Mapping the interaction of B cell Leukemia 3 (BCL-3) and nuclear factor κB (NF-κB) p50 identifies a BCL-3-mimetic anti-inflammatory peptide

The NF-κB transcriptional response is tightly regulated by a number of processes including the phosphorylation, ubiquitination, and subsequent proteasomal degradation of NF-κB subunits. The IκB family protein BCL-3 stabilizes a NF-κB p50 homodimer·DNA complex through inhibition of p50 ubiquitination. This complex inhibits the binding of the transcriptionally active NF-κB subunits p65 and c-Rel on the promoters of NF-κB target genes and functions to suppress inflammatory gene expression. We have previously shown that the direct interaction between p50 and BCL-3 is required for BCL-3-mediated inhibition of pro-inflammatory gene expression. In this study we have used immobilized peptide array technology to define regions of BCl-3 that mediate interaction with p50 homodimers. Our data show that BCL-3 makes extensive contacts with p50 homodimers and in particular with ankyrin repeats (ANK) 1, 6, and 7, and the N-terminal region of Bcl-3. Using these data we have designed a BCL-3 mimetic peptide based on a region of the ANK1 of BCL-3 that interacts with p50 and shares low sequence similarity with other IκB proteins. When fused to a cargo carrying peptide sequence this BCL-3-derived peptide, but not a mutated peptide, inhibited Toll-like receptor-induced cytokine expression in vitro. The BCL-3 mimetic peptide was also effective in preventing inflammation in vivo in the carrageenan-induced paw edema mouse model. This study demonstrates that therapeutic strategies aimed at mimicking the functional activity of BCL-3 may be effective in the treatment of inflammatory disease

Generation and transmission of interlineage recombinants in the SARS-CoV-2 pandemic.

We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single-nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances, there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of 2 months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7's set of mutations.The COG-UK Consortium is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) (MC_PC_19027), and Genome Research Limited, operating as the Wellcome Sanger Institute. O.G.P. was supported by the Oxford Martin School. J.T.M., R.M.C., N.J.L., and A.R. acknowledge the support of the Wellcome Trust (Collaborators Award 206298/Z/17/Z – ARTIC network). D.L.R. acknowledges the support of the MRC (MC_UU_12014/12) and the Wellcome Trust (220977/Z/20/Z). E.S. and A.R. are supported by the European Research Council (grant agreement no. 725422 – ReservoirDOCS). T.R.C. and N.J.L. acknowledge the support of the MRC, which provided the funding for the MRC CLIMB infrastructure used to analyze, store, and share the UK sequencing dataset (MR/L015080/1 and MR/T030062/1). The samples sequenced in Wales were sequenced partly using funding provided by the Welsh Government

Control of NF-κB subunits by ubiquitination

NF-κB is an essential regulator of inflammation and is also required for normal immune development and homeostasis. The inducible activation of NF-κB by a wide range of immuno-receptors such as the toll-like receptors (TLR), Tumour Necrosis Factor receptor (TNFR), and antigen T cell and B cell receptors requires the ubiquitin-triggered proteasomal degradation of IκBα to promote the nuclear translocation and transcriptional activity of NF-κB dimers. More recently, an additional role for ubiquitination and proteasomal degradation in the control of NF-κB activity has been uncovered. In this case, it is the ubiquitination and proteasomal degradation of the NF-κB subunits that play a critical role in the termination of the NF-κB-dependent transcriptional response induced by receptor activation. The primary trigger of NF-κB ubiquitination is DNA binding by NF-κB dimers and is further controlled by specific phosphorylation events which regulate the interaction of NF-κB with the E3 ligase complex and the deubiquitinase enzyme USP7. It is the balance between ubiquitination and deubiquitination that shapes the NF-κB-mediated transcriptional response. This chapter describes methods for the analysis of NF-κB ubiquitination

Using surface-enhanced Raman scattering for simultaneous multiplex detection and quantification of thiols associated to axillary malodour.

