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BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.
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Comportamento de Doença , Malária Cerebral , Camundongos , Animais , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Inibidores do Fator de Necrose Tumoral , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Neurônios/metabolismo , Transdução de SinaisRESUMO
The National Institute of Allergy and Infectious Diseases (NIAID) established the Bioinformatics Resource Center (BRC) program to assist researchers with analyzing the growing body of genome sequence and other omics-related data. In this report, we describe the merger of the PAThosystems Resource Integration Center (PATRIC), the Influenza Research Database (IRD) and the Virus Pathogen Database and Analysis Resource (ViPR) BRCs to form the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) https://www.bv-brc.org/. The combined BV-BRC leverages the functionality of the bacterial and viral resources to provide a unified data model, enhanced web-based visualization and analysis tools, bioinformatics services, and a powerful suite of command line tools that benefit the bacterial and viral research communities.
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Genômica , Software , Vírus , Humanos , Bactérias/genética , Biologia Computacional , Bases de Dados Genéticas , Influenza Humana , Vírus/genéticaRESUMO
Ultrahigh resolution mass spectrometry (UHR-MS) coupled with direct infusion (DI) electrospray ionization offers a fast solution for accurate untargeted profiling. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers have been shown to produce a wealth of insights into complex chemical systems because they enable unambiguous molecular formula assignment even if the vast majority of signals is of unknown identity. Interlaboratory comparisons are required to apply this type of instrumentation in quality control (for food industry or pharmaceuticals), large-scale environmental studies, or clinical diagnostics. Extended comparisons employing different FT-ICR MS instruments with qualitative direct infusion analysis are scarce since the majority of detected compounds cannot be quantified. The extent to which observations can be reproduced by different laboratories remains unknown. We set up a preliminary study which encompassed a set of 17 laboratories around the globe, diverse in instrumental characteristics and applications, to analyze the same sets of extracts from commercially available standard human blood plasma and Standard Reference Material (SRM) for blood plasma (SRM1950), which were delivered at different dilutions or spiked with different concentrations of pesticides. The aim of this study was to assess the extent to which the outputs of differently tuned FT-ICR mass spectrometers, with different technical specifications, are comparable for setting the frames of a future DI-FT-ICR MS ring trial. We concluded that a cluster of five laboratories, with diverse instrumental characteristics, showed comparable and representative performance across all experiments, setting a reference to be used in a future ring trial on blood plasma.
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The Natriuretic Peptide Receptors (NPRs) regulate vascular sodium levels and have been of significant interest for the potential treatment of hypertension and related cardiovascular complications. The peptidomimetic antagonist M372049 is a valuable probe for the study of NPR-C signaling, unfortunately it is presently not commercially available. Described is a detailed protocol for its synthesis that does not require specialized apparatus and builds upon a prior patent from Veale and colleagues. Key steps include a base-mediated lactam formation and a solid-supported peptide synthetic sequence. An X-ray crystal structure of a key lactam intermediate was obtained to confirm the structure and relative stereochemistry of the compound.
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A set of novel Kv7.2/7.3 (KCNQ2/3) channel blockers was synthesized to address several liabilities of the known compounds XE991 (metabolic instability and CYP inhibition) and the clinical compound DMP 543 (acid instability, insolubility, and lipophilicity). Using the anthrone scaffold of the prior channel blockers, alternative heteroarylmethyl substituents were installed via enolate alkylation reactions. Incorporation of a pyridazine and a fluorinated pyridine gave an analog (compound 18, JDP-107) with a promising combination of potency (IC50â¯=â¯0.16⯵M in a Kv7.2 thallium flux assay), efficacy in a Kv7.2/7.3 patch clamp assay, and drug-like properties.
