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PubMed2026

MetaStrainer: accurate reconstruction of bacterial strain genotypes from short-read metagenomic samples.

MOTIVATION: Metagenomics provides broad insights from microbial communities, but more biological relevant phenotypes are attributed to subtle changes at the strain-level rather than species. Despite development of several tools using different algorithms, resolving individual strains from short-read pair-end sequencing data remains challenging. RESULTS: Here we present MetaStrainer, a tool capable of reconstructing strain genotypes from metagenomic data. Compared with existing approaches, MetaStrainer substantially increases genotype accuracy, correctly identifies the number of strains, and accurately estimates their relative abundances. Accuracy of reconstructed genotypes is robust to choice of mapping reference. AVAILABILITY: MetaStrainer is implemented in Python 3. Source code and instructions are available on GitHub at www.github.com/lbobay/MetaStrainer and on Zenodo: 10.5281/zenodo.17872331.

Metagenomics↗
PubMed1993

FISH ordering of reference markers and of the gene for the alpha 5 subunit of the gamma-aminobutyric acid receptor (GABRA5) within the Angelman and Prader-Willi syndrome chromosomal regions.

We have established a probe order within the Angelman/Prader-Willi chromosomal regions by multicolor fluorescence in situ hybridization (FISH). The probe [locus] order extending distally from the centromere is 34[D15S9]-IR4-3R[D15S11]-189-1[D15S13]-PW71++ + [D15S63]-3-21[D15S10]-28 beta 3-H3[GABRB3]-IR10-1 [D15S12]. This order agrees with that recently reported (1) with the exception of PW71 [D15S63]. In addition, a second gamma-aminobutyric acid (GABAA) receptor, the alpha 5 subunit, has been localized within the reference map to between GABRB3 and D15S12. The locus order was further confirmed by DNA hybridization analysis of two patients, one with Angelman syndrome and one with Prader-Willi syndrome, with different unbalanced translocations and molecular extents of deletion. Our results provide a framework map of chromosome 15q11-q13 into which additional markers can be oriented and allow a further differentiation of the critical genetic regions of the two syndromes.

Amino Acid Sequence↗
PubMed2005

DynaProt 2D: an advanced proteomic database for dynamic online access to proteomes and two-dimensional electrophoresis gels.

DynaProt 2D presents an advanced online database for dynamic access to proteomes and two-dimensional (2D) gels. The database was designed to administer complete in silico proteomes and links them with experimental proteomic data in the manner of 2D electrophoresis gels (IPG-Dalt). The 2D gels serve as reference maps in 2D gel analysis as well as tools for navigation of the database to switch between experimental and predicted data. Therefore, all identified spots in the gels are clickable and linked with summarized protein information. The protein information tables contain calculated characteristics, which are often used in proteomics, such as the molecular weight, isoelectric point, codon adaptation index, grand average of hydropathicity, etc. The design of the database permits online extension of gel data and protein attributes without knowledge of any software language. Besides navigation via 2D gels, the clear graphical user interface permits quick and intuitive searching throughout complete proteomes and supports, e.g. the search for proteins with isoelectric points within pH ranges of interest or protein classes (e.g. ribosomal proteins or transporters). The first organism implemented in the database is Lactococcus lactis. The database is available at www.wzw.tum.de/proteomik/lactis.

Bacterial Proteins↗
PubMedDate not supplied

The mitotic, polytene, and meiotic chromosomes of Drosophila ananassae.

The mitotic chromosome complement of D. ananassae consists of four structurally distinguishable submetacentric pairs and all four have been identified with their linkage groups. For the polytene chromosome complement of six arms representing the X, second and third chromosomes, an improved reference map has been constructed and used to describe selected cytogenetically useful rearrangements. In meiotic prophase of spermatocytes, chromosomes 2 and 3 form pachytene-diplotene bivalents whose arms may be associated by chiasmata in postdiplotene stages, but the X, Y and fourth chromosomes participate in a complex multivalent. No correlation was detected between meiotic chromosome behavior and specific genes that regulate crossing over in males. In male inversion heterozygotes having high levels of genetically monitored crossing over, no unequivocal evidence was found for formation of either pachytene inversion loops or anaphase bridges and fragments.

Animals↗
PubMed2004

Protein patterns of pig oocytes during in vitro maturation.

