Publisher Correction: Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

In the version of this article initially published, the first affiliation lacked 'MRC'; the correct name of the institution is 'MRC Weatherall Institute of Molecular Medicine'. Two designations (SP110Y and ST110H) were incorrect in the legend to Fig. 6f,h,i. The correct text is as follows: for panel f, "...loaded with either the CdtB(105-125)SP110Y (DRB4*SP110Y) or the CdtB(105-125)ST110H (DRB4*ST110H) peptide variants..."; for panel h, "...decorated by the DRB4*SP110Y tetramer (lower-right quadrant), the DRB4*ST110H (upper-left quadrant)..."; and for panel i, "...stained ex vivo with DRB4*SP110Y, DRB4*ST110H...". In Fig. 8e, the final six residues (LTEAFF) of the sequence in the far right column of the third row of the table were missing; the correct sequence is 'CASSYRRTPPLTEAFF'. In the legend to Fig. 8d, a designation (HLyE) was incorrect; the correct text is as follows: "(HlyE?)." Portions of the Acknowledgements section were incorrect; the correct text is as follows: "This work was supported by the UK Medical Research Council (MRC) (MR/K021222/1) (G.N., M.A.G., A.S., V.C., A.J.P.),...the Oxford Biomedical Research Centre (A.J.P., V.C.),...and core funding from the Singapore Immunology Network (SIgN) (E.W.N.) and the SIgN immunomonitoring platform (E.W.N.)." Finally, a parenthetical element was phrased incorrectly in the final paragraph of the Methods subsection "T cell cloning and live fluorescence barcoding"; the correct phrasing is as follows: "...(which in all cases included HlyE, CdtB, Ty21a, Quailes, NVGH308, and LT2 strains and in volunteers T5 and T6 included PhoN)...". Also, in Figs. 3c and 4a, the right outlines of the plots were not visible; in the legend to Fig. 3, panel letter 'f' was not bold; and in Fig. 8f, 'ND' should be aligned directly beneath DRB4 in the key and 'ND' should be removed from the diagram at right, and the legend should be revised accordingly as follows: "...colors indicate the HLA class II restriction (gray indicates clones for which restriction was not determined (ND)). Clonotypes are grouped on the basis of pathogen selectivity (continuous line), protein specificity (dashed line) and epitope specificity; for ten HlyE-specific clones (pixilated squares), the epitope specificity was not determined...". The errors have been corrected in the HTML and PDF versions of the article.

Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.

Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial.

Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically 'cold' tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.

On the coupling of intracellular K + ${{\rm{K}}}

We have investigated the interplay between glycolytic oscillations and intracellular K + ${{\rm{K}}}^{+}$ concentration in the yeast Saccharomyces cerevisiae. Intracellular K + ${{\rm{K}}}^{+}$ concentration was measured using the fluorophore potassium-binding benzofuranisophthalate (PBFI). We found that K + ${{\rm{K}}}^{+}$ is an essential ion for the occurrence of glycolytic oscillations and that intracellular K + ${{\rm{K}}}^{+}$ concentration oscillates synchronously with other variables such as nicotinamide adenine dinucleotide hydride (NADH), intracellular adenosine triphosphate (ATP), and mitochondrial membrane potential. We also investigated if glycolysis and intracellular K + ${{\rm{K}}}^{+}$ concentration oscillate in a number of yeast strains with mutations in K + ${{\rm{K}}}^{+}$ transporters in the plasma membrane, mitochondrial membrane and in the vacuolar membrane. Most of these strains are still capable of showing glycolytic oscillations, but two strains are not: (i) a strain with a deletion in the mitochondrial Mdm38p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ transporter and (ii) a strain with deletion of the late endosomal Nhx1p K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ( Na + ∕ H + ${\text{Na}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ ) transporter. In these two mutant strains intracellular K + ${{\rm{K}}}^{+}$ concentration seems to be low, indicating that the two transporters may be involved in transport of K + ${{\rm{K}}}^{+}$ into the cytosol. In the strain, Mdm38p Δ ${\rm{\Delta }}$ oscillations in glycolysis could be restored by addition of the K + ∕ H + ${{\rm{K}}}^{+}\unicode{x02215}{{\rm{H}}}^{+}$ exchange ionophore nigericin. Furthermore, in two nonoscillating mutant strains with a defective V-ATPase and deletion of the Arp1p protein the intracellular K + ${{\rm{K}}}^{+}$ is relatively high, suggesting that the V-ATPase is essential for transport of K + ${{\rm{K}}}^{+}$ out of the cytosol and that the cytoskeleton may be involved in binding K + ${{\rm{K}}}^{+}$ to reduce the concentration of free ion in the cytosol. Analyses of the time series of oscillations of NADH, ATP, mitochondrial membrane potential, and potassium concentration using data-driven modeling corroborate the conjecture that K + ${{\rm{K}}}^{+}$ ion is essential for the emergence of oscillations and support the experimental findings using mutant strains.

