Dental caries is the most prevalent disease in humans globally. Efforts to control it have been invigorated by an increasing knowledge of the oral microbiome composition. This study aimed to evaluate ...the bacterial diversity in occlusal biofilms and its relationship with clinical surface diagnosis and dietary habits. Anamneses were recorded from thirteen 12-year-old children. Biofilm samples collected from occlusal surfaces of 46 permanent second molars were analyzed by 16S rRNA amplicon sequencing combined with the BLASTN-based search algorithm for species identification. The overall mean decayed, missing and filled surfaces modified index DMFSm Index, including active white spot lesions (AWSL) value was 8.77±7.47. Biofilm communities were highly polymicrobial collectively, representing 10 bacterial phyla, 25 classes, 29 orders, 58 families, 107 genera, 723 species. Streptococcus sp_Oral_Taxon_065, Corynebacterium matruchotii, Actinomyces viscosus, Actinomyces sp_Oral_Taxon_175, Actinomyces sp_Oral_Taxon_178, Actinomyces sp_Oral_Taxon_877, Prevotella nigrescens, Dialister micraerophilus, Eubacterium_XI G 1 infirmum were more abundant among surfaces with AWSL, and Streptococcus gordonii, Streptococcus sp._Oral_Taxon_058, Enterobacter sp._str._638 Streptococcus australis, Yersinia mollaretii, Enterobacter cloacae, Streptococcus sp._Oral_Taxon_71, Streptococcus sp._Oral_Taxon_F11, Centipeda sp._Oral_Taxon_D18 were more abundant among sound surfaces. Streptococcus mutans was detected on all surfaces in all patients, while Streptococcus sobrinus was detected only in three patients (mean relative abundances 7.1% and 0.6%, respectively). Neither species differentiated healthy from diseased sites. Diets of nine of the subjects were scored as high in fermentable carbohydrates (≧2X/day between meals). A direct association between relative abundances of bacteria and carbohydrate consumption was observed among 18 species. High consumption of fermentable carbohydrates and sound surfaces were associated with a reduction in bacterial diversity. PCoA plots displayed differences in bacterial community profiles between sound and diseased surfaces. Our study showed that, in addition to mutans streptococci, other species may be associated with the initiation of dental caries on occlusal surfaces, and that biofilm diversity of tooth surfaces is influenced by carbohydrate consumption and a surface's health status.
Gepard provides a user-friendly, interactive application for the quick creation of dotplots. It utilizes suffix arrays to reduce the time complexity of dotplot calculation to Θ(m*log n). A ...client-server mode, which is a novel feature for dotplot creation software, allows the user to calculate dotplots and color them by functional annotation without any prior downloading of sequence or annotation data.
Availability: Both source codes and executable binaries are available at http://mips.gsf.de/services/analysis/gepard
Contact:
krumsiek@in.tum.de
Efforts to map the Escherichia coli interactome have identified several hundred macromolecular complexes, but direct binary protein-protein interactions (PPIs) have not been surveyed on a large ...scale. Here we performed yeast two-hybrid screens of 3,305 baits against 3,606 preys (∼70% of the E. coli proteome) in duplicate to generate a map of 2,234 interactions, which approximately doubles the number of known binary PPIs in E. coli. Integration of binary PPI and genetic-interaction data revealed functional dependencies among components involved in cellular processes, including envelope integrity, flagellum assembly and protein quality control. Many of the binary interactions that we could map in multiprotein complexes were informative regarding internal topology of complexes and indicated that interactions in complexes are substantially more conserved than those interactions connecting different complexes. This resource will be useful for inferring bacterial gene function and provides a draft reference of the basic physical wiring network of this evolutionarily important model microbe.
The localization of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with Pseudomonas aeruginosa, is poorly understood. We performed ...immunohistochemistry for Bcc and P. aeruginosa bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined the growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as "biofilm-like structures." In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a P. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibit P. aeruginosa biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.