In vitro maturation (IVM) of fully grown mammalian oocytes is characterized by initial germinal vesicle (GV) breakdown and rearrangement of microtubule network during the first meiosis (MI), followed by extrusion of the first polar body and block of the oocytes in metaphase of the second meiosis (MII). Only fully matured oocytes are capable of undergoing fertilization and the initiation of zygotic development. These observations are mostly based on morphological evaluation; however, the molecular events responsible for these processes are not known. In this study, we have launched the analysis of pig oocytes during in vitro maturation using a proteomics approach. First, oocyte proteins have been separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Remarkably, several proteins, including peroxiredoxins, ubiquitin carboxyl-terminal hydrolase isozyme L1, and spermine synthase, are even more abundant than actin, usually the most abundant protein in somatic cells. Furthermore, we have initiated comparative analysis of the oocytes at different stages of maturation to characterize candidate proteins, which are differentially expressed during in vitro maturation. To date, we have identified antiquitin (D7A1), the member of aldehyde dehydrogenase family7 that has been significantly increased in MI and MII stages compared with GV oocytes. To our knowledge, this is the first pig oocyte proteome available so far that may be used as a reference map. The proteins that are differentially regulated during IVM may present potential biomarkers of oocyte maturation and quality. It is a useful inventory toward a deeper understanding of the mechanisms underlying reproduction and development.

Animals↗
PubMed2002

Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100.

Proteomics is a powerful tool for analysing differences in gene expression between bacterial strains with alternate phenotypes. Staphylococcus aureus strains are grouped on the basis of their sensitivity to methicillin. Two-dimensional gel electrophoresis was combined with MS to compare the protein profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 'hypothetical' proteins was confirmed, and eight of these appeared to be unique to S. aureus. Strain COL displayed 12 protein spots, which included alkaline-shock protein 23 (Asp23) and cold-shock proteins CspABC, which either were not present in strain 8325 or were present at a significantly lower intensity in this strain. Comparative maps were used to characterize the S. aureus response to treatment with Triton X-100 (TX-100), a detergent that has been shown to reduce methicillin resistance independently of an interaction with the mecA-encoded penicillin-binding protein 2a. In response to growth of the bacteria in the presence of TX-100, 44 protein spots showed altered levels of abundance, and 11 of these spots were found only in COL. The products of genes regulated by sigma(B) (the alternative sigma factor), including Asp23 and three proteins of unknown function, and SarA (a regulator of virulence genes) were shown to be present at significantly altered levels. SarA production was induced in TX-100-treated cultures. A protein of the sigma(B) operon, RsbV, was only detected in COL and its production was down-regulated in COL when the strain was treated with TX-100, whereas RsbW was present at reduced levels in both strains. Upon growth of both strains in the presence of TX-100, no effects on the production of the essential methicillin-resistance factor FemA were detected, whereas phosphoglucosamine mutase (GlmM) production was reduced in COL alone. This study suggests that proteins of the sigma(B) and sarA regulons, as well as other factors, are involved in methicillin resistance in S. aureus.

Bacterial Proteins↗
PubMed1999

Neurofuzzy adaptive controlling of selective stimulation for FES: a case study.

A controller was designed for the selective stimulation of the sciatic nerve with a multiple contact cuff electrode to generate a desired torque in the ankle joint of cat. The design integrates three approaches, artificial neural network (ANN) modeling, fuzzy logical adaptation, and geometrical mapping. The geometrical mapping refers to the vector transformation from the joint coordinates to the virtual muscle coordinates which have been conceptually developed to represent the major recruitment features of contact-based functional units in the physical plant. This method reduces the complexity of generating a data set for training the neural network in the feedforward path and implementing the on-line learning algorithm embedded in the feedback loop. The controller was evaluated by computer simulation with the experimental data obtained from the torque generation in five acute cats. The results show that the ANN-based feedforward is capable of predicting 65% of a given desired isometric torque, and the fuzzy logical machine is able to provide suitable gains for feedback modulation to reduce the error from 35 to 8.5% and produce a robust control.

Animals↗
PubMed2002

Two-dimensional analysis of exoproteins of methicillin-resistant Staphylococcus aureus (MRSA) for possible epidemiological applications.

