Scholarly Works - Biochemistry
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Item Open Access New PomBase Website 2017, Group Leader Survey Summary(2017-12-01) Wood, Valerie; Harris, Midori; Lock, Antonia; Rutherford, Kim; Wood, Valerie [0000-0001-6330-7526]; Harris, Midori [0000-0003-4148-4606]PomBase launched a new version of its website on 25th October 2017. Shortly thereafter, 100 fission yeast research group leaders were surveyed to gauge the impact of the site redesign.Item Open Access Accepted version Peer-reviewed Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.(Nature, 2018-02) Luis, Ana S; Briggs, Jonathon; Zhang, Xiaoyang; Farnell, Benjamin; Ndeh, Didier; Labourel, Aurore; Baslé, Arnaud; Cartmell, Alan; Terrapon, Nicolas; Stott, Katherine; Lowe, Elisabeth C; McLean, Richard; Shearer, Kaitlyn; Schückel, Julia; Venditto, Immacolata; Ralet, Marie-Christine; Henrissat, Bernard; Martens, Eric C; Mosimann, Steven C; Abbott, D Wade; Gilbert, Harry J; Stott, Katherine [0000-0002-4014-1188]The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms.Item Open Access Published version Peer-reviewed SRSF3 maintains transcriptome integrity in oocytes by regulation of alternative splicing and transposable elements(Springer Nature, 2018) Dang Do, Vinh; Strauss, Bernhard; Cukuroglu, Engin; Macaulay, Iain; Boon Wee, Keng; Xiamoming, Tim; Ruiz De Los Mozos, Igor; Lee, Caroline; Harrison, Andrew; Butler, Richard; Dietmann, Sabine; Jernej, Ule; Marioni, John; Smith, Christopher WJ; Goke, Jonathan; Surani, Azim M; Harrison, Andrew [0000-0002-3778-8415]; Butler, Richard [0000-0002-3885-1332]; Marioni, John [0000-0001-9092-0852]; Smith, Chris [0000-0002-2753-3398]; Surani, Azim [0000-0002-8640-4318]The RNA-binding protein SRSF3 (also known as SRp20) has critical roles in the regulation of pre-mRNA splicing. Zygotic knockout of Srsf3 results in embryo arrest at the blastocyst stage. However, SRSF3 is also present in oocytes, suggesting that it might be critical as a maternally inherited factor. Here, we identify SRSF3 as an essential regulator of alternative splicing and of transposable elements to maintain transcriptome integrity in mouse oocyte. Using 3D time-lapse confocal live imaging, we show that conditional deletion of Srsf3 in fully-grown germinal vesicle oocytes substantially compromises the capacity of germinal vesicle breakdown (GVBD), and consequently entry into meiosis. By combining single cell RNA-seq, and oocyte micromanipulation with steric blocking antisense oligonucleotides and RNAse-H inducing gapmers, we found that the GVBD defect in mutant oocytes is due to both aberrant alternative splicing and de-repression of B2 SINE transposable elements. Together, our study highlights how control of transcriptional identity of the maternal transcriptome by the RNA-binding protein SRSF3 is essential to the development of fertilized-competent oocytes.Item Open Access Accepted version Peer-reviewed Sub-minute kinetics of human red cell fumarase: 1 H spin-echo NMR spectroscopy and 13 C rapid-dissolution dynamic nuclear polarization.(Wiley-Blackwell, 2018-03) Shishmarev, Dmitry; Wright, Alan J; Rodrigues, Tiago B; Pileio, Giuseppe; Stevanato, Gabriele; Brindle, Kevin M; Kuchel, Philip W; Wright, Alan [0000-0002-4577-5681]; Brindle, Kevin [0000-0003-3883-6287]Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues. Thus, we aimed to evaluate the sub-minute kinetics of human red blood cell fumarase using nuclear magnetic resonance (NMR) spectroscopy, and to provide a quantitative description of the enzyme that is relevant to the use of fumarate as a probe of cell rupture. The fumarase reaction was studied using time courses of 1 H spin-echo and 13 C-NMR spectra. 