOE cross-peak which avoids local minima by treating each possible assignment as if it were an independent restraint; and a probabilistic method to permit both inactivation and reactivation of all NOE restraints on the fly during the course of simulated annealing. NOE restraints are never removed permanently, thereby significantly reducing the likelihood of becoming trapped in a false minimum of NOE assignment space. The effectiveness of the algorithm is demonstrated using completely automatically peak-picked experimental NOE data from two proteins: interleukin-4 (136 residues) and cyanovirin-N (101 residues). The limits of the method are explored using simulated data on the 56-residue B1 domain of Streptococcal protein G.

Algorithms↗

PubMed2000

Binaural sluggishness in the perception of tone sequences and speech in noise.

The binaural system is well-known for its sluggish response to changes in the interaural parameters to which it is sensitive. Theories of binaural unmasking have suggested that detection of signals in noise is mediated by detection of differences in interaural correlation. If these theories are correct, improvements in the intelligibility of speech in favorable binaural conditions is most likely mediated by spectro-temporal variations in interaural correlation of the stimulus which mirror the spectro-temporal amplitude modulations of the speech. However, binaural sluggishness should limit the temporal resolution of the representation of speech recovered by this means. The present study tested this prediction in two ways. First, listeners' masked discrimination thresholds for ascending vs descending pure-tone arpeggios were measured as a function of rate of frequency change in the NoSo and NoSpi binaural configurations. Three-tone arpeggios were presented repeatedly and continuously for 1.6 s, masked by a 1.6-s burst of noise. In a two-interval task, listeners determined the interval in which the arpeggios were ascending. The results showed a binaural advantage of 12-14 dB for NoSpi at 3.3 arpeggios per s (arp/s), which reduced to 3-5 dB at 10.4 arp/s. This outcome confirmed that the discrimination of spectro-temporal patterns in noise is susceptible to the effects of binaural sluggishness. Second, listeners' masked speech-reception thresholds were measured in speech-shaped noise using speech which was 1, 1.5, and 2 times the original articulation rate. The articulation rate was increased using a phase-vocoder technique which increased all the modulation frequencies in the speech without altering its pitch. Speech-reception thresholds were, on average, 5.2 dB lower for the NoSpi than for the NoSo configuration, at the original articulation rate. This binaural masking release was reduced to 2.8 dB when the articulation rate was doubled, but the most notable effect was a 6-8 dB increase in thresholds with articulation rate for both configurations. These results suggest that higher modulation frequencies in masked signals cannot be temporally resolved by the binaural system, but that the useful modulation frequencies in speech are sufficiently low (<5 Hz) that they are invulnerable to the effects of binaural sluggishness, even at elevated articulation rates.

Auditory Threshold↗

PubMed1991

Optimized strategies for sequence-tagged-site selection in genome mapping.

The physical mapping of complex genomes is based on the construction of a genomic library and the determination of the overlaps between the inserts of the mapping clones in order to generate an ordered, cloned representation of nearly all the sequences present in the target genome. Evaluation of the relative efficiency of experimental procedures used to accomplish this goal must minimally include a comparison of the fraction of the genome covered by the ordered arrays (or "contigs"), the average size of the contigs, and the cost, in terms of time and resources, required to generate the map. Sequence-tagged-site (STS) content mapping is one strategy that has been proposed and is being utilized for this type of experiment. This paper describes three STS selection schemes and presents computer simulations of contig-building experiments based on these procedures. The results of these simulations suggest that a nonrandom STS strategy that uses paired probes requires one-third to one-fourth as many STS assays as are required in random and nonpaired approaches, and also results in a map that has both greater genome coverage and a larger average contig size. This strategy promises to reduce the time and cost required to build a high-quality physical map.

Base Sequence↗

PubMed2006

Differential mitochondrial DNA and gene expression in inherited retinal dysplasia in miniature Schnauzer dogs.

PURPOSE: To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers. METHODS: Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy. RESULTS: RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense. CONCLUSIONS: Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

Animals↗

PubMed2005

Analysis of T-cell receptor V beta gene repertoires after immune stimulation and in malignancy by use of padlock probes and microarrays.

BACKGROUND: Detection of expanded T-cell clones, identified by their receptor (TCR) repertoires, can assist diagnosis and guide therapy in infectious, inflammatory, and autoimmune conditions as well as in tumor immunotherapy. Analysis of tumor-infiltrating lymphocytes often reveals preferential use of one or a few TCR V beta genes, compared with peripheral blood, indicative of a clonal response against tumor antigens. METHODS: To simultaneously measure the relative expression of all V beta gene families, we combined highly specific and sensitive oligonucleotide reagents, called padlock probes, with a microarray read-out format. T-Cell cDNA was combined with a pool of V beta subfamily-specific padlock probes. Reacted probes were selectively amplified and the products hybridized to a microarray, from which the V beta subfamily distribution in each sample could be determined relative to a control sample. RESULTS: In lymphocytes stimulated with the superantigen staphylococcal enterotoxin B, we detected expansions at the mRNA level of TCR subfamilies previously shown to respond to staphylococcal enterotoxin B. Expansions of the same V beta families could also be detected by flow cytometry. In samples from two bladder cancer patients, we detected predominant representations of specific V beta subfamilies in both tumor-infiltrating lymphocytes and in the draining lymph nodes, but not in non-tumor-draining lymph nodes or peripheral blood. Several expression profiles from draining lymph nodes in patients with malignant melanoma were divergent from profiles seen in non-tumor-draining lymph nodes. CONCLUSION: Padlock probe-based parallel analysis of TCR V beta gene distributions provides an efficient method for screening multiple samples for T-cell clonal expansions with reduced labor and time of analysis compared with traditional methods.

