Global Surveillance of Emerging Influenza Virus Genotypes by Mass Spectrometry

Effective influenza surveillance requires new methods capable of rapid and inexpensive genomic analysis of evolving viral species for pandemic preparedness, to understand the evolution of circulating viral species, and for vaccine strain selection. We have developed one such approach based on previously described broad-range reverse transcription PCR/electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology.Analysis of base compositions of RT-PCR amplicons from influenza core gene segments (PB1, PB2, PA, M, NS, NP) are used to provide sub-species identification and infer influenza virus H and N subtypes. Using this approach, we detected and correctly identified 92 mammalian and avian influenza isolates, representing 30 different H and N types, including 29 avian H5N1 isolates. Further, direct analysis of 656 human clinical respiratory specimens collected over a seven-year period (1999-2006) showed correct identification of the viral species and subtypes with >97% sensitivity and specificity. Base composition derived clusters inferred from this analysis showed 100% concordance to previously established clades. Ongoing surveillance of samples from the recent influenza virus seasons (2005-2006) showed evidence for emergence and establishment of new genotypes of circulating H3N2 strains worldwide. Mixed viral quasispecies were found in approximately 1% of these recent samples providing a view into viral evolution.Thus, rapid RT-PCR/ESI-MS analysis can be used to simultaneously identify all species of influenza viruses with clade-level resolution, identify mixed viral populations and monitor global spread and emergence of novel viral genotypes. This high-throughput method promises to become an integral component of influenza surveillance

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at its optimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radiodurans genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis

Complexity in transcription control at the activation domain-mediator interface.

International audienceTranscript elongation by polymerase II paused at the Egr1 promoter is activated by mitogen-activated protein kinase phosphorylation of the ternary complex factor (TCF) ELK1 bound at multiple upstream sites and subsequent phospho-ELK1 interaction with mediator through the MED23 subunit. Consequently, Med23 knockout (KO) nearly eliminates Egr1 (early growth response factor 1) transcription in embryonic stem (ES) cells, leaving a paused polymerase at the promoter. Med23 KO did not, however, eliminate Egr1 transcription in fibroblasts. Chromatin immunoprecipitation analysis and direct visualization of fluorescently labeled TCF derivatives and mediator subunits revealed that three closely related TCFs bound to the same control regions. The relative amounts of these TCFs, which responded differently to the loss of MED23, differed in ES cells and fibroblasts. Transcriptome analysis suggests that most genes expressed in both cell types, such as Egr1, are regulated by alternative transcription factors in the two cell types that respond differently to the same signal transduction pathways

Multivariate indicators of disease severity in COVID-19

Abstract The novel coronavirus pandemic continues to cause significant morbidity and mortality around the world. Diverse clinical presentations prompted numerous attempts to predict disease severity to improve care and patient outcomes. Equally important is understanding the mechanisms underlying such divergent disease outcomes. Multivariate modeling was used here to define the most distinctive features that separate COVID-19 from healthy controls and severe from moderate disease. Using discriminant analysis and binary logistic regression models we could distinguish between severe disease, moderate disease, and control with rates of correct classifications ranging from 71 to 100%. The distinction of severe and moderate disease was most reliant on the depletion of natural killer cells and activated class-switched memory B cells, increased frequency of neutrophils, and decreased expression of the activation marker HLA-DR on monocytes in patients with severe disease. An increased frequency of activated class-switched memory B cells and activated neutrophils was seen in moderate compared to severe disease and control. Our results suggest that natural killer cells, activated class-switched memory B cells, and activated neutrophils are important for protection against severe disease. We show that binary logistic regression was superior to discriminant analysis by attaining higher rates of correct classification based on immune profiles. We discuss the utility of these multivariate techniques in biomedical sciences, contrast their mathematical basis and limitations, and propose strategies to overcome such limitations

Nanolock–Nanopore Facilitated Digital Diagnostics of Cancer Driver Mutation in Tumor Tissue

Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock–nanopore method for single-molecule detection of a serine/threonine protein kinase gene <i>BRAF</i> V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the <i>BRAF</i> V600E mutant DNA lower than 1% in the tumor tissues. The nanolock–nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy

The Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by E. coli RcnR

The RcnR metalloregulator represses the transcription of the Co(II) and Ni(II) exporter, RcnAB. Previous studies have shown that Co(II) and Ni(II) bind to RcnR in six-coordinate sites, resulting in de-repression. Here, the roles of His60, His64, and His67 in specific metal recognition are examined. His60 and His64 correspond to ligands that are important for Cu(I) binding in the homologous Cu(I)-responsive metalloregulator, CsoR. These residues are known to be functionally important in RcnR transcriptional regulation. XAS was used to examine the structure of bound cognate and non-cognate metal ions, and lacZ reporter assays were used to assess the transcription of rcnA in response to metal binding in the three His → Cys mutations, H60C, H64C and H67C. These studies confirm that both Ni(II) and Co(II) use His64 as a ligand. H64C-RcnR is also the only known mutation that retains a Co(II) response while eliminating the response to Ni(II) binding. XAS data indicate that His60 and His67 are potential Co(II) ligands. The effects of the mutations of His60, His64, and His67 residues on the structures of the non-cognate metal ions (Zn(II) and Cu(I)) reveals that these residues have distinctive roles in binding non-cognate metals. None of the His → Cys mutants in RcnR confer any response to Cu(I) binding, including H64C-RcnR, where the ligands involved in Cu(I) binding in CsoR are present. These data indicate that while the secondary, tertiary and quaternary structures of CsoR and RcnR are quite similar, small changes in primary sequence reveal that the specific mechanisms involved in metal recognition are quite different

Role of the N-terminus in Determining Metal-Specific Responses in the E. coli

RcnR (resistance to cobalt and nickel regulator) is a 40 kDa homotetrameric protein and metalloregulator that controls the transcription of the Co(II) and Ni(II) exporter, RcnAB, by binding to DNA as an apo-protein and releasing DNA in response to specifically binding Co(II) and Ni(II) ions. Using x-ray absorption spectroscopy (XAS) to examine the structure of metals bound, and lacZ reporter assays of the transcription of RcnAB in response to metal binding, in WT- and mutant proteins, the roles of coordination number, ligand selection and residues in the N-terminus of the protein were examined as determinants in metal ion recognition. The studies show that the cognate metal ions, Co(II) and Ni(II), which bind in (N/O)(5)S six-coordinate sites, are distinguished from non-cognate metal ions (Cu(I) and Zn(II)), which bind only three protein ligands and one anion from the buffer, by coordination number and ligand selection. Using mutations of residues near the N-terminus, the N-terminal amine is shown to be a ligand of the cognate metal ions that is missing in the complexes with non-cognate metal ions. The side chain of His3 is also shown to play an important role in distinguishing metal ions. The imidazole group is shown to be a ligand in the Co(II) RcnR complex, but not in the Zn(II) complex. Further, His3 does not appear to bind to Ni(II), providing a structural basis for the differential regulation of RcnAB by the two cognate ions. The Zn(II) complexes change coordination number in response to the residue in position three. In H3C-RcnR, the Zn(II) complex is five-coordinate, and in H3E-RcnR the Zn(II) ion is bound to six protein ligands. The metric parameters of this unusual Zn(II) structure resemble those of the WT-Ni(II) complex, and the mutant protein is able to regulate expression of RcnAB in response to binding the non-cognate ion. The results are discussed within a protein allosteric model for gene regulation by metalloregulators

Adenovirus Type 5 Early Region 1B 55K Oncoprotein-Dependent Degradation of Cellular Factor Daxx Is Required for Efficient Transformation of Primary Rodent Cells▿

Early region 1B 55K (E1B-55K) from adenovirus type 5 (Ad5) is a multifunctional regulator of lytic infection and contributes in vitro to complete cell transformation of primary rodent cells in combination with Ad5 E1A. Inhibition of p53 activated transcription plays a key role in processes by which E1B-55K executes its oncogenic potential. Nevertheless, additional functions of E1B-55K or further protein interactions with cellular factors of DNA repair, transcription, and apoptosis, including Mre11, PML, and Daxx, may also contribute to the transformation process. In line with previous results, we performed mutational analysis to define a Daxx interaction motif within the E1B-55K polypeptide. The results from these studies showed that E1B-55K/Daxx binding is not required for inhibition of p53-mediated transactivation or binding and degradation of cellular factors (p53/Mre11). Surprisingly, these mutants lost the ability to degrade Daxx and showed reduced transforming potential in primary rodent cells. In addition, we observed that E1B-55K lacking the SUMO-1 conjugation site (SCS/K104R) was sufficient for Daxx interaction but no longer capable of E1B-55K-dependent proteasomal degradation of the cellular factor Daxx. These results, together with the observation that E1B-55K SUMOylation is required for efficient transformation, provides evidence for the idea that SUMO-1-conjugated E1B-55K-mediated degradation of Daxx plays a key role in adenoviral oncogenic transformation. We assume that the viral protein contributes to cell transformation through the modulation of Daxx-dependent pathways. This further substantiates the assumption that further mechanisms for efficient transformation of primary cells can be separated from functions required for the inhibition of p53-stimulated transcription