ALBERT

Description: Background: Propionibacteria are part of the human microbiota. Many studies have addressed the predominant colonizer of sebaceous follicles of the skin, Propionibacterium acnes, and investigated its association with the skin disorder acne vulgaris, and lately with prostate cancer. Much less is known about two other propionibacterial species frequently found on human tissue sites, Propionibacterium granulosum and Propionibacterium avidum. Here we analyzed two and three genomes of P. granulosum and P. avidum, respectively, and compared them to two genomes of P. acnes; we further highlight differences among the three cutaneous species with proteomic and microscopy approaches. Results: Electron and atomic force microscopy revealed an exopolysaccharide (EPS)-like structure surrounding P. avidum cells, that is absent in P. acnes and P. granulosum. In contrast, P. granulosum possesses pili-like appendices, which was confirmed by surface proteome analysis. The corresponding genes were identified; they are clustered with genes encoding sortases. Both, P. granulosum and P. avidum lack surface or secreted proteins for predicted host-interacting factors of P. acnes, including several CAMP factors, sialidases, dermatan-sulphate adhesins, hyaluronidase and a SH3 domain-containing lipoprotein; accordingly, only P. acnes exhibits neuraminidase and hyaluronidase activities. These functions are encoded on previously unrecognized island-like regions in the genome of P. acnes. Conclusions: Despite their omnipresence on human skin little is known about the role of cutaneous propionibacteria. All three species are associated with a variety of diseases, including postoperative and device-related abscesses and infections. We showed that the three organisms have evolved distinct features to interact with their human host. Whereas P. avidum and P. granulosum produce an EPS-like surface structure and pili-like appendices, respectively, P. acnes possesses a number of unique surface-exposed proteins with host-interacting properties. The different surface properties of the three cutaneous propionibacteria are likely to determine their colonizing ability and pathogenic potential on the skin and at non-skin sites.

Description: Background Propionibacteria are part of the human microbiota. Many studies have addressed the predominant colonizer of sebaceous follicles of the skin, Propionibacterium acnes, and investigated its association with the skin disorder acne vulgaris, and lately with prostate cancer. Much less is known about two other propionibacterial species frequently found on human tissue sites, Propionibacterium granulosum and Propionibacterium avidum. Here we analyzed two and three genomes of P. granulosum and P. avidum, respectively, and compared them to two genomes of P. acnes; we further highlight differences among the three cutaneous species with proteomic and microscopy approaches. Results Electron and atomic force microscopy revealed an exopolysaccharide (EPS)-like structure surrounding P. avidum cells, that is absent in P. acnes and P. granulosum. In contrast, P. granulosum possesses pili-like appendices, which was confirmed by surface proteome analysis. The corresponding genes were identified; they are clustered with genes encoding sortases. Both, P. granulosum and P. avidum lack surface or secreted proteins for predicted host-interacting factors of P. acnes, including several CAMP factors, sialidases, dermatan-sulphate adhesins, hyaluronidase and a SH3 domain-containing lipoprotein; accordingly, only P. acnes exhibits neuraminidase and hyaluronidase activities. These functions are encoded on previously unrecognized island-like regions in the genome of P. acnes. Conclusions Despite their omnipresence on human skin little is known about the role of cutaneous propionibacteria. All three species are associated with a variety of diseases, including postoperative and device-related abscesses and infections. We showed that the three organisms have evolved distinct features to interact with their human host. Whereas P. avidum and P. granulosum produce an EPS-like surface structure and pili-like appendices, respectively, P. acnes possesses a number of unique surface-exposed proteins with host-interacting properties. The different surface properties of the three cutaneous propionibacteria are likely to determine their colonizing ability and pathogenic potential on the skin and at non-skin sites.

Description: Background: Apoptosis is a highly controlled process of cell death that can be induced by periodontopathogens. The present study aimed to investigate the expression of Fas and Bcl-2 proteins by CD3+ T cells in vitro under stimulation by total Porphyromonas gingivalis antigens and purified recombinant P. gingivalis HmuY protein. Results: CD3+ T cells derived from CP patients and stimulated with HmuY expressed higher levels of Bcl-2 compared to identical cells stimulated with P. gingivalis crude extract or cells derived from NP control subjects (p = 0.043). Conclusion: The authors hypothesize that P. gingivalis HmuY plays a role in the pathogenesis of chronic periodontitis, possibly by reducing or delaying apoptosis in T cells through a pathway involving the Bcl-2 protein.