We applied two-dimensional gel electrophoresis (2-DE) to the total exoproteins secreted from pathogenic MRSA strains and identified major protein spots by N-terminal amino acid sequence analysis. In approximately 300 to 500 spots visualized on each gel, various exoproteins and cell-associated proteins were identified and their sites on the gels confirmed for construction of a reference map. Major exotoxins such as enterotoxins SEA, SEB, and SEC,, toxic shock syndrome toxin-1 (TSST-1), and hemolysins were distributed in the region of pI 6.8 to 8.1 and MW 21 to 35 kDa. Although the differences between calculated and observed values of pI and MW were relatively small in each exoprotein, those of several proteins including alpha-hemolysin and SEB were considerably deviated from the positions of the expected values. Some exoproteins were detected as multiple spots. These included beta-hemolysin, enterotoxins SEA, SEB, and SEC3, glutamic acid-specific endopeptidase, glycerophosphoryl diester phosphodiesterase and triacylglycerol lipase. The multiple spots of these exoproteins may be generated by the action of own proteases. Certain similarities of 2-DE patterns among strains belonging to the same coagulase types were observed. On the basis of 2-DE image analysis, coagulase type II strains secreted somewhat larger amounts of SEB and SEC3 as well as TSST-1 than the strains belonging to other coagulase types. Taken together, 2-DE analysis of exoproteins is applicable to epidemiological studies for MRSA, as compared with pulsed field gel electrophoresis of restricted chromosomal DNA.

Amino Acid Sequence↗
PubMed1997

Role of Escherichia coli histone-like nucleoid-structuring protein in bacterial metabolism and stress response--identification of targets by two-dimensional electrophoresis.

The histone-like nucleoid-structuring protein, H-NS, is a major bacterial chromatin component which influences DNA structure and gene expression. Mutations in hns, the structural gene of H-NS protein, have been shown to result in highly pleiotropic effects in Escherichia coli cells. In this study, we have initiated an index of the proteins whose synthesis is, directly or indirectly regulated by H-NS. Using two-dimensional gel electrophoresis, we have examined the global changes in gene expression which occured in an hns background compared with its wild-type parent. In addition, we analysed the effects of mutations in two other genes i.e. lrp and pta, which are also involved in global regulatory pathways. Although these comparative analyses revealed several common differences, thus suggesting possible interactions between these regulatory mechanisms, i.e. H-NS, Lrp (leucine-responsive regulatory protein) and acetylphosphate, the most extensive modifications occurred in an hns mutant. Among the polypeptides whose level of synthesis was specifically altered in an hns mutant, several corresponded to H-NS targets previously identified by classical selection methods. Moreover, the present study allows us to characterize several H-NS targets, which were identified either by comparison with the E. coli two-dimensional reference maps or by microsequencing procedure. Many of these newly identified polypeptides are involved in adaptation of E. coli cells to environmental challenges, and one of them could be involved in bacterial virulence. Finally, synthesis of several proteins belonging to the heat-shock regulon, more particularly molecular chaperones, was induced in an hns mutant.

Amino Acid Sequence↗
PubMed2005

Shifting from preconceptions to pure wonderment.

The author reflects upon her role as a public health nurse striving to attain practice authenticity. Client assessment and nursing interventions were seemingly sufficient until she became curious about 'Who is this person sitting across from me?' and 'What are her experiences in the world as a lone parent living in poverty at the margins of society?' The author begins to think that she could shift from mere client investigation to pure wonderment about the Other by imagining herself as a researcher, an explorer of another's life world. Ultimately this process enables her to enhance the 'caring' in her practice with the knowledge gained of the perceptions and meanings impoverished clients assigned to their everyday lives. Jurgen Habermas' theory of communicative competence serves as the reference map guiding exploration. The author uses Habermas' theoretical principles of intersubjective mutuality--the validity claims of comprehensibility, truth, sincerity, and legitimacy. Comprehensibility embodies understanding, an attitude of unconditional acceptance, and care respect of another's individual person and self-defined reality. Intersubjective mutuality also requires that one dwell in the moment with the Other, satisfied that communication is founded on truth. Sincerity implies fostering the Other's expression of authentic self apart from oppressive distracters. Lastly, legitimacy reconciles the author's altruistic pursuit to know the Other's ontological truth with the reality of the present world.

Empathy↗
PubMed2002

Proteomic patterns of cultured breast cancer cells and epithelial mammary cells.

Breast cancer is one of the leading causes of death from cancer among women in western countries. The different types of breast cancer are grouped into invasive and noninvasive forms. Among the invasive types, ductal infiltrating carcinoma (DIC) is the most common and aggressive form. Using an in vitro model consisting of a DIC-derived cell line (8701-BC) and a nontumoral mammary epithelial cell line (HB2), we used the proteomics approach to search for homology and differences in protein expression patterns between tumoral and nontumoral phenotypes. Within an analysis window comprising 1,750 discernible spots we have currently catalogued 140 protein spots of potential interest. Fifty-eight of them were identified by gel matching with reference maps, immunodetection, or N-terminal microsequencing and classified into four functional groups. Twelve proteins were found differentially expressed in two cell lines: four were uniquely present in the neoplastic cell proteome and eight in epithelial cells. In addition, 53 proteins displayed different relative expression levels between the two cell lines, that is, 44 were more elevated in cancer cells and 9 in HB2 cells. Among proteins with greater relative abundance in cancer cells we identified glycolytic enzymes (or their isoforms), which may indicate that the known metabolic dysregulation in cancer can reflect oncogenic-related defects of glycolytic gene expression.

