African swine fever is a devastating viral disease of domestic and wild pigs against which no vaccine or therapy is available. Therefore, we applied the CRISPR (clustered regularly interspaced short ...palindromic repeats) - Cas9 nuclease system to target the double-stranded DNA genome of African swine fever virus (ASFV). To this end, a permissive wild boar lung (WSL) cell line was modified by stable transfection with a plasmid encoding Cas9 and a guide RNA targeting codons 71 to 78 of the phosphoprotein p30 gene (CP204L) of ASFV. Due to targeted Cas9 cleavage of the virus genome, plaque formation of ASFV was completely abrogated and virus yields were reduced by four orders of magnitude. The specificity of these effects could be demonstrated by using a natural ASFV isolate and escape mutants possessing nucleotide exchanges within the target sequence, which were not inhibited in the Cas9-expressing cell line. Growth of the cell line was not affected by transgene expression which, as well as virus inhibition, proved to be stable over at least 50 passages. Thus, CRISPR-Cas9 mediated targeting of the ASFV p30 gene is a valid strategy to convey resistance against ASF infection, which may also be applied in its natural animal host.
Emergent Properties of Reduced-Genome Escherichia coli Pósfai, György; Plunkett, Guy III; Fehér, Tamás ...
Science (American Association for the Advancement of Science),
05/2006, Letnik:
312, Številka:
5776
Journal Article
Recenzirano
With the use of synthetic biology, we reduced the Escherichia coli K-12 genome by making planned, precise deletions. The multiple-deletion series (MDS) strains, with genome reductions up to 15%, were ...designed by identifying nonessential genes and sequences for elimination, including recombinogenic or mobile DNA and cryptic virulence genes, while preserving good growth profiles and protein production. Genome reduction also led to unanticipated beneficial properties: high electroporation efficiency and accurate propagation of recombinant genes and plasmids that were unstable in other strains. Eradication of stress-induced transposition evidently stabilized the MDS genomes and provided some of the new properties.
The RNA world scenario posits replication by RNA polymerases. On early Earth, a geophysical setting is required to separate hybridized strands after their replication and to localize them against ...diffusion. We present a pointed heat source that drives exponential, RNA-catalyzed amplification of short RNA with high efficiency in a confined chamber. While shorter strands were periodically melted by laminar convection, the temperature gradient caused aggregated polymerase molecules to accumulate, protecting them from degradation in hot regions of the chamber. These findings demonstrate a size-selective pathway for autonomous RNA-based replication in natural nonequilibrium conditions.
African swine fever (ASF) is a viral disease that affects members of the Suidae family such as African bush pigs, warthogs, but also domestic pigs, and wild boar. It is transmitted by direct contact ...of naïve with infected animals, by soft ticks of the Ornithodoros genus, or indirectly by movement of infected animals, improper disposal of contaminated animal products or other sources related to human activity. The recent spread of ASF into Eastern and Central European countries is currently threatening the European pig industry. The situation is aggravated as to-date no efficient vaccine is available. African swine fever virus (ASFV) is a large enveloped ds DNA-virus encoding at least 150 open reading frames. Many of the deduced gene products have not been described, less functionally characterized. We have analysed ASFV protein expression in three susceptible mammalian cell lines representing a susceptible host (wild boar) and two non-susceptible species (human and green monkey) by mass spectrometry and provide first evidence for the expression of 23 so far uncharacterized ASFV ORFs. Expression levels of several newly identified ASFV proteins were remarkably high indicating importance in the viral replication cycle. Moreover, expression profiles of ASFV proteins in the three cell lines differed markedly.
The brain is a genomic mosaic shaped by cellular responses to genome damage. Here, we manipulate somatic genome stability by conditional Knl1 deletion from embryonic mouse brain. KNL1 mutations cause ...microcephaly and KNL1 mediates the spindle assembly checkpoint, a safeguard against chromosome missegregation and aneuploidy. We find that following Knl1 deletion, segregation errors in mitotic neural progenitor cells give rise to DNA damage on the missegregated chromosomes. This triggers rapid p53 activation and robust apoptotic and microglial phagocytic responses that extensively eliminate cells with somatic genome damage, thus causing microcephaly. By leaving only karyotypically normal progenitors to continue dividing, these mechanisms provide a second safeguard against brain somatic aneuploidy. Without Knl1 or p53-dependent safeguards, genome-damaged cells are not cleared, alleviating microcephaly, but paradoxically leading to total pre-weaning lethality. Thus, mitotic genome damage activates robust responses to eliminate somatic mutant cells, which if left unpurged, can impact brain and organismal fitness.
Stratakis CA, Tichomirowa MA, Boikos S, Azevedo MF, Lodish M, Martari M, Verma S, Daly AF, Raygada M, Keil MF, Papademetriou J, Drori‐Herishanu L, Horvath A, Tsang KM, Nesterova M, Franklin S, ...Vanbellinghen J‐F, Bours V, Salvatori R, Beckers A. The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.