Axillary malodour is caused by the microbial conversion of human-derived precursors to volatile organic compounds. Thiols strongly contribute to this odour but are hard to detect as they are present at low concentrations. Additionally, thiols are highly volatile and small making sampling and quantification difficult, including by gas chromatography-mass spectrometry. In this study, surface-enhanced Raman scattering (SERS), combined with chemometrics, was utilised to simultaneously quantify four malodourous thiols associated with axillary odour, both in individual and multiplex solutions. Univariate and multivariate methods of partial least squares regression (PLS-R) were used to calculate the limit of detection (LoD) and results compared. Both methods yielded comparable LoD values, with LoDs using PLS-R ranging from 0.0227 ppm to 0.0153 ppm for the thiols studied. These thiols were then examined and quantified simultaneously in 120 mixtures using PLS-R. The resultant models showed high linearity (Q2 values between 0.9712 and 0.9827 for both PLS-1 and PLS-2) and low values of root mean squared error of predictions (0.0359 ppm and 0.0459 ppm for PLS-1 and PLS-2, respectively). To test this approach further, these models were challenged with 15 new blind test samples, collected independently from the initial samples. This test demonstrated that SERS combined with PLS-R could be used to predict the unknown concentrations of these thiols in a mixture. These results display the ability of SERS for the simultaneous multiplex detection and quantification of analytes and its potential for future development for detecting gaseous thiols produced from skin and other body sites

Autophagosomal iκbα degradation plays a role in the long term control of tumor necrosis factor-α-induced nuclear factor-κb (nf-κb) activity

Transcription factor NF-kappa B is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-kappa B is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-alpha on NF-kappa B activity for up to 48 h in intestinal epithelial cells. Results show that NF-kappa B remained persistently activated up to 48 h after TNF-alpha and that the long term activation of NF-kappa B was accompanied by a biphasic degradation of I kappa B alpha. The first phase of I kappa B alpha degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-alpha stimulated formation of autophagosomes in intestinal epithelial cells and that I kappa B alpha co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-alpha-induced degradation of I kappa B alpha and lowered NF-kappa B target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-kappa B activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-kappa B may mediate this effect

Regulation of nf- b responses by epigenetic suppression of i b expression in hct116 intestinal epithelial cells

O'Gorman A, Colleran A, Ryan A, Mann J, Egan LJ. Regulation of NF-kappa B responses by epigenetic suppression of I kappa B alpha expression in HCT116 intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol 299: G96-G105, 2010. First published April 8, 2010; doi:10.1152/ajpgi.00460.2009.-Intestinal epithelial cells play critical roles in regulating mucosal immunity. Since epigenetic factors such as DNA methylation and histone modifications are implicated in aging, carcinogenesis, and immunity, we set out to assess any role for epigenetic factors in the regulation of intestinal epithelial cell immune responses. Experiments were conducted using the HCT116 cell line, and a subclone was genetically engineered to lack DNA methyltransferases (DNMT). The induction of the chemokine interleukin-8 and the antiapoptotic protein cFLIP by tumor necrosis factor-alpha were markedly less in HCT116 cells lacking DNMT than in parental cells. These effects were accompanied by lower monocyte chemotaxis and higher caspase signaling in HCT116 cells lacking DNMT than parental cells. Tumor necrosis factor-alpha-induced NF-kappa B activation was blocked and I kappa B alpha expression was higher in HCT116 cells lacking DNMT than in parental cells. A CpG island in the I kappa B alpha gene promoter region was found to contain variable levels of methylation in parental HCT116 cells. Chromatin immunoprecipitation analysis of histone proteins bound to the I kappa B alpha gene promoter revealed that higher levels of I kappa B alpha expression in HCT116 cells lacking DNMT compared with parental cells were accompanied by more chromatin marks permissive to gene transcription. These findings show that epigenetic factors influence the NF-kappa B system in intestinal epithelial cells, resulting in a previously unrecognized mechanism of innate immune regulation