Glycolysis inhibition affects proliferation and cytotoxicity of Vγ9Vδ2 T cells expanded for adoptive cell therapy.

BACKGROUND AIMS: Vγ9Vδ2 T cells are under investigation as alternative effector cells for adoptive cell therapy (ACT) in cancer. Despite promising in vitro results, anti-tumor efficacies in early clinical studies have been lower than expected, which could be ascribed to the complex interplay of tumor and immune cell metabolism competing for the same nutrients in the tumor microenvironment. METHODS: To contribute to the scarce knowledge regarding gamma delta T-cell metabolism, we investigated the metabolic phenotype of 25-day-expanded Vγ9Vδ2 T cells and how it is intertwined with functionality. RESULTS: We found that Vγ9Vδ2 T cells displayed a quiescent metabolism, utilizing both glycolysis and oxidative phosphorylation (OXPHOS) for energy production, as measured in Seahorse assays. Upon T-cell receptor activation, both pathways were upregulated, and inhibition with metabolic inhibitors showed that Vγ9Vδ2 T cells were dependent on glycolysis and the pentose phosphate pathway for proliferation. The dependency on glucose for proliferation was confirmed in glucose-free conditions. Cytotoxicity against malignant melanoma was reduced by glycolysis inhibition but not OXPHOS inhibition. CONCLUSIONS: These findings lay the groundwork for further studies on manipulation of Vγ9Vδ2 T-cell metabolism for improved ACT outcome.

JAK2V617F drives gut microbiota differences in patients with myeloproliferative neoplasms.

BACKGROUND: Essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (MF) are myeloproliferative neoplasms (MPN). Inflammation is involved in the initiation, progression, and symptomology of the diseases. The gut microbiota impacts the immune system, infection control, and steady-state hematopoiesis. METHODS: We analyzed the gut microbiota of 227 MPN patients and healthy controls (HCs) using next-generation sequencing. We expanded our previous results in PV and ET patients with additional PV, pre-MF, and MF patients which allowed us to compare MPN patients collectively, MPN sub-diagnoses, and MPN mutations (separately and combined) vs. HCs (N = 42) and compare within MPN sub-diagnoses and MPN mutation. RESULTS: MPN patients had a higher observed richness (median, 245 [range, 49-659]) compared with HCs (191.5 [range, 111-300; p = .003]) and a lower relative abundance of taxa within the Firmicutes phylum; for example, Faecalibacterium (6% vs. 14%, p < .001). The microbiota of CALR-positive patients (N = 30) resembled that of HCs more than that of patients with JAK2V617F (N = 177). In JAK2V617F-positive patients, only minor differences in the gut microbiota were observed between MPN sub-diagnoses, illustrating the importance of this mutation. CONCLUSION: The gut microbiota in MPN patients differs from HCs and is driven by JAK2V617F, whereas the gut microbiota in CALR patients resembles HCs more.

Expression and splicing mediate distinct biological signals.

BACKGROUND: Through alternative splicing, most human genes produce multiple isoforms in a cell-, tissue-, and disease-specific manner. Numerous studies show that alternative splicing is essential for development, diseases, and their treatments. Despite these important examples, the extent and biological relevance of splicing are currently unknown. RESULTS: To solve this problem, we developed pairedGSEA and used it to profile transcriptional changes in 100 representative RNA-seq datasets. Our systematic analysis demonstrates that changes in splicing, on average, contribute to 48.1% of the biological signal in expression analyses. Gene-set enrichment analysis furthermore indicates that expression and splicing both convey shared and distinct biological signals. CONCLUSIONS: These findings establish alternative splicing as a major regulator of the human condition and suggest that most contemporary RNA-seq studies likely miss out on critical biological insights. We anticipate our results will contribute to the transition from a gene-centric to an isoform-centric research paradigm.

