186,414 research outputs found
The development and application of protemics to the analysis of Chlamydia Trachomatis
No full textThe bacterial pathogen Chlamydia trachomatis causes Trachoma, the worlds leading cause of preventable blindness and is also responsible for the most common curable sexually transmitted disease in the UK and United States. C. trachomatis is an obligate intracellular organism characterised by a unique and complex growth cycle. Its study presents many challenges since it has historically been recalcitrant to genetic manipulation and growth in the absence of a host cell. Nevertheless, the sequencing of the C. trachomatis genome and its relatively small size by comparison to genomes from other bacterial pathogens, has paved the way for studies at the proteomic level.This thesis describes the development and application of proteomic approaches to study C. trachomatis L2. To survey the expressed chlamydial proteome, a combination of the qualitative approaches, 2-DGE, MudPIT and GeLC-MS/MS; and the quantitative approaches AQUA, iTRAQ and LC-MSE were used. Collectively, the approaches efficiently identified 648 expressed proteins, representing ~72% of the predicted proteome of C. trachomatis L2, from both the infectious (elementary body, EB) and replicating (reticulate body, RB) form of the pathogen. In the infectious EB, the entire set of predicted glycolytic enzymes were detected, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. Further, proteomic analysis of the RB form also uncovered biosynthetic enzymes for chlamydial cell wall synthesis, indicating that peptidoglycan is produced in some form during growth in host cells. Comparison of the quantitative approaches iTRAQ and LC-MSE demonstrated that LC-MSE quantitative data was significantly more robust and extensive relative to iTRAQ data. In addition to information on relative amounts of these proteins between the two forms, LC-MSE data also yielded the cellular concentration (molecules per cell) for 489 proteins.This extensive set of absolute quantitation data permits estimates of the energy invested in the synthesis of various classes of proteins. The results indicate that C. trachomatis devotes most of its energy into maintenance of the translational machinery. However, it also expends significant amounts of energy into making cell envelope components and a set of hitherto hypothetical proteins. These proteins, which account for the bulk of the energy invested by the intracellular RB form of the pathogen as it converts to the extracellular EB form, highlight the importance of absolute quantitation data for understanding the biological processing status of the cell. The datasets also revealed a large number of proteins that were differentially expressed between replicating RBs and infectious EBs, ranging from 8.4-fold down-regulation to 3.5-fold up-regulation. Consistent with transcriptomic studies (Belland et al., 2003), proteins involved in protein synthesis, ATP generation, central metabolism, secretion and nutrient uptake were predominant in the metabolically active RB at 15 h PI. Although many of the proteins in these functional categories were down-regulated in EBs, proteins required for glycolysis, central metabolism, protein synthesis, and type III secretion were present in significant amounts in EBs suggesting that the infectious EB is primed ‘ready-to-go’ upon contact with the host cell
What do cancer-specific CD8+ T cells see?: the contribution of immunopeptidomics
No full textImmunopeptidomics is the survey of all peptides displayed on a cell or tissue when bound to human leukocyte antigen (HLA) molecules using tandem mass spectrometry. When attempting to determine the targets of tumour-specific CD8+ T cells, a survey of the potential ligands in tumour tissues is invaluable, and, in comparison with in-silico predictions, provides greater certainty of the existence of individual epitopes, as immunopeptidomics-confirmed CD8+ T-cell epitopes are known to be immunogenic, and direct observation should avoid the risk of autoreactivity which could arise following immunisation with structural homologues. The canonical sources of CD8+ T-cell tumour specific epitopes, such as tumour associated antigens, may be well conserved between patients and tumour types, but are often only weakly immunogenic. Direct observation of tumour-specific neoantigens by immunopeptidomics is rare, although valuable. Thus, there has been increasing interest in the non-canonical origins of tumour-reactive CD8+ T-cell epitopes, such as those arising from proteasomal splicing events, translational/turnover defects and alternative open reading frame reads. Such epitopes can be identified in silico, although validation is more challenging. Non-self CD8+ T-cell epitopes such as viral epitopes may be useful in certain cancer types with known viral origins, however these have been relatively unexplored with immunopeptidomics to date, possibly due to the paucity of source viral proteins in tumour tissues. This review examines the latest evidence for canonical, non-canonical and non-human CD8+ T-cell epitopes identified by immunopeptidomics, and concludes that the relative contribution for each of these sources to anti-tumour CD8+ T-cell reactivity is currently uncertain.</p
Increased expression of glial fibrillary acidic protein fragments and mu-calpain activation within the hippocampus of prion-infected mice
No full textPrion diseases are characteristically accompanied by marked astrocytic activation, which is initiated relatively early in the disease process. Using the intracerebrally injected ME7 strain of prion agent to model disease, we identified an expected increase in GFAP (glial fibrillary acidic protein) but additionally noted an accumulation of GFAP cleavage fragments in hippocampal homogenates. A time-dependent increase in hippocampal m-calpain immunoreactivity within astrocytes suggests that its proteolytic activity may account for the cleavage of GFAP that is observed in the ME7 model. It may therefore contribute to the reactive gliosis that is characteristic of prion diseases
Characterizing the beta-catenin interactome using inhibitor screens and novel interaction proteomics techniques
