One of the main challenges in cancer treatment is the selection of treatment resistant clones which leads to the emergence of resistance to previously efficacious therapies. Identifying ...vulnerabilities in the form of cellular trade-offs constraining the phenotypic possibility space could allow to avoid the emergence of resistance by simultaneously targeting cellular processes that are involved in different alternative phenotypic strategies linked by trade-offs. The Pareto optimality theory has been proposed as a framework allowing to identify such trade-offs in biological data from its prediction that it would lead to the presence of specific geometrical patterns (polytopes) in, e.g., gene expression space, with vertices representing specialized phenotypes. We tested this approach in diffuse large B-cell lymphoma (DLCBL) transcriptomic data. As predicted, there was highly statistically significant evidence for the data forming a tetrahedron in gene expression space, defining four specialized phenotypes (archetypes). These archetypes were significantly enriched in certain biological functions, and contained genes that formed a pattern of shared and unique elements among archetypes, as expected if trade-offs between essential functions underlie the observed structure. The results can be interpreted as reflecting trade-offs between aerobic energy production and protein synthesis, and between immunotolerant and immune escape strategies. Targeting genes on both sides of these trade-offs simultaneously represent potential promising avenues for therapeutic applications.
Antibiotic resistance is a worldwide health issue spreading quickly among human and animal pathogens, as well as environmental bacteria. Misuse of antibiotics has an impact on the selection of ...resistant bacteria, thus contributing to an increase in the occurrence of resistant genotypes that emerge via spontaneous mutation or are acquired by horizontal gene transfer. There is a specific and urgent need not only to detect antimicrobial resistance but also to predict antibiotic resistance in silico. We now have the capability to sequence hundreds of bacterial genomes per week, including assembly and annotation. Novel and forthcoming bioinformatics tools can predict the resistome and the mobilome with a level of sophistication not previously possible. Coupled with bacterial strain collections and databases containing strain metadata, prediction of antibiotic resistance and the potential for virulence are moving rapidly toward a novel approach in molecular epidemiology. Here, we present a model system in antibiotic‐resistance prediction, along with its promises and limitations. As it is commonly multidrug resistant, Pseudomonas aeruginosa causes infections that are often difficult to eradicate. We review novel approaches for genotype prediction of antibiotic resistance. We discuss the generation of microbial sequence data for real‐time patient management and the prediction of antimicrobial resistance.
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BFBNIB, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
In contrast to the large amount of ecological information supporting the role of natural selection as a main cause of population divergence and speciation, an understanding of the genomic basis ...underlying those processes is in its infancy. In this paper, we review the main findings of a long-term research programme that we have been conducting on the ecological genomics of sympatric forms of whitefish (Coregonus spp.) engaged in the process of speciation. We present this system as an example of how applying a combination of approaches under the conceptual framework of the theory of adaptive radiation has yielded substantial insight into evolutionary processes in a non-model species. We also discuss how the joint use of recent biotechnological developments will provide a powerful means to address issues raised by observations made to date. Namely, we present data illustrating the potential offered by combining next generation sequencing technologies with other genomic approaches to reveal the genomic bases of adaptive divergence and reproductive isolation. Given increasing access to these new genomic tools, we argue that non-model species studied in their ecological context such as whitefish will play an increasingly important role in generalizing knowledge of speciation.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Gene expression divergence is one of the mechanisms thought to be involved in the emergence of incipient species. Next‐generation sequencing has become an extremely valuable tool for the study of ...this process by allowing whole transcriptome sequencing, or RNA‐Seq. We have conducted a 454 GS‐FLX pyrosequencing experiment to refine our understanding of adaptive divergence between dwarf and normal lake whitefish species (Coregonus clupeaformis spp.). The objectives were to: (i) investigate transcriptomic divergence as measured by liver RNA‐Seq; (ii) test the correlation between divergence in expression and sequence polymorphism; and (iii) investigate the extent of allelic imbalance. We also compared the results of RNA‐seq with those of a previous microarray study performed on the same fish. Following de novo assembly, results showed that normal whitefish overexpressed more contigs associated with protein synthesis while dwarf fish overexpressed more contigs related to energy metabolism, immunity and DNA replication and repair. Moreover, 63 SNPs showed significant allelic imbalance, and this phenomenon prevailed in the recently diverged dwarf whitefish. Results also showed an absence of correlation between gene expression divergence as measured by RNA‐Seq and either polymorphism rate or sequence divergence between normal and dwarf whitefish. This study reiterates an important role for gene expression divergence, and provides evidence for allele‐specific expression divergence as well as evolutionary decoupling of regulatory and coding sequences in the adaptive divergence of normal and dwarf whitefish. It also demonstrates how next‐generation sequencing can lead to a more comprehensive understanding of transcriptomic divergence in a young species pair.
