In Conversation with Mubin Shaikh: From Salafi Jihadist to Undercover Agent inside the "Toronto 18" Terrorist Group

This interview with former undercover agent Mubin Shaikh can help academics and security practitioners understand the key role played and the challenges faced by covert human intelligence sources within domestic terrorist groups. The interview highlights the identity crisis, the personal factors, and the allure of jihadi militancy that initially drove Shaikh to join a Salafi jihadist group. It investigates Shaikh’s process of disengagement from the Salafi jihadist belief system and his rediscovery of a moderate, inclusive, and benevolent form of Islam. It explores his work as an undercover agent for the Canadian Security Intelligence Service, the Royal Canadian Mounted Police, and the Integrated National Security Enforcement Team responsible for disrupting domestic terrorist groups. The “Toronto 18” terrorist cell, the key role played by undercover agents in preventing terrorist action, and the challenges posed by entrapment are also discussed

Discovery and application of colorectal cancer protein markers for disease stratification

Colorectal cancer (CRC) is a major cause of cancer mortality. Whereas some patients respond well to therapy, others do not, and thus more precise methods of CRC stratification are needed. The intracellular protein expression from 28 CRC primary tumours and corresponding normal intestinal mucosa was analysed using saturation-DIGE/MS and Explorer antibody microarrays. Changes in protein abundance were identified at each stage of CRC. Proteins associated with proliferation, glycolysis, reduced adhesion, endoplasmic reticulum stress, angiogenesis, and response to hypoxia represent changes to CRC and its microenvironment during development. Molecular changes in CRC cells and their microenvironment can be incorporated into clinic-pathological data to help sub-classify tumours and personalise treatment. DotScan antibody microarray analysis was used to profile the surface proteome of cells derived from 50 CRC samples and corresponding normal intestinal mucosa. Fluorescence multiplexing enabled the analysis of two different sub-populations of cells from each sample: EpCAM+ cells (CRC cells or normal epithelial cells in normal mucosa) and CD3+ T-cells (tumour-infiltrating lymphocytes). Unsupervised hierarchical clustering of the CRC and T-cell surface profiles defined four clinically relevant clusters, which showed some correlation with histopathological and clinical characteristics such as cancer cell differentiation, peri-tumoural inflammation and stimulation of infiltrating T-cells. The observed relationship between the surface antigen expression profiles of patients’ CRC cells and their corresponding tumour infiltrating T-cells suggests that CRC surface proteins may play a direct role in influencing the activity (and hence surface protein expression) of neighbouring T-cells and/or vice versa. We conclude that the application of surface profiling may provide improved patient stratification, allowing more reliable prediction of disease progression and patient outcome

Exploring the versatility of the covalent thiol-alkyne reaction with substituted propargyl warheads: a deciding role for the cysteine protease

Terminal unactivated alkynes are nowadays considered the golden standard for cysteine-reactive warheads in activity-based probes (ABPs) targeting cysteine deubiquitinating enzymes (DUBs). In this work, we study the versatility of the thiol-alkyne addition reaction in more depth. Contrary to previous findings with UCHL3, we now show that covalent adduct formation can progress with substituents on the terminal or internal alkyne position. Strikingly, acceptance of alkyne substituents is strictly DUB-specific as this is not conserved among members of the same subfamily. Covalent adduct formation with the catalytic cysteine residue was validated by gel analysis and mass spectrometry of intact ABP-treated USP16CD(WT) and catalytically inactive mutant USP16CD(C205A). Bottom-up mass spectrometric analysis of the covalent adduct with a deuterated propargyl ABP provides mechanistic understanding of the in situ thiol-alkyne reaction, identifying the alkyne rather than an allenic intermediate as the reactive species. Furthermore, kinetic analysis revealed that introduction of (bulky/electron-donating) methyl substituents on the propargyl moiety decreases the rate of covalent adduct formation, thus providing a rational explanation for the commonly lower level of observed covalent adduct compared to unmodified alkynes. Altogether, our work extends the scope of possible propargyl derivatives in cysteine targeting ABPs from unmodified terminal alkynes to internal and substituted alkynes, which we anticipate will have great value in the development of ABPs with improved selectivity profiles.Proteomic

Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Phosphorylation is a posttranslational modification that can affect both housekeeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile, the extent and nature of phosphorylation events are poorly characterized, though a protein kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine, and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of the stationary growth phase. Our approach identifies expected targets and phosphorylation sites among the more than 1,500 phosphosites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV), and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. IMPORTANCE In this paper, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in the regulation of this organism has been published; the current study is expected to form the basis for research on this posttranslational modification in C. difficile.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc

The lncRNA LETS1 promotes TGF-β-induced EMT and cancer cell migration by transcriptionally activating a TβR1-stabilizing mechanism