No full textBackgroundWnt signalling is a critical developmental pathway and associated aberrant signaling drives oncogenesis including colorectal and bone cancer. β-catenin, a central protein forms a range of protein-protein interactions(PPIs), fundamental to driving the transcription of oncogenes inducing proliferation and tumour formation. To further elucidate these interactions, we have sought to i) identify inhibitors of β-catenin PPIs and ii) employ a proximity dependent labelling technique (MiniTurbo) to characterize novel PPIs.MethodsMiniTurbo-Mutant Biotin ligase BirA is genetically fused to β-catenin and upon addition of Biotin, tagging is induced of proteins within 20nm in as quick as 10 minutes enabling streptavidin pull down and tagging to Mass spectrometry. CTNNB1 gene was successfully inserted into the Miniturbo plasmid construct using SLICE cloning. DH5alpha bacteria were transformed with plasmid and single colony selected with Ampicillin. Following plasmid purification, it was added with Retroviral components for viral transfection and production in HEK293 cells. U2OS adenocarcinoma cells transduced and selected using puromycin. Validated with western blot and immunofluorescence. 24-hour application of MSAB derivatives following induction of Miniturbo with Doxycycline. Streptavidin pulldown was carried out and samples were subjected to Mass Spectrometry following on-bead trypsin digestion. Proteomic analysis was carried out to elucidate novel PPI.Results-MSAB derivatives influence levels of β-catenin and Wnt target genes at protein and transcriptional level. -Successful introduction and selection of the miniturbo plasmid into U2OS cells. Western blots and immunofluorescence verification. -Novel Proteomic networks of Beta-catenin protein interactions in U2OS cells. The results illustrate the dynamic PPI network of β-catenin in a 2D cancer model and how those interactions are modulated in the presence of small molecule inhibitors for therapeutic indications.ConclusionsTargeting β-catenin directly for Oncotherapy has been difficult with very little success. Further characterization and understanding of the dynamic β-catenin interactome may pave an avenue for addressing this challenge
Personalising a writing course with a flipped classroom approach
No full textWe present a writing course that uses a flipped classroom approach for personalised learning (Deroey & Skipp, 2023). The student-centred course design has at its core non-instructor-led activities and allows us to accommodate linguistically and disciplinary heterogeneous groups of PhD students. It considers practical issues such as PhD students’ busy schedules (Casanave, 2010), their ability to work independently and limited staff resources. The course has been very successfully delivered several times, both fully and partly online.
This writing course provides insight into the structural, stylistic and rhetorical features of research articles as well as the writing and publication process. Students first submit an ‘independent learning task’ in which they apply theory from the book to their writing. Teachers use this output to illustrate key theory points and to provide further practice in the subsequent workshop. Additionally, students submit article drafts with their reflections (Yasuda, 2011) and receive feedback through peer review and consultations (Cho & MacArthur, 2010).
Our course design has several advantages. First, it allows personalised learning and differentiated teaching. Second, it limits the number of workshops and maximises their value. Third, it stimulates novice research writers to become writing researchers, promoting continuous learning.
References
Casanave, C. P. (2010). Dovetailing under impossible circumstances. In C. Aitchison, B. Kamler, & A. Lee (Eds.), Publishing pedagogies for the doctorate and beyond (pp. 47-63). Routledge.
Cho, K., & MacArthur, C. (2010). Student revision with peer and expert reviewing. Learning and Instruction, 20, 328-338.
Deroey, K. L. B., & Skipp, J. (2023). Designing and delivering an online research article writing course for doctoral students in Luxembourg during Covid-19. In B. Fenton-Smith, J. Gimenez, K. Mansfield, M. Percy, & M. Spinillo (Eds.), International perspectives on teaching academic English in turbulent times (pp. 81-94). Routledge. https://doi.org/10.4324/9781003283409-10
Yasuda, S. (2011). Genre-based tasks in foreign language writing: Developing writers’ genre awareness, linguistic knowledge, and writing competence. Journal of Second Language Writing, 20(2), 111-133
Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers
No full textThe risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MS(E) approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MS(E) approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemical
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Shotgun proteomic analysis of human induced sputum
No full textSputum is increasingly recognised as a biologically relevant sample of the environment of the airways when collected under controlled conditions. As such, it has been used for the analysis of bacterial load, tumour and inflammatory cell content, and of biomarkers of airways diseases such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. While related biofluids such as broncho-alveolar lavage1 fluid, nasal lavage fluid,2 and in particular saliva3 have been well-characterised using standard 2-D gel based proteomics, sputum has not. This is probably due to the presence of mucin glycoproteins that compromise the separation of proteins during the IEF stage of 2-D gel analysis, preventing good resolution.We have used a combination of 2-D gel electrophoresis (fig 1BGo) and a multidimensional mass-spectrometric technique (GeLC-MS/MS) (fig 1AGo) to examine the proteome of induced sputum. 191 individual human proteins were confidently assigned using these techniques. In addition to expected components, several hitherto unreported proteins were discovered, including three members of the annexin family, kallikreins 1 and 11, and peroxiredoxins 1, 2, and 5. The resultant data represent the first detailed survey of the human induced sputum proteome and allow a comparison of the induced sputum proteome with other biologically related fluids such as broncho-alveolar lavage fluid and saliva. They also provide a platform for the future identification of biomarkers of airways disease
- …
