RESUMO
Detection of genetic variants in clinically relevant genomic hot-spot regions has become a promising application of next-generation sequencing technology in precision oncology. Effective personalized diagnostics requires the detection of variants with often very low frequencies. This can be achieved by targeted, short-read sequencing that provides high sequencing depths. However, rare genetic variants can contain crucial information for early cancer detection and subsequent treatment success, an inevitable level of background noise usually limits the accuracy of low frequency variant calling assays. To address this challenge, we developed DEEPGENTM, a variant calling assay intended for the detection of low frequency variants within liquid biopsy samples. We processed reference samples with validated mutations of known frequencies (0%-0.5%) to determine DEEPGENTM's performance and minimal input requirements. Our findings confirm DEEPGENTM's effectiveness in discriminating between signal and noise down to 0.09% variant allele frequency and an LOD(90) at 0.18%. A superior sensitivity was also confirmed by orthogonal comparison to a commercially available liquid biopsy-based assay for cancer detection.
Assuntos
Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Neoplasias/diagnóstico , Biomarcadores Tumorais/genética , Detecção Precoce de Câncer , Frequência do Gene , Predisposição Genética para Doença , Humanos , Biópsia Líquida , Neoplasias/genética , Medicina de PrecisãoRESUMO
Codon pair deoptimization is an efficient virus attenuation strategy, but the mechanism that leads to attenuation is unknown. The strategy involves synthetic recoding of viral genomes that alters the positions of synonymous codons, thereby increasing the number of suboptimal codon pairs and CpG dinucleotides in recoded genomes. Here we identify the molecular mechanism of codon pair deoptimization-based attenuation by studying recoded influenza A viruses. We show that suboptimal codon pairs cause attenuation, whereas the increase of CpG dinucleotides has no effect. Furthermore, we show that suboptimal codon pairs reduce both mRNA stability and translation efficiency of codon pair-deoptimized genes. Consequently, reduced protein production directly causes virus attenuation. Our study provides evidence that suboptimal codon pairs are major determinants of mRNA stability. Additionally, it demonstrates that codon pair bias can be used to increase mRNA stability and protein production of synthetic genes in many areas of biotechnology.
Assuntos
Códon , Vírus de DNA/genética , Genoma Viral/genética , Proteínas Virais/metabolismo , Animais , Humanos , CamundongosRESUMO
The self-assembly of the 42-residue amyloid-ß peptide, Aß42, into fibrillar aggregates is associated with neuronal dysfunction and toxicity in Alzheimer's disease (AD) patient brains, suggesting that small molecules acting on this process might interfere with pathogenesis. Here, we present experimental evidence that the small molecule sclerotiorin (SCL), a natural product belonging to the group of azaphilones, potently delays both seeded and nonseeded Aß42 polymerization in cell-free assays. Mechanistic biochemical studies revealed that the inhibitory effect of SCL on fibrillogenesis is caused by its ability to kinetically stabilize small Aß42 oligomers. These structures exhibit low ß-sheet content and do not possess seeding activity, indicating that SCL acts very early in the amyloid formation cascade before the assembly of seeding-competent, ß-sheet-rich fibrillar aggregates. Investigations with NMR WaterLOGSY experiments confirmed the association of Aß42 assemblies with SCL in solution. Furthermore, using ion mobility-mass spectrometry, we observed that SCL directly interacts with a small fraction of Aß42 monomers in the gas phase. In comparison to typical amyloid fibrils, small SCL-stabilized Aß42 assemblies are inefficiently taken up into mammalian cells and have low toxicity in cell-based assays. Overall, these mechanistic studies support a pathological role of stable, ß-sheet-rich Aß42 fibrils in AD, while structures with low ß-sheet content may be less relevant.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/antagonistas & inibidores , Benzopiranos/farmacologia , Proliferação de Células , Neuroblastoma/tratamento farmacológico , Fragmentos de Peptídeos/química , Multimerização Proteica/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Células PC12 , Fragmentos de Peptídeos/metabolismo , Conformação Proteica em Folha beta , Ratos , Células Tumorais CultivadasRESUMO
The telomerase RNA subunit (TR) is overexpressed in many tumors; however, the contribution of TR in cancer formation remains elusive. The most frequent clinically diagnosed cancer in the animal kingdom is caused by the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV encodes a TR (vTR) that plays an important role in virus-induced tumorigenesis and shares 88% sequence identity with its cellular homologue. To determine if the cellular TR possesses pro-oncogenic activity, we replaced vTR with the cellular homologue in the virus genome. Insertion of cellular TR resulted in a strong overexpression in virus infected cells, while virus replication was not affected. Strikingly, cellular TR promoted tumor formation as efficient as vTR, while tumorigenesis was severely impaired in the absence of vTR. Our data provide the first evidence that overexpression of cellular TR can contribute to tumor formation in vivo using this natural virus-host model for herpesvirus-induced oncogenesis.
Assuntos
Transformação Celular Neoplásica/genética , Herpesvirus Galináceo 3/fisiologia , Doença de Marek/virologia , RNA/genética , Telomerase/genética , Animais , Células Cultivadas , Embrião de Galinha , Feminino , Herpesvirus Galináceo 3/genética , Masculino , Doença de Marek/enzimologia , RNA/metabolismo , Telomerase/metabolismo , Replicação ViralRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
RNA virus populations are composed of highly diverse individuals that form a cloud of related sequences commonly referred to as a 'quasispecies'1-3. This diversity arises as a consequence of low-fidelity genome replication4,5. By contrast, DNA virus populations contain more uniform individuals with similar fitness6. Genome diversity is often correlated with increased fitness in RNA viruses, while DNA viruses are thought to require more faithful genome replication. During DNA replication, erroneously incorporated bases are removed by a 3'-5' exonuclease, a highly conserved enzymatic function of replicative DNA but not RNA polymerases. This proofreading process enhances replication fidelity and ensures the genome integrity of DNA organisms, including large DNA viruses7. Here, we show that a herpesvirus can tolerate impaired exonucleolytic proofreading, resulting in DNA virus populations, which, as in RNA viruses8, are composed of highly diverse genotypes of variable individual fitness. This indicates that herpesvirus mutant diversity may compensate for individual fitness loss. Notably, in vivo infection with diverse virus populations results in a marked increase in virulence compared to genetically homogenous parental virus. While we cannot exclude that the increase in virulence is caused by selection of and/or interactions between individual genotypes, our findings are consistent with quasispecies dynamics. Our results contrast with traditional views of DNA virus replication and evolution, and indicate that a substantial increase in population diversity can lead to higher virulence.