Differential contributions of the proteasome, autophagy, and chaperones to the clearance of arsenite-induced protein aggregates in yeast.

The poisonous metalloid arsenite induces widespread misfolding and aggregation of nascent proteins in vivo, and this mode of toxic action might underlie its suspected role in the pathology of certain protein misfolding diseases. Evolutionarily conserved protein quality-control systems protect cells against arsenite-mediated proteotoxicity, and herein, we systematically assessed the contribution of the ubiquitin-proteasome system, the autophagy-vacuole pathway, and chaperone-mediated disaggregation to the clearance of arsenite-induced protein aggregates in Saccharomyces cerevisiae. We show that the ubiquitin-proteasome system is the main pathway that clears aggregates formed during arsenite stress and that cells depend on this pathway for optimal growth. The autophagy-vacuole pathway and chaperone-mediated disaggregation both contribute to clearance, but their roles appear less prominent than the ubiquitin-proteasome system. Our in vitro assays with purified components of the yeast disaggregating machinery demonstrated that chaperone binding to aggregates formed in the presence of arsenite is impaired. Hsp104 and Hsp70 chaperone activity was unaffected by arsenite, suggesting that this metalloid influences aggregate structure, making them less accessible for chaperone-mediated disaggregation. We further show that the defect in chaperone-mediated refolding of a model protein was abrogated in a cysteine-free version of the substrate, suggesting that arsenite directly modifies cysteines in non-native target proteins. In conclusion, our study sheds novel light on the differential contributions of protein quality-control systems to aggregate clearance and cell proliferation and extends our understanding of how these systems operate during arsenite stress.

Complex dynamics in an unexplored simple model of the peroxidase-oxidase reaction.

A previously overlooked version of the so-called Olsen model of the peroxidase-oxidase reaction has been studied numerically using 2D isospike stability and maximum Lyapunov exponent diagrams and reveals a rich variety of dynamic behaviors not observed before. The model has a complex bifurcation structure involving mixed-mode and bursting oscillations as well as quasiperiodic and chaotic dynamics. In addition, multiple periodic and non-periodic attractors coexist for the same parameters. For some parameter values, the model also reveals formation of mosaic patterns of complex dynamic states. The complex dynamic behaviors exhibited by this model are compared to those of another version of the same model, which has been studied in more detail. The two models show similarities, but also notable differences between them, e.g., the organization of mixed-mode oscillations in parameter space and the relative abundance of quasiperiodic and chaotic oscillations. In both models, domains with chaotic dynamics contain apparently disorganized subdomains of periodic attractors with dinoflagellate-like structures, while the domains with mainly quasiperiodic behavior contain subdomains with periodic attractors organized as regular filamentous structures. These periodic attractors seem to be organized according to Stern-Brocot arithmetics. Finally, it appears that toroidal (quasiperiodic) attractors develop into first wrinkled and then fractal tori before they break down to chaotic attractors.

Prospects of Improving Molecular Solar Energy Storage of the Norbornadiene/Quadricyclane System through Bridgehead Modifications.

We have investigated novel bicyclic diene molecular solar thermal energy storage systems that presently are the ones with the highest predicted energy density. Using a variety of different ab initio quantum chemical methods, we report storage energies, absorption spectra, and reaction barriers for the release of stored energy for a series of bicyclic dienes. The bicyclic dienes are all constructed by modifying the bridgehead of the well-known norbornadiene/quadricyclane (NBD/QC) system. In conclusion, we find it promising that it is possible to significantly amplify the storage energy of the NBD/QC system without seriously compromising other crucial properties by introducing simple modifications to the bridgehead.

Complexity in subnetworks of a peroxidase-oxidase reaction model.