1 H-NMR experiments showed that the fumarase reaction in hemolysates is sufficiently rapid to make its kinetics amenable to study in a period of approximately 3 min, a timescale characteristic of hyperpolarized 13 C-NMR spectroscopy. The rapid-dissolution dynamic nuclear polarization (RD-DNP) technique was used to hyperpolarize [1,4-13 C]fumarate, which was injected into concentrated hemolysates. The kinetic data were analyzed using recently developed FmRα analysis and modeling of the enzymatic reaction using Michaelis-Menten equations. In RD-DNP experiments, the decline in the 13 C-NMR signal from fumarate, and the concurrent rise and fall of that from malate, were captured with high spectral resolution and signal-to-noise ratio, which allowed the robust quantification of fumarase kinetics. The kinetic parameters obtained indicate the potential contribution of hemolysis to the overall rate of the fumarase reaction when 13 C-NMR RD-DNP is used to detect necrosis in animal models of implanted tumors. The analytical procedures developed will be applicable to studies of other rapid enzymatic reactions using conventional and hyperpolarized substrate NMR spectroscopy.Item Open Access Accepted version Peer-reviewed The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.(Springer Nature, 2018-01-22) Monzón-Casanova, Elisa; Screen, Michael; Díaz-Muñoz, Manuel D; Coulson, Richard MR; Bell, Sarah E; Lamers, Greta; Solimena, Michele; Smith, Christopher WJ; Turner, Martin; Bell, Sarah Elizabeth [0000-0002-3249-707X]; Smith, Chris [0000-0002-2753-3398]; Turner, Martin [0000-0002-3801-9896]Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.Item Open Access Accepted version Peer-reviewed Characterization of a 5-HT3-ELIC Chimera Revealing the Sites of Action of Modulators.(American Chemical Society (ACS), 2018-06-20) Price, Kerry L; Lummis, Sarah CR; Lummis, Sarah [0000-0001-9410-9805]Cys-loop receptors are major sites of action for many important therapeutically active compounds, but the sites of action of those that do not act at the orthosteric binding site or at the pore are mostly poorly understood. To help understand these, we here describe a chimeric receptor consisting of the extracellular domain of the 5-HT3A receptor and the transmembrane domain of a prokaryotic homologue, ELIC. Alterations of some residues at the coupling interface are required for function, but the resulting receptor expresses well and responds to 5-HT with a lower EC50 (0.34 μM) than that of the 5-HT3A receptor. Partial agonists and competitive antagonists of the 5-HT3A receptor activate and inhibit the chimera as expected. Examination of a range of receptor modulators, including ethanol, thymol, 5-hydroxyindole, and 5-chloroindole, which can affect the 5-HT3A receptor and ELIC, suggest that these compounds act via the transmembrane domain, except for 5-hydroxyindole, which can compete with 5-HT at the orthosteric binding site. The data throw further light on the importance of coupling interface in Cys-loop receptors and provide a platform for examining the mechanism of action of compounds that act in the extracellular domain of the 5-HT3A receptor and the transmembrane domain of ELIC.Item Open Access Published version Peer-reviewed FRET-enhanced photostability allows improved single-molecule tracking of proteins and protein complexes in live mammalian cells.(Springer Science and Business Media LLC, 2018-06-28) Basu, Srinjan; Needham, Lisa-Maria; Lando, David; Taylor, Edward JR; Wohlfahrt, Kai J; Shah, Devina; Boucher, Wayne; Tan, Yi Lei; Bates, Lawrence E; Tkachenko, Olga; Cramard, Julie; Lagerholm, B Christoffer; Eggeling, Christian; Hendrich, Brian; Klenerman, Dave; Lee, Steven F; Laue, Ernest D; Lando, David [0000-0001-5783-8769]; Bates, Lawrence E [0000-0002-2675-309X]; Hendrich, Brian [0000-0002-0231-3073]; Laue, Ernest D [0000-0002-7476-4148]A major challenge in single-molecule imaging is tracking the dynamics of proteins or complexes for long periods of time in the dense environments found in living cells. Here, we introduce the concept of using FRET to enhance the photophysical properties of photo-modulatable (PM) fluorophores commonly used in such studies. By developing novel single-molecule FRET pairs, consisting of a PM donor fluorophore (either mEos3.2 or PA-JF549) next to a photostable acceptor dye JF646, we demonstrate that FRET competes with normal photobleaching kinetic pathways to increase the photostability of both donor fluorophores. This effect was further enhanced using a triplet-state quencher. Our approach allows us to significantly improve single-molecule tracking of chromatin-binding proteins in live mammalian cells. In addition, it provides a novel way to track the localization and dynamics of protein complexes by labeling one protein with the PM donor and its interaction partner with the acceptor dye.Item Open Access Published version Peer-reviewed Ciprofloxacin binding to GyrA causes global changes in the proteome of Pseudomonas aeruginosa.