Transforming growth factor–β (TGF-β) signaling is a critical driver of epithelial-to-mesenchymal transition (EMT) and cancer progression. In SMAD-dependent TGF-β signaling, activation of the TGF-β receptor complex stimulates the phosphorylation of the intracellular receptor-associated SMADs (SMAD2 and SMAD3), which translocate to the nucleus to promote target gene expression. SMAD7 inhibits signaling through the pathway by promoting the polyubiquitination of the TGF-β type I receptor (TβRI). We identified an unannotated nuclear long noncoding RNA (lncRNA) that we designated LETS1 (lncRNA enforcing TGF-β signaling 1) that was not only increased but also perpetuated by TGF-β signaling. Loss of LETS1 attenuated TGF-β–induced EMT and migration in breast and lung cancer cells in vitro and extravasation of the cells in a zebrafish xenograft model. LETS1 potentiated TGF-β–SMAD signaling by stabilizing cell surface TβRI, thereby forming a positive feedback loop. Specifically, LETS1 inhibited TβRI polyubiquitination by binding to nuclear factor of activated T cells (NFAT5) and inducing the expression of the gene encoding the orphan nuclear receptor 4A1 (NR4A1), a component of a destruction complex for SMAD7. Overall, our findings characterize LETS1 as an EMT-promoting lncRNA that potentiates signaling through TGF-β receptor complexes.Proteomic

The alkyne moiety as a latent electrophile in irreversible covalent small molecule inhibitors of Cathepsin K

Molecular Physiolog

Glycoproteomic Analysis of MGL-Binding Proteins on Acute T-Cell Leukemia Cells

C-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of which are pivotal for innate immune responses against pathogens. Other C-type lectins, such as the macrophage galactose-type lectin (MGL), have been shown to induce immunosuppressive responses upon the recognition of aberrant glycosylation on cancer cells. MGL is known to recognize terminal N-acetylgalactosamine (GalNAc), such as the Tn antigen, which is commonly found on malignant cells. Even though this glycan specificity of MGL is well described, there is a lack of understanding of the actual glycoproteins that bind MGL. We present a glycoproteomic workflow for the identification of MGL-binding proteins, which we applied to study MGL ligands on the human Jurkat leukemia cell line. In addition to the known MGL ligands and Tn antigen-carrying proteins CD43 and CD45 on these cells, we have identified a set of novel cell-surface ligands for MGL. Importantly, for several of these, O-glycosylation has hitherto not been described. Altogether, our data provide new insight into the identification and structure of novel MGL ligands that presumably act as modulatory molecules in cancer immune responses.Proteomic

Acetylated bacterial proteins as potent antigens inducing an anti-modified protein antibody response

Objective Gut-residing bacteria, such as Escherichia coli, can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses crossreactive to modified self-proteins and be recognised by human AMPA (hAMPA). Methods E.coli bacteria were grown under amine starvation to generate endogenously acetylated bacterial proteins. Furthermore, E.coli proteins were acetylated chemically. Recognition of these proteins by hAMPA was analysed by western blotting and ELISA; recognition by B cells carrying a modified protein-reactive B cell receptor (BCR) was analysed by pSyk (Syk phosphorylation) activation assay. C57BL/6 mice were immunised with (modified) bacterial protein fractions, and sera were analysed by ELISA. Results Chemically modified bacterial protein fractions contained high levels of acetylated proteins and were readily recognised by hAMPA and able to activate B cells carrying modified protein-reactive BCRs. Likely due to substantially lower levels of acetylation, endogenously acetylated protein fractions were not recognised by hAMPA or hAMPA-expressing B cells. Immunising mice with chemically modified protein fractions induced a strong cross-reactive AMPA response, targeting various modified antigens including citrullinated proteins. Conclusions Acetylated bacterial proteins are recognisable by hAMPA and are capable of inducing crossreactive AMPA in mice. These observations provide the first conceptual evidence for a novel mechanism involving the (endogenous) acetylation of the bacterial proteome, allowing a breach of tolerance to modified proteins and the formation of cross-reactive AMPA.</p

A flexible MHC class I multimer loading system for large-scale detection of antigen-specific T cells

Tumorimmunolog

Revised model for the type A glycan biosynthetic pathway in Clostridioides difficile strain 630Δerm based on quantitative proteomics of cd0241-cd0244 mutant strains

The bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the Clostridioides difficile strain 630 Delta erm, the main filamentous component, FliC, is post-translationally modified with an O-linked Type A glycan structure. This modification is essential for flagellar function, since motility is seriously impaired in gene mutants with improper biosynthesis of the Type A glycan. The cd0240-cd0244 gene cluster encodes the Type A biosynthetic proteins, but the role of each gene, and the corresponding enzymatic activity, have not been fully elucidated. Using quantitative mass spectrometry-based proteomics analyses, we determined the relative abundance of the observed glycan variations of the Type A structure in cd0241, cd0242, cd0243, and cd0244 mutant strains. Our data not only confirm the importance of CD0241, CD0242, and CD0243 but, in contrast to previous data, also show that CD0244 is essential for the biosynthesis of the Type A modification. Combined with additional bioinformatic analyses, we propose a revised model for Type A glycan biosynthesis.Molecular basis of bacterial pathogenesis, virulence factors and antibiotic resistanc