Description: Background: The identification of the loci and specific alleles underlying variation in quantitative traits is an important goal for evolutionary biologists and breeders. Despite major advancements in genomics technology, moving from QTL to causal alleles remains a major challenge in genetics research. Near-isogenic lines are the ideal raw material for QTL validation, refinement of QTL location and, ultimately, gene discovery. Results: In this study, a population of 75 Arabidopsis thaliana near-isogenic lines was developed from an existing recombinant inbred line (RIL) population derived from a cross between physiologically divergent accessions Kas-1 and Tsu-1. First, a novel algorithm was developed to utilize genome-wide marker data in selecting RILs fully isogenic to Kas-1 for a single chromosome. Seven such RILs were used in 2 generations of crossing to Tsu-1 to create BC1 seed. BC1 plants were genotyped with SSR markers so that lines could be selected that carried Kas-1 introgressions, resulting in a population carrying chromosomal introgressions spanning the genome. BC1 lines were genotyped with 48 genome-wide SSRs to identify lines with a targeted Kas-1 introgression and the fewest genomic introgressions elsewhere. 75 such lines were selected and genotyped at an additional 41 SNP loci and another 930 tags using 2b-RAD genotyping by sequencing. The final population carried an average of 1.35 homozygous and 2.49 heterozygous introgressions per line with average introgression sizes of 5.32 and 5.16 Mb, respectively. In a simple case study, we demonstrate the advantage of maintaining heterozygotes in our library whereby fine-mapping efforts are conducted simply by self-pollination. Crossovers in the heterozygous interval during this single selfing generation break the introgression into smaller, homozygous fragments (sub-NILs). Additionally, we utilize a homozygous NIL for validation of a QTL underlying stomatal conductance, a low heritability trait. Conclusions: The present results introduce a new and valuable resource to the Brassicaceae research community that enables rapid fine-mapping of candidate loci in parallel with QTL validation. These attributes along with dense marker coverage and genome-wide chromosomal introgressions make this population an ideal starting point for discovery of genes underlying important complex traits of agricultural and ecological significance.

Description: Background: In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. Results: The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. Conclusions: The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure.

Description: Background: The epithelial-to-mesenchymal transition is an important mechanism in cancer metastasis. Although transcription factors including SNAIL, SLUG, and TWIST1 regulate the epithelial-to-mesenchymal transition, other unknown transcription factors could also be involved. Identification of the full complement of transcription factors is essential for a more complete understanding of gene regulation in this process. Chromatin immunoprecipitation-sequencing (ChIP-Seq) technologies have been used to detect genome-wide binding of transcription factors; here, we developed a systematic approach to integrate existing ChIP-Seq and transcriptome data. We scanned multiple transcription factors to investigate their functional impact on the epithelial-to-mesenchymal transition in the human A549 lung adenocarcinoma cell line. Results: Among the transcription factors tested, impact scores identified the forkhead box protein A1 (FOXA1) as the most significant transcription factor in the epithelial-to-mesenchymal transition. FOXA1 physically associates with the promoters of its predicted target genes. Several critical epithelial-to-mesenchymal transition effectors involved in cellular adhesion and cellular communication were identified in the regulatory network of FOXA1, including FOXA2, FGA, FGB, FGG, and FGL1. The implication of FOXA1 in the epithelial-to-mesenchymal transition via its regulatory network indicates that FOXA1 may play an important role in the initiation of lung cancer metastasis. Conclusions: We identified FOXA1 as a potentially important transcription factor and negative regulator in the initial stages of lung cancer metastasis. FOXA1 may modulate the epithelial-to-mesenchymal transition via its transcriptional regulatory network. Further, this study demonstrates how ChIP-Seq and expression data could be integrated to delineate the impact of transcription factors on a specific biological process.

Description: Background: Mutations that alter chromosomal structure play critical roles in evolution and disease, including in the origin of new lifestyles and pathogenic traits in microbes. Large-scale rearrangements in genomes are often mediated by recombination events involving new or existing copies of mobile genetic elements, recently duplicated genes, or other repetitive sequences. Most current software programs for predicting structural variation from short-read DNA resequencing data are intended primarily for use on human genomes. They typically disregard information in reads mapping to repeat sequences, and significant post-processing and manual examination of their output is often required to rule out false-positive predictions and precisely describe mutational events. Results: We have implemented an algorithm for identifying structural variation from DNA resequencing data as part of the breseq computational pipeline for predicting mutations in haploid microbial genomes. Our method evaluates the support for new sequence junctions present in a clonal sample from split-read alignments to a reference genome, including matches to repeat sequences. Then, it uses a statistical model of read coverage evenness to accept or reject these predictions. Finally, breseq combines predictions of new junctions and deleted chromosomal regions to output biologically relevant descriptions of mutations and their effects on genes. We demonstrate the performance of breseq on simulated Escherichia coli genomes with deletions generating unique breakpoint sequences, new insertions of mobile genetic elements, and deletions mediated by mobile elements. Then, we reanalyze data from an E. coli K-12 mutation accumulation evolution experiment in which structural variation was not previously identified. Transposon insertions and large-scale chromosomal changes detected by breseq account for ~25% of spontaneous mutations in this strain. In all cases, we find that breseq is able to reliably predict structural variation with modest read-depth coverage of the reference genome (〉40-fold). Conclusions: Using breseq to predict structural variation should be useful for studies of microbial epidemiology, experimental evolution, synthetic biology, and genetics when a reference genome for a closely related strain is available. In these cases, breseq can discover mutations that may be responsible for important or unintended changes in genomes that might otherwise go undetected.