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Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted ...among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung.
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is a bacterial species that is a major cause of illness in humans and food-producing animals.
exhibits considerable inter-serovar diversity, as evidenced by the large number of host adapted serovars ...that have been identified. The development of methods to assess genome diversity in
will help to further define the limits of diversity in this foodborne pathogen. Thus, we evaluated a PCR assay, which targets prophage integrase genes, as a rapid method to investigate
genome diversity. To evaluate the PCR prophage integrase assay, 49 isolates of
were selected, including 19 clinical isolates from clonal serovars (Enteritidis and Heidelberg) that commonly cause human illness, and 30 isolates from food-associated
serovars that rarely cause human illness. The number of integrase genes identified by the PCR assay was compared to the number of integrase genes within intact prophages identified by whole genome sequencing and phage finding program PHASTER. The PCR assay identified a total of 147 prophage integrase genes within the 49
genomes (79 integrase genes in the food-associated
isolates, 50 integrase genes in
. Enteritidis, and 18 integrase genes in
. Heidelberg). In comparison, whole genome sequencing and PHASTER identified a total of 75 prophage integrase genes within 102 intact prophages in the 49
genomes (44 integrase genes in the food-associated
isolates, 21 integrase genes in
. Enteritidis, and 9 integrase genes in
. Heidelberg). Collectively, both the PCR assay and PHASTER identified the presence of a large diversity of prophage integrase genes in the food-associated isolates compared to the clinical isolates, thus indicating a high degree of diversity in the food-associated isolates, and confirming the clonal nature of
. Enteritidis and
. Heidelberg. Moreover, PHASTER revealed a diversity of 29 different types of prophages and 23 different integrase genes within the food-associated isolates, but only identified four different phages and integrase genes within clonal isolates of
Enteritidis and
Heidelberg. These results demonstrate the potential usefulness of PCR based detection of prophage integrase genes as a rapid indicator of genome diversity in
.
Non-typhoidal
is a leading cause of foodborne illness worldwide. Prompt and accurate identification of the sources of
responsible for disease outbreaks is crucial to minimize infections and eliminate ...ongoing sources of contamination. Current subtyping tools including single nucleotide polymorphism (SNP) typing may be inadequate, in some instances, to provide the required discrimination among epidemiologically unrelated
strains. Prophage genes represent the majority of the accessory genes in bacteria genomes and have potential to be used as high discrimination markers in
. In this study, the prophage sequence diversity in different
serovars and genetically related strains was investigated. Using whole genome sequences of 1,760 isolates of
representing 151
serovars and 66 closely related bacteria, prophage sequences were identified from assembled contigs using PHASTER. We detected 154 different prophages in
genomes. Prophage sequences were highly variable among
serovars with a median ± interquartile range (IQR) of 5 ± 3 prophage regions per genome. While some prophage sequences were highly conserved among the strains of specific serovars, few regions were lineage specific. Therefore, strains belonging to each serovar could be clustered separately based on their prophage content. Analysis of
. Enteritidis isolates from seven outbreaks generated distinct prophage profiles for each outbreak. Taken altogether, the diversity of the prophage sequences correlates with genome diversity. Prophage repertoires provide an additional marker for differentiating
subtypes during foodborne outbreaks.