Assuntos
Variação Genética , Genoma Viral , Herpesvirus Galináceo 2/genética , Quase-Espécies/genética , Replicação Viral , Animais , Galinhas/virologia , Aptidão Genética , Genótipo , Herpesvirus Galináceo 2/patogenicidade , Herpesvirus Galináceo 2/fisiologia , Doença de Marek/virologia , Mutação , Organismos Livres de Patógenos Específicos , Virulência/genéticaRESUMO
Self-propagating amyloid-ß (Aß) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aß42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aß42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aß42 peptides and decreases the seeding activity of Aß aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aß plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aß structures with small molecules as a valid therapeutic strategy.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Compostos Azo/farmacologia , Corantes/farmacologia , Inflamação/tratamento farmacológico , Placa Amiloide/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Compostos Azo/química , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Corantes/química , Relação Dose-Resposta a Droga , Feminino , Inflamação/genética , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estrutura Molecular , Placa Amiloide/genética , Placa Amiloide/metabolismo , Polimerização/efeitos dos fármacos , Agregados Proteicos/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Polar bears in captivity can be exposed to opportunistic pathogens not present in their natural environments. A 4-month-old polar bear (Ursus maritimus) living in an isolated enclosure with his mother in the Tierpark Berlin, Berlin, Germany, was suffering from severe abdominal pain, mild diarrhea, and loss of appetite and died in early 2017. Histopathology revealed severe hepatic degeneration and necrosis without evidence of inflammation or inclusion bodies, although a viral infection had been suspected on the basis of the clinical signs. We searched for nucleic acids of pathogens by shotgun high-throughput sequencing (HTS) from genomic DNA and cDNA extracted from tissue and blood. We identified a novel Mastadenovirus and assembled a nearly complete genome from the shotgun sequences. Quantitative PCR (qPCR) revealed that viral DNA was present in various concentrations in all tissues examined and that the highest concentrations were found in blood. Viral culture did not yield cytopathic effects, but qPCR suggested that virus replication was sustained for up to three passages. Positive immunofluorescence staining confirmed that the virus was able to replicate in the cells during early passage. Phylogenetic analysis demonstrated that the virus is highly divergent compared to other previously identified Mastadenovirus members and basal to most known viral clades. The virus was found only in the 4-month-old bear and not in other captive polar bears tested. We surmised, therefore, that the polar bear was infected from an unknown reservoir, illustrating that adenoviral diversity remains underestimated and that cross-species transmission of viruses can occur even under conditions of relative isolation.IMPORTANCE Cross-species transmission of viral pathogens is becoming an increasing problem for captive-animal facilities. This study highlights how animals in captivity are vulnerable to novel opportunistic pathogens, many of which do not result in straightforward diagnosis from symptoms and histopathology. In this study, a novel pathogen was suspected to have contributed to the death of a juvenile polar bear. HTS techniques were employed, and a novel Mastadenovirus was isolated. The virus was present in both the tissue and blood samples. Phylogenetic analysis of the virus at both the gene and genome levels revealed that it is highly divergent to other known mastadenoviruses. Overall, this study shows that animals in isolated conditions still come into contact with novel pathogens, and for many of these pathogens, the host reservoir and mode of transmission are yet to be determined.
Assuntos
Infecções por Adenoviridae/veterinária , Mastadenovirus/classificação , Mastadenovirus/isolamento & purificação , Ursidae/virologia , Infecções por Adenoviridae/virologia , Estruturas Animais/virologia , Animais , Animais de Zoológico , Berlim , Genoma Viral , Mastadenovirus/genética , Mastadenovirus/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Cultura de Vírus , Replicação ViralRESUMO
Virulence determines the impact a pathogen has on the fitness of its host, yet current understanding of the evolutionary origins and causes of virulence of many pathogens is surprisingly incomplete. Here, we explore the evolution of Marek's disease virus (MDV), a herpesvirus commonly afflicting chickens and rarely other avian species. The history of MDV in the 20th century represents an important case study in the evolution of virulence. The severity of MDV infection in chickens has been rising steadily since the adoption of intensive farming techniques and vaccination programs in the 1950s and 1970s, respectively. It has remained uncertain, however, which of these factors is causally more responsible for the observed increase in virulence of circulating viruses. We conducted a phylogenomic study to understand the evolution of MDV in the context of dramatic changes to poultry farming and disease control. Our analysis reveals evidence of geographical structuring of MDV strains, with reconstructions supporting the emergence of virulent viruses independently in North America and Eurasia. Of note, the emergence of virulent viruses appears to coincide approximately with the introduction of comprehensive vaccination on both continents. The time-dated phylogeny also indicated that MDV has a mean evolutionary rate of ~1.6 × 10-5 substitutions per site per year. An examination of gene-linked mutations did not identify a strong association between mutational variation and virulence phenotypes, indicating that MDV may evolve readily and rapidly under strong selective pressures and that multiple genotypic pathways may underlie virulence adaptation in MDV.