(Oxford University Press (OUP), 2018-07-01) Jedrey, Hannah; Lilley, Kathryn S; Welch, Martin; Lilley, Kathryn [0000-0003-0594-6543]; Welch, Martin [0000-0003-3646-1733]Ciprofloxacin is one of the most widely-used antibiotics, and has proven especially effective at controlling infections associated with the opportunistic human pathogen, Pseudomonas aeruginosa. In this work, we show that sub-inhibitory concentrations of ciprofloxacin induce discrete changes in the intracellular proteome. Central metabolism and cell envelope-associated functions are particularly affected. In spite of the low magnitude of the intracellular proteomic changes, we found that sub-lethal concentrations of ciprofloxacin had substantial effects on motility and exoprotein secretion. Crucially, the proteomic and phenotypic modulations that we observed were absolutely dependent upon the presence of wild-type GyrA; an isogenic strain of P. aeruginosa carrying a ciprofloxacin-insensitive form of GyrA (a T83→I mutant) did not display ciprofloxacin-dependent changes unless complemented with wild-type gyrA in trans. These results show that the diverse effects of sub-inhibitory ciprofloxacin on the cell are routed through its primary target in the cell, DNA gyrase.Item Open Access Published version Peer-reviewed Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males.(Elsevier BV, 2018-06-01) Sousa, Elsa J; Stuart, Hannah T; Bates, Lawrence E; Ghorbani, Mohammadmersad; Nichols, Jennifer; Dietmann, Sabine; Silva, José CR; Bates, Lawrence [0000-0002-2675-309X]; Nichols, Jennifer [0000-0002-8650-1388]; Rebelo Da Silva, Jose [0000-0001-5487-1117]A hallmark of naive pluripotency is the presence of two active X chromosomes in females. It is not clear whether prevention of X chromosome inactivation (XCI) is mediated by gene networks that preserve the naive state. Here, we show that robust naive pluripotent stem cell (nPSC) self-renewal represses expression of Xist, the master regulator of XCI. We found that nPSCs accumulate Xist on the male X chromosome and on both female X chromosomes as they become NANOG negative at the onset of differentiation. This is accompanied by the appearance of a repressive chromatin signature and partial X-linked gene silencing, suggesting a transient and rapid XCI-like state in male nPSCs. In the embryo, Xist is transiently expressed in males and in females from both X chromosomes at the onset of naive epiblast differentiation. In conclusion, we propose that XCI initiation is gender independent and triggered by destabilization of naive identity, suggesting that gender-specific mechanisms follow, rather than precede, XCI initiation.Item Open Access Published version Peer-reviewed Detection of functional protein domains by unbiased genome-wide forward genetic screening.(Springer Science and Business Media LLC, 2018-04-18) Herzog, Mareike; Puddu, Fabio; Coates, Julia; Geisler, Nicola; Forment, Josep V; Jackson, Stephen P; Puddu, Fabio [0000-0002-2033-5209]; Jackson, Stephen [0000-0001-9317-7937]Establishing genetic and chemo-genetic interactions has played key roles in elucidating mechanisms by which certain chemicals perturb cellular functions. In contrast to gene disruption/depletion strategies to identify mechanisms of drug resistance, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations has been limited. Here, by demonstrating its utility in identifying chemical-genetic suppressors of sensitivity to the DNA topoisomerase I poison camptothecin or the poly(ADP-ribose) polymerase inhibitor olaparib, we detail an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast or haploid mammalian cells in a short timeframe, and with potential applications in other haploid systems. In addition to applications in molecular biology research, this protocol can be used to identify drug targets and predict drug-resistance mechanisms. Mapping suppressor mutations on the primary or tertiary structures of protein suppressor hits provides insights into functionally relevant protein domains. Importantly, we show that olaparib resistance is linked to missense mutations in the DNA binding regions of PARP1, but not in its catalytic domain. This provides experimental support to the concept of