Month: May 2018

Given the importance of transcription factors in facilitating vital aspects of cell biology, mutations in -or aberrant regulation oftranscription factors have been associated with human disease. The identification of inhibitors or activators of transcription factors will therefore not only illuminate the signaling pathways that regulate them, but could also identify targets that may prove to be better drug targets than transcription factors themselves, or whose 2-Pyridylethylamine dihydrochloride inhibition may provide a more selective therapeutic effect. We chose to screen for inhibitors of NF-��B, a family of transcription factors that in mammals plays a central role in regulating immune responses, development, cell proliferation, and survival. They form dimers and are normally kept inactive in the cytoplasm. Activation of a wide variety of receptors, including antigen receptors, patternrecognition receptors and cytokine receptors leads to translocation of NF-��B dimers into the nucleus. Here the dimers bind to DNA ��B sites in promoters and enhancers of target genes. Activation of NF-��B needs to be tightly controlled and rapidly curtailed following the initial stimulus to prevent uncontrolled tissue damage and/or disease. Here we performed the first reporter screen in KBM7 cells to identify constitutive inhibitors of NF-��B. The identification of CYLD, a known negative regulator of NF-��B, demonstrates the utility of using human haploid cells to dissect a variety of biological processes. All screens in human haploid cells performed to date have relied on intrinsic phenotypes, such as sensitivity to A 839977 toxins or protein surface expression, both of which can be easily observed at a cellular level. To provide a clear phenotypic readout for abrogation of NF-��B inhibitor function -and thus improper activation of NF-��B-we generated a NF-��B reporter cell line. We transduced KBM7 cells, which are haploid for all chromosomes but chromosome 8, with a reporter construct that contains a NF-��B transcriptional response element and a minimum cytomegalovirus promoter upstream of the blasticidin S resistance gene from Bacillus cereus. Thus, insertional inactivation of genes that normally repress activation of NF-��B would render the reporter cells resistant to blasticidin and provide an easy means to distinguish them from wild-type cells. To ensure that the selected clonal reporter cell line had intact NF-��B regulation, we stimulated both KBM7 cells and the NF-��B reporter cell line with TNF. We saw that both cells displayed similar degradation kinetics. The selected clonal reporter cell line survived in the presence of blasticidin only when stimulated with NF-��B activators, demonstrating that the reporter functioned properly. The NF-��B reporter cell line was then mutagenized with a retroviral gene-trap vector, using an established protocol that generally yields a library containing mutations in approximately 98% of genes expressed in KBM7 cells. Mutagenized NF-��B reporter cells were exposed to blasticidin and the survivors were pooled and expanded. The selected mutant population was markedly more resistant to blasticidin than the parental reporter cell line and wild-type KBM7 cells in the absence of any stimulus, suggesting that the survivors contain mutations that cause constitutive activation of NF-��B. To identify the mutations in the selected mutant population, genomic DNA was harvested from the survivors. The DNA sequences that flank gene-trap insertion sites were amplified, sequenced in parallel, and mapped to the human genome.

In two clusters, possible interdepartment spread was observed. The heterogeneous cluster 1 contained sequences from four different departments over a time span of nearly half a year. One of the later samples in this cluster was taken from a patient without earlier hospital contact within 48 hours after admission, thus indicating a new introduction of the virus from the community into the hospital. However, inter-department spread could have occurred in temporarily related samples from this cluster. An example of this could be the simultaneously occurring norovirus infections in the cardiology department and the internal medicine department 1 in week 5�C7/2008. In other temporary and/or locally related cases with sequences differing by no more than one nucleotide the transmission route was unclear. Identical sequences belonging to cluster 1 were for example found in three patients on three different wards in week 13�C15/2008. Both nosocomial transmission and repeated introduction from the community could ACET explain these infections. The same is true for the first two samples from cluster 2 which were from patients from two different departments. In the nephrology department and both internal medicine departments, consecutive 22-Oxacalcitriol outbreaks with distinct clusters of the GII.4 variant Den Haag 2006b were detected, indicating repeated introductions of different strains from the community. As opposed to this, a prolonged outbreak over 12 weeks with the same cluster occurred in the hematology department. Sequencing of the capsid gene has previously been shown to be helpful for investigating outbreaks. Comparable to our study, which found a presumed larger hospital outbreak to consist of several distinct smaller outbreaks, Xerry et al. could differentiate two distinct simultaneous outbreaks in different departments of the same hospital and Sukhrie et al. found that one of four epidemiological outbreaks involved several unrelated strains. However, in the study of Xerry et al., P2 sequences also detected links between presumably unrelated outbreaks in different departments and even two different hospitals. Similarly, in another study by Sukhrie and coworkers only three out of 14 molecular clusters found by sequencing of the P2 region had been identified through epidemiological investigations, even though the conservative approach of defining a molecular cluster by 100% sequence identity was chosen. The frequency of nucleotide changes found in our study is in concordance with previous studies investigating nosocomial NoV GII.4 outbreaks: Dingle et al. showed a diversity of six nucleotide changes in the entire NoV genome in a 1-week outbreak with presumed person-to-person transmission, whereas the strains of an outbreak with a presumed point source only differed by one substitution. Sukhrie found a similar sequence variation of two to 22 nucleotide changes in the P2 domain within prospectively monitored nosocomial outbreaks, where patients and health care workers were sampled once weekly for one to two months.