Breast↗
PubMed1998

Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins.

Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP.

Amino Acid Sequence↗
PubMed2005

Childhood cancers and atmospheric carcinogens.

STUDY OBJECTIVES: To retest previous findings that childhood cancers are probably initiated by prenatal exposures to combustion process gases and to volatile organic compounds (VOCs); and to identify specific chemical hazards. DESIGN: Birth and death addresses of fatal child cancers in Great Britain between 1966 and 1980, were linked with high local atmospheric emissions of different chemical species. Among migrant children, distances from each address to the nearest emissions "hotspot" were compared. Excesses of outward over inward migrations show an increased prenatal or early infancy risk. SETTING AND SUBJECTS: Maps of emissions of many different substances were published on the internet by the National Atmospheric Emissions Inventory and "hotspots" for 2001 were translated to map coordinates. Child cancer addresses were extracted from an earlier inquiry into the carcinogenic effects of obstetric radiographs; and their postcodes translated to map references. MAIN RESULTS: Significant birth proximity relative risks were found within 1.0 km of hotspots for carbon monoxide, PM10 particles, VOCs, nitrogen oxides, benzene, dioxins, 1,3-butadiene, and benz(a)pyrene. Calculated attributable risks showed that most child cancers and leukaemias are probably initiated by such exposures. CONCLUSIONS: Reported associations of cancer birth places with sites of industrial combustion, VOCs uses, and associated engine exhausts, are confirmed. Newly identified specific hazards include the known carcinogens 1,3-butadiene, dioxins, and benz(a)pyrene. The mother probably inhales these or related materials and passes them to the fetus across the placenta.

Adolescent↗
PubMed2005

Oil combustion and childhood cancers.

STUDY OBJECTIVES: To identify specific toxic atmospheric emissions and their industrial sources in Great Britain. To link them with each other and with the birth addresses of children dying from cancer. To identify specific causal agents and sources. DESIGN: Birth and death addresses of children dying from cancer were linked to emissions hotspots for specific chemicals: and to related source installations. Among those who moved house, distances from each address to the nearest hazard were compared. Relative excesses of close-to-hazard birth addresses showed high prenatal or early postnatal risks. Relative risks for individual and for combined exposures were measured. SETTING AND SUBJECTS: Atmospheric emissions hotspots (UK, 2001) published as maps on the internet, were converted to coordinates. Industrial sites were identified through trade directories and map inspections. Child cancer addresses for 1955-80 births were extracted from an earlier inquiry and their postcodes converted to map references. MAIN RESULTS: There were excess relative risks (RR) within 0.3 km of hotspots for carbon monoxide, PM10 particles, nitrogen oxides, 1,3-butadiene, benzene, dioxins, benzo(a)pyrene, and volatiles; and within 1.0 km of bus stations, hospitals, heavy transport centres, railways, and oil installations. Some excesses were attributable to mutual confounding, but 1,3-butadiene and carbon monoxide, mainly derived from engine exhausts, were powerful independent predictors. They were strongly reinforced when associated with bus stations, hospitals, railways, oil installations, and industrial transport centres; RR = 12.6 for joint <0.5 km exposure to bus stations and 1,3-butadiene. CONCLUSIONS: Childhood cancers are strongly determined by prenatal or early postnatal exposures to oil based combustion gases, especially from engine exhausts. 1,3-butadiene, a known carcinogen, may be directly causal.

Air Pollutants↗
PubMed1992

Leukaemia clusters in Great Britain. 1. Space-time interactions.