PARP1 trapping on DNA as the prime source of toxicity to PARP inhibitors, and points to a novel olaparib resistance mechanism with potential therapeutic implications.Item Open Access Accepted version Peer-reviewed Electrophysiological stimulation of excised rat muscle elicits a measurable change in tissue sodium concentration using 23Na-MRI(Wiley, 2018-06-01) Riemer, F; Kaggie, Josh; O'Neill, Cormac; McLean, Mary; Grist, James; Hill, Myfanwy; Guy, Joe; Schulte, Rolf; Graves, Martin; Fraser, James; Gallagher, Ferdia; Riemer, Frank [0000-0002-3805-5221]; Kaggie, Joshua [0000-0001-6706-3442]; McLean, Mary [0000-0002-3752-0179]; Grist, James [0000-0001-7223-4031]; Graves, Martin [0000-0003-4327-3052]; Fraser, James [0000-0002-6505-1883]; Gallagher, Ferdia [0000-0003-4784-5230]Changes in the tissue sodium gradient play an important role in cell signalling such as at the neuromuscular junction and as part of neuronal action potentials. 23Na-MRI has the ability to measure the macroscopic sodium distribution. In this study we investigated the changes in tissue sodium in an electrically stimulated and freshly excised rat leg muscle.Item Open Access Published version Peer-reviewed SCNS: a graphical tool for reconstructing executable regulatory networks from single-cell genomic data.(BioMed Central, 2018-05-25) Woodhouse, Steven; Piterman, Nir; Wintersteiger, Christoph M; Gottgens, Berthold; Fisher, Jasmin; Wintersteiger, Christoph M [0000-0003-0102-4381]; Göttgens, Berthold [0000-0001-6302-5705]; Fisher, Yasmin [0000-0003-4477-9047]Background Reconstruction of executable mechanistic models from single-cell gene expression data represents a powerful approach to understanding developmental and disease processes. New ambitious efforts like the Human Cell Atlas will soon lead to an explosion of data with potential for uncovering and understanding the regulatory networks which underlie the behaviour of all human cells. In order to take advantage of this data, however, there is a need for general-purpose, user-friendly and efficient computational tools that can be readily used by biologists who do not have specialist computer science knowledge. Results The Single Cell Network Synthesis toolkit (SCNS) is a general-purpose computational tool for the reconstruction and analysis of executable models from single-cell gene expression data. Through a graphical user interface, SCNS takes single-cell qPCR or RNA-sequencing data taken across a time course, and searches for logical rules that drive transitions from early cell states towards late cell states. Because the resulting reconstructed models are executable, they can be used to make predictions about the effect of specific gene perturbations on the generation of specific lineages. Conclusions SCNS should be of broad interest to the growing number of researchers working in single-cell genomics and will help further facilitate the generation of valuable mechanistic insights into developmental, homeostatic and disease processes.Item Open Access Published version Peer-reviewed DynaMut: predicting the impact of mutations on protein conformation, flexibility and stability.(Oxford University Press (OUP), 2018-07-02) Rodrigues, Carlos Hm; Pires, Douglas Ev; Ascher, David B; Ascher, David [0000-0003-2948-2413]Proteins are highly dynamic molecules, whose function is intrinsically linked to their molecular motions. Despite the pivotal role of protein dynamics, their computational simulation cost has led to most structure-based approaches for assessing the impact of mutations on protein structure and function relying upon static structures. Here we present DynaMut, a web server implementing two distinct, well established normal mode approaches, which can be used to analyze and visualize protein dynamics by sampling conformations and assess the impact of mutations on protein dynamics and stability resulting from vibrational entropy changes. DynaMut integrates our graph-based signatures along with normal mode dynamics to generate a consensus prediction of the impact of a mutation on protein stability. We demonstrate our approach outperforms alternative approaches to predict the effects of mutations on protein stability and flexibility (P-value < 0.001), achieving a correlation of up to 0.70 on blind tests. DynaMut also provides a comprehensive suite for protein motion and flexibility analysis and visualization via a freely available, user friendly web server at http://biosig.unimelb.edu.au/dynamut/.Item Open Access Published version Peer-reviewed Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii.(Public Library of Science (PLoS), 2018-02) Biddau, Marco; Bouchut, Anne; Major, Jack; Saveria, Tracy; Tottey, Julie; Oka, Ojore; van-Lith, Marcel; Jennings, Katherine Elizabeth; Ovciarikova, Jana; DeRocher, Amy; Striepen, Boris; Waller, Ross Frederick; Parsons, Marilyn; Sheiner, Lilach; Biddau, Marco [0000-0002-9992-0418]; Tottey, Julie [0000-0001-6500-1117]; Oka, Ojore [0000-0003-3482-115X]; Jennings, Katherine Elizabeth [0000-0003-2881-7306]; DeRocher, Amy [0000-0003-1205-7920]; Waller, Ross Frederick [0000-0001-6961-9344]; Parsons, Marilyn [0000-0003-3994-8096]; Sheiner, Lilach [0000-0001-5909-2307]Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites.Item Open Access Published version Peer-reviewed Validating the concept of mutational signatures with isogenic cell models.(Springer Science and Business Media LLC, 2018-05-01) Zou, Xueqing; Owusu, Michel; Harris, Rebecca; Jackson, Stephen P; Loizou, Joanna I; Nik-Zainal, Serena; Zou, Xueqing [0000-0003-1143-1028]; Jackson, Stephen [0000-0001-9317-7937]; Nik-Zainal, Serena [0000-0001-5054-1727]The diversity of somatic mutations in human cancers can be decomposed into individual mutational signatures, patterns of mutagenesis that arise because of DNA damage and DNA repair processes that have occurred in cells as they evolved towards malignancy. Correlations between mutational signatures and environmental exposures, enzymatic activities and genetic defects have been described, but human cancers are not ideal experimental systems-the exposures to different mutational processes in a patient's lifetime are uncontrolled and any relationships observed can only be described as an association. Here, we demonstrate the proof-of-principle that it is possible to recreate cancer mutational signatures in vitro using CRISPR-Cas9-based gene-editing experiments in an isogenic human-cell system. We provide experimental and algorithmic methods to discover mutational signatures generated under highly experimentally-controlled conditions. Our in vitro findings strikingly recapitulate in vivo observations of cancer data, fundamentally validating the concept of (particularly) endogenously-arising mutational signatures.Item Open Access Accepted version Peer-reviewed RNase E and the High-Fidelity Orchestration of RNA Metabolism.(American Society for Microbiology, 2018-04) Bandyra, Katarzyna J; Luisi, Ben F; Bandyra, Katarzyna [0000-0003-2607-6700]; Luisi, Ben [0000-0003-1144-9877]The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced. Early in its evolutionary history, RNase E acquired a natively unfolded appendage that recruits accessory proteins and RNA. These accessory factors facilitate the activity of RNase E and include helicases that remodel RNA and RNA-protein complexes, and polynucleotide phosphorylase, a relative of the archaeal and eukaryotic exosomes. RNase E also associates with enzymes from central metabolism, such as enolase and aconitase. RNase E-based complexes are diverse in composition, but generally bear mechanistic parallels with eukaryotic machinery involved in RNA-induced gene regulation and transcript quality control. That these similar processes arose independently underscores the universality of RNA-based regulation in life. Here we provide a synopsis and perspective of the contributions made by RNase E to sustain robust gene regulation with speed and accuracy.Item Open Access Published version Peer-reviewed Trans-acting translational regulatory RNA binding proteins.