The peroxidase-oxidase (PO) reaction is a paradigmatic (bio)chemical system well suited to study the organization and stability of self-sustained oscillatory phases typically present in nonlinear systems. The PO reaction can be simulated by the state-of-the-art Bronnikova-Fedkina-Schaffer-Olsen model involving ten coupled ordinary differential equations. The complex and dynamically rich distribution of self-sustained oscillatory stability phases of this model was recently investigated in detail. However, would it be possible to understand aspects of such a complex model using much simpler models? Here, we investigate stability phases predicted by three simple four-variable subnetworks derived from the complete model. While stability diagrams for such subnetworks are found to be distorted compared to those of the complete model, we find them to surprisingly preserve significant features of the original model as well as from the experimental system, e.g., period-doubling and period-adding scenarios. In addition, return maps obtained from the subnetworks look very similar to maps obtained in the experimental system under different conditions. Finally, two of the three subnetwork models are found to exhibit quint points, i.e., recently reported singular points where five distinct stability phases coalesce. We also provide experimental evidence that such quint points are present in the PO reaction.

TANTIGEN 2.0: a knowledge base of tumor T cell antigens and epitopes.

We previously developed TANTIGEN, a comprehensive online database cataloging more than 1000 T cell epitopes and HLA ligands from 292 tumor antigens. In TANTIGEN 2.0, we significantly expanded coverage in both immune response targets (T cell epitopes and HLA ligands) and tumor antigens. It catalogs 4,296 antigen variants from 403 unique tumor antigens and more than 1500 T cell epitopes and HLA ligands. We also included neoantigens, a class of tumor antigens generated through mutations resulting in new amino acid sequences in tumor antigens. TANTIGEN 2.0 contains validated TCR sequences specific for cognate T cell epitopes and tumor antigen gene/mRNA/protein expression information in major human cancers extracted by Human Pathology Atlas. TANTIGEN 2.0 is a rich data resource for tumor antigens and their associated epitopes and neoepitopes. It hosts a set of tailored data analytics tools tightly integrated with the data to form meaningful analysis workflows. It is freely available at http://projects.met-hilab.org/tadb .

Complexity of a peroxidase-oxidase reaction model.

The peroxidase-oxidase oscillating reaction was the first (bio)chemical reaction to show chaotic behaviour. The reaction is rich in bifurcation scenarios, from period-doubling to peak-adding mixed mode oscillations. Here, we study a state-of-the-art model of the peroxidase-oxidase reaction. Using the model, we report systematic numerical experiments exploring the impact of changing the enzyme concentration on the dynamics of the reaction. Specifically, we report high-resolution phase diagrams predicting and describing how the reaction unfolds over a quite extended range of enzyme concentrations. Surprisingly, such diagrams reveal that the enzyme concentration has a huge impact on the reaction evolution. The highly intricate dynamical behaviours predicted here are difficult to establish theoretically due to the total absence of an adequate framework to solve nonlinearly coupled differential equations. But such behaviours may be validated experimentally.

Chaos in the peroxidase-oxidase oscillator.

The peroxidase-oxidase (PO) reaction involves the oxidation of reduced nicotinamide adenine dinucleotide by molecular oxygen. When both reactants are supplied continuously to a reaction mixture containing the enzyme and a phenolic compound, the reaction will exhibit oscillatory behavior. In fact, the reaction exhibits a zoo of dynamical behaviors ranging from simple periodic oscillations to period-doubled and mixed mode oscillations to quasiperiodicity and chaos. The routes to chaos involve period-doubling, period-adding, and torus bifurcations. The dynamic behaviors in the experimental system can be simulated by detailed semiquantitative models. Previous models of the reaction have omitted the phenolic compound from the reaction scheme. In the current paper, we present new experimental results with the oscillating PO reaction that add to our understanding of its rich dynamics, and we describe a new variant of a previous model, which includes the chemistry of the phenol in the reaction mechanism. This new model can simulate most of the experimental behaviors of the experimental system including the new observations presented here. For example, the model reproduces the two main routes to chaos observed in experiments: (i) a period-doubling scenario, which takes place at low pH, and a period-adding scenario involving mixed mode oscillations (MMOs), which occurs at high pH. Our simulations suggest alternative explanations for the pH-sensitivity of the dynamics. We show that the MMO domains are separated by narrow parameter regions of chaotic behavior or quasiperiodicity. These regions start as tongues of secondary quasiperiodicity and develop into strange attractors through torus breakdown.