The web server probeCheck, freely accessible at http://www.microbial-ecology.net/probecheck, provides a pivotal forum for rapid specificity and coverage evaluations of probes and primers against ...selected databases of phylogenetic and functional marker genes. Currently, 24 widely used sequence collections including the Ribosomal Database Project (RDP) II, Greengenes, SILVA and the Functional Gene Pipeline/Repository can be queried. For this purpose, probeCheck integrates a new online version of the popular ARB probe match tool with free energy (ΔG) calculations for each perfectly matched and mismatched probe-target hybrid, allowing assessment of the theoretical binding stabilities of oligo-target and non-target hybrids. For each output sequence, the accession number, the GenBank taxonomy and a link to the respective entry at GenBank, EMBL and, if applicable, the query database are displayed. Filtering options allow customizing results on the output page. In addition, probeCheck is linked with probe match tools of RDP II and Greengenes, NCBI blast, the Oligonucleotide Properties Calculator, the two-state folding tool of the DINAMelt server and the rRNA-targeted probe database probeBase. Taken together, these features provide a multifunctional platform with maximal flexibility for the user in the choice of databases and options for the evaluation of published and newly developed probes and primers.
Aerosols and spatter are concerns in health care owing to their potential adverse health effects. The Isolite illuminated isolation system (Isolite Systems) and a saliva ejector were compared for ...aerosol and spatter reduction during and after ultrasonic scaling.
Fifty participants were randomized to control (n = 25, saliva ejector) or test (n = 25, Isolite) groups and received a prophylaxis with an ultrasonic scaler. Aerosols were collected in a petri dish containing transport media, dispersed, and plated to anaerobic blood agar to determine colony-forming units (CFUs). The authors analyzed the data using a t test.
No significant difference occurred between groups in aerosol and spatter reduction (P = .25). Mean (standard deviation) of log10 CFUs per milliliter collected during ultrasonic scaling in the control and test groups were 3.61 (0.95) and 3.30 (0.88), respectively. All samples contained α-hemolytic streptococci, and many samples contained strictly oral anaerobes.
A significant amount of contamination occurred during ultrasonic scaling in both groups, as indicated by high numbers of CFUs and the identification of strictly oral anaerobes in all plates.
Neither device reduced aerosols and spatter effectively, and there was no significant difference in reduction between the 2 devices. Additional measures should be taken with these devices to reduce the likelihood of disease transmission.
The human proteome contains a plethora of short linear motifs (SLiMs) that serve as binding interfaces for modular protein domains. Such interactions are crucial for signaling and other cellular ...processes, but are difficult to detect because of their low to moderate affinities. Here we developed a dedicated approach, proteomic peptide-phage display (ProP-PD), to identify domain–SLiM interactions. Specifically, we generated phage libraries containing all human and viral C-terminal peptides using custom oligonucleotide microarrays. With these libraries we screened the nine PSD-95/Dlg/ZO-1 (PDZ) domains of human Densin-180, Erbin, Scribble, and Disks large homolog 1 for peptide ligands. We identified several known and putative interactions potentially relevant to cellular signaling pathways and confirmed interactions between full-length Scribble and the target proteins β-PIX, plakophilin-4, and guanylate cyclase soluble subunit α-2 using colocalization and coimmunoprecipitation experiments. The affinities of recombinant Scribble PDZ domains and the synthetic peptides representing the C termini of these proteins were in the 1- to 40-μM range. Furthermore, we identified several well-established host–virus protein–protein interactions, and confirmed that PDZ domains of Scribble interact with the C terminus of Tax-1 of human T-cell leukemia virus with micromolar affinity. Previously unknown putative viral protein ligands for the PDZ domains of Scribble and Erbin were also identified. Thus, we demonstrate that our ProP-PD libraries are useful tools for probing PDZ domain interactions. The method can be extended to interrogate all potential eukaryotic, bacterial, and viral SLiMs and we suggest it will be a highly valuable approach for studying cellular and pathogen–host protein–protein interactions.
Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal ...transduction proteins (
, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (
,
-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours.
We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups.
Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of
may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (
,
) occurring even in CIMP-negative LD cancers.
mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93).
Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential
expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.
The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a ...molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of approximately 71% and selectivity of approximately 85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will facilitate further studies on and improve our understanding of type III secretion and its role in pathogen-host interactions.
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