Adenocarcinoma↗

PubMed2003

Comparative analysis of the Arabidopsis pollen transcriptome.

We present a genome-wide view of the male gametophytic transcriptome in Arabidopsis based on microarray analysis. In comparison with the transcriptome of the sporophyte throughout development, the pollen transcriptome showed reduced complexity and a unique composition. We identified 992 pollen-expressed mRNAs, nearly 40% of which were detected specifically in pollen. Analysis of the functional composition of the pollen transcriptome revealed the over-representation of mRNAs encoding proteins involved in cell wall metabolism, cytoskeleton, and signaling and under-representation of mRNAs involved in transcription and protein synthesis. For several gene families, we observed a common pattern of mutually exclusive gene expression between pollen and sporophytic tissues for different gene family members. Our results provide a 50-fold increase in the knowledge of genes expressed in Arabidopsis pollen. Moreover, we also detail the extensive overlap (61%) of the pollen transcriptome with that of the sporophyte, which provides ample potential to influence sporophytic fitness through gametophytic selection.

Arabidopsis↗

PubMed2001

Differential expression of a novel serine protease homologue in squamous cell carcinoma of the head and neck.

Differential gene expression between squamous cell carcinoma of the head and neck and matched normal tissue was studied by utilizing Representational Difference Analysis. Using this methodology, a novel gene, DESC1 was isolated. DESC1 possesses strong identity to the serine protease super-family. Comparison of DESC1 expression between primary squamous cell carcinoma and matched normal tissue shows that the level of DESC1 expression is reduced or absent in 11/12 SCC tissue specimens when compared to specimens of matched normal tissue. Tissue-specific expression studies further show that DESC1 expression can only be detected in tissues derived from the head and neck, and in skin, prostate and testes. Cell line studies demonstrate that DESC1 expression is epithelial-specific. Chromosomal localization studies indicate that DESC1 is located on the long arm of chromosome 4 at position q12-13.

Amino Acid Sequence↗

PubMed2006

A two-stage approach for improved prediction of residue contact maps.

BACKGROUND: Protein topology representations such as residue contact maps are an important intermediate step towards ab initio prediction of protein structure. Although improvements have occurred over the last years, the problem of accurately predicting residue contact maps from primary sequences is still largely unsolved. Among the reasons for this are the unbalanced nature of the problem (with far fewer examples of contacts than non-contacts), the formidable challenge of capturing long-range interactions in the maps, the intrinsic difficulty of mapping one-dimensional input sequences into two-dimensional output maps. In order to alleviate these problems and achieve improved contact map predictions, in this paper we split the task into two stages: the prediction of a map's principal eigenvector (PE) from the primary sequence; the reconstruction of the contact map from the PE and primary sequence. Predicting the PE from the primary sequence consists in mapping a vector into a vector. This task is less complex than mapping vectors directly into two-dimensional matrices since the size of the problem is drastically reduced and so is the scale length of interactions that need to be learned. RESULTS: We develop architectures composed of ensembles of two-layered bidirectional recurrent neural networks to classify the components of the PE in 2, 3 and 4 classes from protein primary sequence, predicted secondary structure, and hydrophobicity interaction scales. Our predictor, tested on a non redundant set of 2171 proteins, achieves classification performances of up to 72.6%, 16% above a base-line statistical predictor. We design a system for the prediction of contact maps from the predicted PE. Our results show that predicting maps through the PE yields sizeable gains especially for long-range contacts which are particularly critical for accurate protein 3D reconstruction. The final predictor's accuracy on a non-redundant set of 327 targets is 35.4% and 19.8% for minimum contact separations of 12 and 24, respectively, when the top length/5 contacts are selected. On the 11 CASP6 Novel Fold targets we achieve similar accuracies (36.5% and 19.7%). This favourably compares with the best automated predictors at CASP6. CONCLUSION: Our final system for contact map prediction achieves state-of-the-art performances, and may provide valuable constraints for improved ab initio prediction of protein structures. A suite of predictors of structural features, including the PE, and PE-based contact maps, is available at http://distill.ucd.ie.

Algorithms↗

PubMed1995

Drastic changes in the peptidoglycan composition of penicillin resistant laboratory mutants of Streptococcus pneumoniae.

The penicillin MIC of 2 Streptococcus pneumoniae clinical isolates was increased 100-fold (from 0.02 to 2.0 micromilligrams) and 20-fold (from 0.5 to 10.0 micromilligrams) through gradual exposure of the bacteria to increasing concentrations of penicillin in the laboratory. In both mutants the affinity of all four high molecular mass penicillin binding proteins (PBPs) for penicillin was drastically reduced accompanied by major changes in the composition of peptidoglycan as resolved by HPLC. The ratio of crosslinked to monomeric peptides became virtually inverted in the resistant cell walls with monomers representing two-thirds of the muropeptide species. The proportion of the crosslinked tri-tetra dimer, a major component of the cell wall of the original isolates, decreased to one-third or one-sixth of its normal representation, while the amounts of tripeptide monomers with an alanyl-serine substitution on the lysine epsilon amino group increased by close to a factor of two. The growth rates of both mutants decreased by a factor of approximately two, as compared to the original bacteria.

Amino Acid Sequence↗