(Wiley, 2018-05) Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner ML; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E; Smith, Tom [0000-0002-0697-8777]; Lilley, Kathryn [0000-0003-0594-6543]; Willis, Anne [0000-0002-1470-8531]The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms.Item Open Access Accepted version Peer-reviewed Analysis of a novel pncA mutation for susceptibility to Pyrazinamide therapy(American Thoracic Society, 2018-08-15) Karmakar; Globan; Fyfe; Stinear; Johnson; Holmes; Denholm; Ascher, DB; Ascher, David [0000-0003-2948-2413]Pyrazinamide (PZA), which is an analogue of nicotinamide, is an important first-line drug used in the short-course treatment of tuberculosis. PZA is a prodrug devoid of significant antibacterial activity. It is metabolized into its active form, pyrazinoic acid (POA), by the amidase activity of the Mycobacterium tuberculosis nicotinamidase/pyrazinamidase, encoded by the pncA gene. Mutations in pncA that prevent activation of the prodrug represent the major mechanism of PZA resistance in M. tuberculosis [1]. This antibiotic plays a key role in shortening the duration of anti-tuberculous treatment due to its activity against the persisting tubercle bacilli at acidic pH.Item Open Access Published version Peer-reviewed Targeting and Recognition of Toll-Like Receptors by Plant and Pathogen Lectins.(Frontiers Media SA, 2017) Ricci-Azevedo, Rafael; Roque-Barreira, Maria-Cristina; Gay, Nicholas J; Gay, Nicholas [0000-0002-2782-7169]We have reported that some lectins act as agonists of toll-like receptors (TLRs) and have immunomodulatory properties. The plant lectin ArtinM, for example, interacts with N-glycans of TLR2, whereas other lectins of microbial origin interact with TLR2 and TLR4. Expression of the receptors on the surface of antigen-presenting cells exposes N-glycans that may be targeted by lectins of different structures, specificities, and origins. In vitro, these interactions trigger cell signaling that leads to NF-κB activation and production of the Th1 polarizing cytokine IL-12. In vivo, a same sequence of events follows the administration of an active lectin to mice infected with an intracellular pathogen, conferring resistance to the pathogen. The lectins of the human pathogens Toxoplasma gondii (TgMIC1 and TgMIC4) and Paracoccidioides brasiliensis (Paracoccin), by recognition and activation of TLR2 and TLR4, induce cell events and in vivo effects comparable to the promoted by the plant lectin ArtinM. In this article, we highlight these two distinct mechanisms for activating antigen-presenting cells. On the one hand, TLRs act as sensors for the presence of conventional pathogen-associated molecular patterns, such as microbial lipids. On the other hand, we showed that TLR-mediated cell activation might be triggered by an alternative way, in which lectins bind to TLRs N-glycans and stimulate cells to increase the expression of pro-inflammatory cytokines. This process may lead to the development of new pharmaceutical tools that promote protective immune responses directed against intracellular pathogens and tumors.Item Open Access Accepted version Peer-reviewed Lipidomics Profiling of Human Adipose Tissue Identifies a Pattern of Lipids Associated with Fish Oil Supplementation.(American Chemical Society (ACS), 2017-09-01) Stanley, Elizabeth G; Jenkins, Benjamin J; Walker, Celia G; Koulman, Albert; Browning, Lucy; West, Annette L; Calder, Philip C; Jebb, Susan A; Griffin, Julian L; Griffin, Julian L [0000-0003-1336-7744]To understand the interaction between diet and health, biomarkers that accurately reflect consumption of foods of perceived health relevance are needed. The aim of this investigation was to use direct infusion-mass spectrometry (DI-MS) lipidomics to determine the effects of fish oil supplementation on lipid profiles of human adipose tissue. Adipose tissue samples from an n-3 polyunsaturated fatty acid (PUFA) supplementation study (n = 66) were analyzed to compare the pattern following supplementation equivalent to zero or four portions of oily fish per week. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were incorporated into highly unsaturated (≥5 double bonds) triglycerides (TGs), phosphocholines, and phosphoethanolamines as well as being detected directly as the nonesterified fatty acid forms. Multivariate statistics demonstrated that phospholipids were the most accurate and sensitive lipids for the assessing EPA and DHA incorporation into adipose tissue. Potential confounding factors (adiposity, age, and sex of the subject) were also considered in the analysis, and adiposity was also associated with an increase in highly unsaturated TGs as a result of incorporation of the n-6 PUFA arachidonic acid. DI-MS provides a high-throughput analysis of fatty acid status that can monitor oily fish consumption, suitable for use in cohort studies.