Some elements for a history of the dynamical systems theory.

Writing a history of a scientific theory is always difficult because it requires to focus on some key contributors and to "reconstruct" some supposed influences. In the 1970s, a new way of performing science under the name "chaos" emerged, combining the mathematics from the nonlinear dynamical systems theory and numerical simulations. To provide a direct testimony of how contributors can be influenced by other scientists or works, we here collected some writings about the early times of a few contributors to chaos theory. The purpose is to exhibit the diversity in the paths and to bring some elements-which were never published-illustrating the atmosphere of this period. Some peculiarities of chaos theory are also discussed.

SMARTCyp 3.0: enhanced cytochrome P450 site-of-metabolism prediction server.

MOTIVATION: Cytochromes P450 are the most important class of drug metabolizing enzymes. Prediction of drug metabolism is important in development of new drugs, to understand and reduce adverse drug reactions and to reduce animal testing. RESULTS: SMARTCyp 3.0 is an updated version of our previous web server for prediction of site-of-metabolism for Cytochrome P450-mediated metabolism, now in Python 3 with increased structural coverage and new features. The SMARTCyp program is a first principle-based method using density functional theory determined activation energies for more than 250 molecules to identify the most likely site-of-metabolism. New features include a similarity measure between the query molecule and the model fragment, a new graphical interface and additional parameters expanding the structural coverage of the SMARTCyp program. AVAILABILITY AND IMPLEMENTATION: The SMARTCyp server is freely available for use on the web at smartcyp.sund.ku.dk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Glycolytic oscillations and intracellular K+ concentration are strongly coupled in the yeast Saccharomyces cerevisiae.

We measured temporal oscillations of intracellular K+ concentration in yeast cells exhibiting glycolytic oscillations using fluorescence spectroscopy and microscopy methods. These oscillations showed the same period as those of glycolytic metabolites (NADH, ATP), indicating a strong coupling between them. We experimentally ruled out that oscillations originate in extra- or intracellular K+ fluxes and conclude that these oscillations arise from fluctuations in free and adsorbed states of K+ in the cell interior. Oscillations in K+ showed a strong dependence on ATP and the organization of the cell cytoskeleton. Our results challenge the widely held view that intracellular K+ predominantly exists in a free state. They can, however, be productively understood in terms of Gilbert Ling's Association-Induction hypothesis.

Discovery of Novel Non-Steroidal Cytochrome P450 17A1 Inhibitors as Potential Prostate Cancer Agents.

The current study presents the design, synthesis, and evaluation of novel cytochrome P450 17A1 (CYP17A1) ligands. CYP17A1 is a key enzyme in the steroidogenic pathway that produces androgens among other steroids, and it is implicated in prostate cancer. The obtained compounds are potent enzyme inhibitors (sub µM) with antiproliferative activity in prostate cancer cell lines. The binding mode of these compounds is also discussed.

BioReader: a text mining tool for performing classification of biomedical literature.

BACKGROUND: Scientific data and research results are being published at an unprecedented rate. Many database curators and researchers utilize data and information from the primary literature to populate databases, form hypotheses, or as the basis for analyses or validation of results. These efforts largely rely on manual literature surveys for collection of these data, and while querying the vast amounts of literature using keywords is enabled by repositories such as PubMed, filtering relevant articles from such query results can be a non-trivial and highly time consuming task. RESULTS: We here present a tool that enables users to perform classification of scientific literature by text mining-based classification of article abstracts. BioReader (Biomedical Research Article Distiller) is trained by uploading article corpora for two training categories - e.g. one positive and one negative for content of interest - as well as one corpus of abstracts to be classified and/or a search string to query PubMed for articles. The corpora are submitted as lists of PubMed IDs and the abstracts are automatically downloaded from PubMed, preprocessed, and the unclassified corpus is classified using the best performing classification algorithm out of ten implemented algorithms. CONCLUSION: BioReader supports data and information collection by implementing text mining-based classification of primary biomedical literature in a web interface, thus enabling curators and researchers to take advantage of the vast amounts of data and information in the published literature. BioReader outperforms existing tools with similar functionalities and expands the features used for mining literature in database curation efforts. The tool is freely available as a web service at http://www.cbs.dtu.dk/services/BioReader.