Description: Background: In agricultural regions, streamside forests have been reduced in age and extent, or removed entirely to maximize arable cropland. Restoring and reforesting such riparian zones to mature forest, particularly along headwater streams (which constitute 90% of stream network length) would both increase carbon storage and improve water quality. Age and management-related cover/condition classes of headwater stream networks can be used to rapidly inventory carbon storage and sequestration potential if carbon storage capacity of conditions classes and their relative distribution on the landscape are known. Results: Based on the distribution of riparian zone cover/condition classes in sampled headwater reaches, current and potential carbon storage was extrapolated to the remainder of the North Carolina Coastal Plain stream network. Carbon stored in headwater riparian reaches is only about 40% of its potential capacity, based on 242 MgC/ha stored in sampled mature riparian forest (forest 〉 50 y old). The carbon deficit along 57,700 km of headwater Coastal Plain streams is equivalent to about 25TgC in 30-m-wide riparian buffer zones and 50 TgC in 60-m-wide buffer zones. Conclusions: Estimating carbon storage in recognizable age-and cover-related condition classes provides a rapid way to better inventory current carbon storage, estimate storage capacity, and calculate the potential for additional storage. In light of the particular importance of buffer zones in headwater reaches in agricultural landscapes in ameliorating nutrient and sediment input to streams, encouraging the restoration of riparian zones to mature forest along headwater reaches worldwide has the potential to not only improve water quality, but also simultaneously reduce atmospheric CO2.

Description: Background: In interferon-gamma-stimulated cells, the dimeric transcription factor STAT1 (signal transducer and activator of transcription 1) recognizes semi-palindromic motifs in the promoter regions of cytokine-driven target genes termed GAS (gamma-activated sites). However, the molecular steps that facilitate GAS binding and the subsequent liberation of STAT1 homodimers from these promoter elements are not well understood. Results: Using a mutational approach, we identified two critical glutamyl residues within the DNA-binding domain adjacent to the phosphodiester backbone of DNA which efficiently release phospho-STAT1 from DNA. The release of STAT1 dimers from DNA enhances transcriptional activity on both interferon-driven reporter and endogenous target genes. A substitution of either of the two glutamic acid residues broadens the repertoire of putative binding sites on DNA and enhances binding affinity to GAS sites. However, despite elevated levels of tyrosine phosphorylation and a prolonged nuclear accumulation period, the STAT1 DNA-binding mutants show a significantly reduced transcriptional activity upon stimulation of cells with interferon-gamma. This reduced transcriptional response may be explained by the deposition of oligomerized STAT1 molecules outside GAS sites. Conclusions: Thus, two negatively charged amino acid residues in the DNA-binding domain are engaged in the liberation of STAT1 from DNA, resulting in a high dissociation rate from non-GAS sites as a key feature of STAT1 signal transduction, which positively regulates cytokine-dependent gene expression probably by preventing retention at transcriptionally inert sites.

Description: Background: Protein structure mediates site-specific patterns of sequence divergence. In particular, residues in the core of a protein (solvent-inaccessible residues) tend to be more evolutionarily conserved than residues on the surface (solvent-accessible residues). Results: Here, we present a model of sequence evolution that explicitly accounts for the relative solvent accessibility of each residue in a protein. Our model is a variant of the Goldman-Yang 1994 (GY94) model in which all model parameters can be functions of the relative solvent accessibility (RSA) of a residue. We apply this model to a data set comprised of nearly 600 yeast genes, and find that an evolutionary-rate ratio omega that varies linearly with RSA provides a better model fit than an RSA-independent omega or an omega that is estimated separately in individual RSA bins. We further show that the branch length t and the transition--transverion ratio kappa also vary with RSA. The RSA-dependent GY94 model performs better than an RSA-dependent Muse-Gaut 1994 (MG94) model in which the synonymous and non-synonymous rates individually are linear functions of RSA. Finally, protein core size affects the slope of the linear relationship between omega and RSA, and gene expression level affects both the intercept and the slope. Conclusions: Structure-aware models of sequence evolution provide a significantly better fit than traditional models that neglect structure. The linear relationship between omega and RSA implies that genes are better characterized by their omega slope and intercept than by just their mean omega.