The emergence of multidrug-resistant bacterial strains worldwide has become a serious problem for public health over recent decades. The increase in antimicrobial resistance has been expanding via ...plasmids as mobile genetic elements encoding antimicrobial resistance (AMR) genes that are transferred vertically and horizontally. This study focuses on Salmonella enterica, one of the leading foodborne pathogens in industrialized countries. S. enterica is known to carry several plasmids involved not only in virulence but also in AMR. In the current paper, we present an integrated strategy to detect plasmid scaffolds in whole genome sequencing (WGS) assemblies. We developed a two-step procedure to predict plasmids based on i) the presence of essential elements for plasmid replication and mobility, as well as ii) sequence similarity to a reference plasmid. Next, to confirm the accuracy of the prediction in 1750 S. enterica short-read sequencing data, we combined Oxford Nanopore MinION long-read sequencing with Illumina MiSeq short-read sequencing in hybrid assemblies for 84 isolates to evaluate the proportion of plasmid that has been detected. At least one scaffold with an origin of replication (ORI) was predicted in 61.3% of the Salmonella isolates tested. The results indicated that IncFII and IncI1 ORIs were distributed in many S. enterica serotypes and were the most prevalent AMR genes carrier, whereas IncHI2A/IncHI2 and IncA/C2 were more serotype restricted but bore several AMR genes. Comparison between hybrid and short-read assemblies revealed that 81.1% of plasmids were found in the short-read sequencing using our pipeline. Through this process, we established that plasmids are prevalent in S. enterica and we also substantially expand the AMR genes in the resistome of this species.
Ciprofloxacin is one of the most widely used antibiotics for treating Pseudomonas aeruginosa infections. However, P. aeruginosa acquires mutations that confer ciprofloxacin resistance, making ...treatment more difficult. Resistance is multifactorial, with mutations in multiple genes influencing the resistance phenotype. However, the contributions of individual mutations and mutation combinations to the amounts of ciprofloxacin that P. aeruginosa can tolerate are not well understood. Engineering P. aeruginosa strain PAO1 to contain mutations in any one of the resistance-associated genes
,
,
,
, and
showed that only
mutations increased the MIC for ciprofloxacin. Mutations in
and
increased the MIC of a
mutant, making the bacteria ciprofloxacin resistant. Mutations in
and
increased the MIC, conferring resistance, only if both were mutated in a
background. Mutations in all of
,
,
, and
further increased the MIC. These findings reveal an epistatic network of gene-gene interactions in ciprofloxacin resistance. We used this information to predict ciprofloxacin resistance/susceptibility for 274 isolates of P. aeruginosa from their genome sequences. Antibiotic susceptibility profiles were predicted correctly for 84% of the isolates. The majority of isolates for which prediction was unsuccessful were ciprofloxacin resistant, demonstrating the involvement of additional as yet unidentified genes and mutations in resistance. Our data show that gene-gene interactions can play an important role in antibiotic resistance and can be successfully incorporated into models predicting resistance phenotype.
A full-length cDNA encoding a triosephosphate isomerase (TPI) was cloned from the wild potato
Solanum chacoense. Sequence analysis showed that the cDNA has high levels of homology to sequences coding ...for cytoplasmic TPIs (cTPIs) and lower homology to plastidic TPI (pTPI) sequences. A specific polyclonal antiserum was generated against recombinant (6 × His) epitope-tagged cTPI expressed in
Escherichia coli. In
Solanum tuberosum this immune-serum recognized a single TPI polypeptide present in crude leaf extracts while an antigenic signal was not evident in purified chloroplasts. Immunoblot analysis demonstrated that cTPI was distributed in all vegetative and reproductive tissues. TPI activity was followed over a 3 months time course encompassing the whole development of potato plants. Activity expressed on a fresh weight basis was significantly higher in expanding leaves compared to mature and senescing leaves during the course of the experiment. Consistent with this, there was a gradient of TPI activity and cTPI protein along the shoot axis with the highest levels found in the youngest tissues. Analysis of TPI isoforms profiles by anion exchange chromatography demonstrated that (i) photosynthetic and non-photosynthetic tissues express 2 TPI isoforms and (ii) cTPI always represents the bulk of extractable TPI activity. Of all tissues surveyed, expanding leaves had the highest cTPI to pTPI ratio. The results are discussed in relation to the hypothesis that leaf cTPI plays a role in growing tissues, where glycolysis and respiration fulfill a key function in production of energy and C skeletons for biosynthetic purposes.
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