STUDY OBJECTIVE: The aim was to test a large set of childhood leukaemia and lymphoma registrations for the presence of clusters in space and in time. DESIGN: The study was a space-time cluster analysis. SETTING: England, Wales and Scotland. PATIENTS: All registrations for leukaemia and lymphoma between 1966 and 1983 in children aged 0 to 14 years were examined. The records included date and age of registration, sex, diagnosis, and the map reference of the postcode of residence. Of the 9411 registrations, 8888 were suitable for inclusion. MAIN RESULTS: There was a statistically significant excess of case pairs occurring jointly within 0.5 km and 60 d of each other: 68 pairs compared with 50.0 expected. The excess was detectable in central England, in the north of England and Scotland, but not in the south west of England. It was concentrated within the age band 4 to 7 years and among the lymphatic leukaemias. Several potential artefacts were considered and excluded, but the possibility remained that clustered detections might be triggered by haematological examinations undertaken for some communicable disease. CONCLUSIONS: There was strong evidence of joint spatial-temporal clustering, with an excess of pairs separated by very short time and distance intervals. The causes are probably biological rather than artefactual, but further work will be necessary in order to exclude the latter.

Adolescent↗
PubMed1992

Leukaemia clusters in Great Britain. 2. Geographical concentrations.

STUDY OBJECTIVE: The aim was to test a large set of childhood leukaemia and lymphoma registrations for the presence of short radius spacial clusters. DESIGN: The study was a geographical cluster analysis. SETTING: England, Wales and Scotland. PATIENTS: All registrations for leukaemia and lymphoma between 1966 and 1983 in children aged 0 to 14 years were examined. The records included date and age of registration, sex, diagnosis, and the map reference of the postcode of residence. Of the 9411 registrations, 8888 were suitable for inclusion. MAIN RESULTS: There was a significant excess of case pair addresses separated by < 0.5 km. There was also a significant excess of pairs sharing the same postcode. Both findings were based upon comparison with random pairs of postcodes drawn from the Central Postcode Directory. Examination for clustering at this very short range was based upon a clear prior hypothesis derived from the results of a study of space-time interaction, reported in a companion paper. CONCLUSIONS: It is postulated that the space-time interaction and the geographical concentrations shown here result from a common epidemic process. The epidemiology of this disease is characterised by short range geographical concentrations, with temporal non-homogeneity superimposed. The findings exclude certain artefacts which remained unresolved in the space-time interaction study. The distributions almost certainly reflect biological processes, and the most probable explanation is in terms of an infective process.

Adolescent↗
PubMed2005

Fluorescence in situ hybridization to chromosomes as a tool to understand human and primate genome evolution.

For the last 15 years molecular cytogenetic techniques have been extensively used to study primate evolution. Molecular probes were helpful to distinguish mammalian chromosomes and chromosome segments on the basis of their DNA content rather than solely on morphological features such as banding patterns. Various landmark rearrangements have been identified for most of the nodes in primate phylogeny while chromosome banding still provides helpful reference maps. Fluorescence in situ hybridization (FISH) techniques were used with probes of different complexity including chromosome painting probes, probes derived from chromosome sub-regions and in the size of a single gene. Since more recently, in silico techniques have been applied to trace down evolutionarily derived chromosome rearrangements by searching the human and mouse genome sequence databases. More detailed breakpoint analyses of chromosome rearrangements that occurred during higher primate evolution also gave some insights into the molecular changes in chromosome rearrangements that occurred in evolution. Hardly any "fusion genes" as known from chromosome rearrangements in cancer cells or dramatic "position effects" of genes transferred to new sites in primate genomes have been reported yet. Most breakpoint regions have been identified within gene poor areas rich in repetitive elements and/or low copy repeats (segmental duplications). The progress in various molecular and molecular-cytogenetic approaches including the recently launched chimpanzee genome project suggests that these new tools will have a significant impact on the further understanding of human genome evolution.

Animals↗
PubMed1987

Antigenic and biochemical characterization of poliovirus type 2 isolated from two cases of paralytic disease.

Three isolates of poliovirus type 2 were obtained from rectal and pharyngeal swabs of two patients with paralytic poliomyelitis from Southern Italy (1981/1983). Neither one of the patients had received oral poliovirus vaccine, although a systematic vaccination campaign is conducted in the area. Neutralization tests with strain-specific antisera and analysis of the structural proteins by SDS-PAGE indicated that the three isolates were typical Sabin-like poliovirus type 2. Moreover, neutralization assays by means of a panel of well-characterized monoclonal antibodies indicated that the three isolates carried an antigenic mosaic where both wild-type and Sabin-specific epitopes were represented. The T1 fingerprints of the RNA of the three isolates diverged slightly from the standard Sabin reference map. A comparison of the oligonucleotide fingerprints of the viruses isolated from the pharynx and the feces of one patient indicated that mutations appeared as early as during replication in the intestinal tract.

Antibodies, Monoclonal↗