The prevalence of germline mutations in MEN1, AIP, PRKAR1A, CDKN1B and CDKN2CI is unknown among pediatric patients with pituitary adenomas (PA). In this study, we screened children with PA for mutations in these genes; somatic GNAS mutations were also studied in a limited number of growth hormone (GH) or prolactin (PRL)‐secreting PA. We studied 74 and 6 patients with either isolated Cushing disease (CD) or GH‐ or PRL‐secreting PA, respectively. We also screened four pediatric patients with CD, and four with GH/PRL‐secreting tumors who had some syndromic features. There was one AIP mutation (p.Lys103Arg) among 74 CD patients. Two MEN1 mutations that occurred in patients with recurrent or difficult‐to‐treat disease were found among patients with CD. There was one MEN1 and three AIP mutations (p.Gln307ProfsX104, p.Pro114fsX, p.Lys241X) among pediatric patients with isolated GH‐ or PRL‐secreting PA and one additional MEN1 mutation in a patient with positive family history. There were no mutations in the PRKAR1A, CDKN1B, CDKN2C or GNAS genes. Thus, germline AIP or MEN1 gene mutations are frequent among pediatric patients with GH‐ or PRL‐secreting PA but are significantly rarer in pediatric CD; PRKAR1A mutations are not present in PA outside of Carney complex.
African swine fever virus (ASFV) is a most devastating pathogen affecting swine. In 2007, ASFV was introduced into Eastern Europe where it continuously circulates and recently reached Western Europe ...and Asia, leading to a socio-economic crisis of global proportion. In Africa, where ASFV was first described in 1921, it is transmitted between warthogs and soft ticks of the genus Ornithodoros in a so-called sylvatic cycle. However, analyses into this virus' evolution are aggravated by the absence of any closely related viruses. Even ancient endogenous viral elements, viral sequences integrated into a host's genome many thousand years ago that have proven extremely valuable to analyse virus evolution, remain to be identified. Therefore, the evolution of ASFV, the only known DNA virus transmitted by arthropods, remains a mystery.
For the identification of ASFV-like sequences, we sequenced DNA from different recent Ornithodoros tick species, e.g. O. moubata and O. porcinus, O. moubata tick cells and also 100-year-old O. moubata and O. porcinus ticks using high-throughput sequencing. We used BLAST analyses for the identification of ASFV-like sequences and further analysed the data through phylogenetic reconstruction and molecular clock analyses. In addition, we performed tick infection experiments as well as additional small RNA sequencing of O. moubata and O. porcinus soft ticks.
Here, we show that soft ticks of the Ornithodoros moubata group, the natural arthropod vector of ASFV, harbour African swine fever virus-like integrated (ASFLI) elements corresponding to up to 10% (over 20 kb) of the ASFV genome. Through orthologous dating and molecular clock analyses, we provide data suggesting that integration could have occurred over 1.47 million years ago. Furthermore, we provide data showing ASFLI-element specific siRNA and piRNA in ticks and tick cells allowing for speculations on a possible role of ASFLI-elements in RNA interference-based protection against ASFV in ticks. We suggest that these elements, shaped through many years of co-evolution, could be part of an evolutionary virus-vector 'arms race', a finding that has not only high impact on our understanding of the co-evolution of viruses with their hosts but also provides a glimpse into the evolution of ASFV.
Bovine respiratory disease complex (BRDC) is the major cause of serious respiratory tract infections in calves. The disease is multifactorial, with either stress or reduced immunity allowing several ...pathogens to emerge. We investigated the susceptibility of bovine airway epithelial cells (BAEC) to infection by the three major viruses associated with the BRDC: bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1 (BHV-1) and bovine parainfluenza virus type 3 (BPIV3). For this purpose, two culture systems for well-differentiated BAEC were used: the air-liquid interface (ALI) system, where filter-grown BAEC differentiate into a pseudostratified respiratory epithelium and precision-cut lung slices (PCLS) where BAEC are maintained in the original tissue organisation. Comparative infection studies demonstrated that entry and release of BPIV3 occurred specifically via the apical membrane with ciliated cells being the major target cells. By contrast, airway epithelial cells were largely resistant to infection by BHV-1. When the epithelial barrier was abolished by opening tight junctions or by injuring the cell monolayer, BHV-1 infected mainly basal cells. Respiratory epithelial cells were also refractory to infection by BRSV. However, this virus infected neither differentiated epithelial cells nor basal cells when the integrity of the epithelial barrier was destroyed. In contrast to cells of the airway epithelium, subepithelial cells were susceptible to infection by BRSV. Altogether, these results indicate that the three viruses of the same disease complex follow different strategies to interact with the airway epithelium. Possible entry mechanisms are discussed.
Two strains of African swine fever virus (ASFV), the high-virulence Lisboa60 (L60) and the low-virulence NH/P68 (NHV), which have previously been used in effective immunization/protection studies, ...were sequenced. Both were isolated in Portugal during the 11-year period after the introduction of ASFV to the European Continent in 1957. The predicted proteins coded by both strains were compared, and where differences were found these were also compared to other strains of known virulence. This highlighted several genes with significant alterations in low-virulence strains of ASFV that may constitute virulence factors, several of which are still uncharacterized regarding their function. Phylogenetic analysis grouped L60 and NHV closest to other P72 genotype I ASFV strains from Europe and West Africa, consistent with the assumed West African origin of all European strains. Interestingly, a relatively lower genomic identity exists between L60 and NHV, both isolated in a similar geographical location 8 years apart, than with other European and west African strains isolated subsequently and in more distant locations. This may reflect the intensive passage in tissue culture, during the early 1960s, of a Portuguese isolate to obtain an attenuated vaccine, which may have led to NHV. This study contributes to a better understanding of the evolution of ASFV, and defines additional potential virulence genes for future studies of pathogenesis towards the development of effective vaccines.
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