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Antracenos/farmacologia , Canal de Potássio KCNQ2/antagonistas & inibidores , Canal de Potássio KCNQ3/antagonistas & inibidores , Transtornos Mentais/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacologia , Antracenos/síntese química , Antracenos/química , Relação Dose-Resposta a Droga , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Estrutura Molecular , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Relação Estrutura-AtividadeRESUMO
In the present work, the advantages of ESI-TIMS-FT-ICR MS to address the isomeric content of dissolved organic matter are studied. While the MS spectra allowed the observation of a high number of peaks (e.g., PAN-L: 5004 and PAN-S: 4660), over 4× features were observed in the IMS-MS domain (e.g., PAN-L: 22 015 and PAN-S: 20 954). Assuming a total general formula of CxHyN0-3O0-19S0-1, 3066 and 2830 chemical assignments were made in a single infusion experiment for PAN-L and PAN-S, respectively. Most of the identified chemical compounds (â¼80%) corresponded to highly conjugated oxygen compounds (O1-O20). ESI-TIMS-FT-ICR MS provided a lower estimate of the number of structural and conformational isomers (e.g., an average of 6-10 isomers per chemical formula were observed). Moreover, ESI-q-FT-ICR MS/MS at the level of nominal mass (i.e., 1 Da isolation) allowed for further estimation of the number of isomers based on unique fragmentation patterns and core fragments; the later suggested that multiple structural isomers could have very closely related CCS. These studies demonstrate the need for ultrahigh resolution TIMS mobility scan functions (e.g., R = 200-500) in addition to tandem MS/MS isolation strategies.
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BACKGROUND: Topologically associating domains (TADs) are considered the structural and functional units of the genome. However, there is a lack of an integrated resource for TADs in the literature where researchers can obtain family classifications and detailed information about TADs. RESULTS: We built an online knowledge base TADKB integrating knowledge for TADs in eleven cell types of human and mouse. For each TAD, TADKB provides the predicted three-dimensional (3D) structures of chromosomes and TADs, and detailed annotations about the protein-coding genes and long non-coding RNAs (lncRNAs) existent in each TAD. Besides the 3D chromosomal structures inferred by population Hi-C, the single-cell haplotype-resolved chromosomal 3D structures of 17 GM12878 cells are also integrated in TADKB. A user can submit query gene/lncRNA ID/sequence to search for the TAD(s) that contain(s) the query gene or lncRNA. We also classified TADs into families. To achieve that, we used the TM-scores between reconstructed 3D structures of TADs as structural similarities and the Pearson's correlation coefficients between the fold enrichment of chromatin states as functional similarities. All of the TADs in one cell type were clustered based on structural and functional similarities respectively using the spectral clustering algorithm with various predefined numbers of clusters. We have compared the overlapping TADs from structural and functional clusters and found that most of the TADs in the functional clusters with depleted chromatin states are clustered into one or two structural clusters. This novel finding indicates a connection between the 3D structures of TADs and their DNA functions in terms of chromatin states. CONCLUSION: TADKB is available at http://dna.cs.miami.edu/TADKB/ .
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Montagem e Desmontagem da Cromatina , Cromossomos de Mamíferos , Regulação da Expressão Gênica , Genoma , Genômica/métodos , Bases de Conhecimento , Algoritmos , Animais , Humanos , Camundongos , Família Multigênica , RNA Longo não CodificanteRESUMO
RATIONALE: DNA quadruplex structures have emerged as novel drug targets due to their role in preventing abnormal gene transcription and maintaining telomere stability. Trapped Ion Mobility Spectrometry-Mass Spectrometry (TIMS-MS), combined with theoretical modeling, is a powerful tool for studying the kinetic intermediates of DNA complexes formed in solution and interrogated in the gas phase after desolvation. METHODS: A TAGGGT ssDNA sequence was purchased and studied in 10 mM ammonium acetate using nanospray electrospray ionization (nESI)-TIMS-MS in positive and negative ion mode. Collisional cross section (CCS) profiles were measured using internal calibration (Tune Mix). Theoretical structures were proposed based on molecular dynamics, charge location and geometry optimization for the most intense IMS bands based on the number of TAGGGT units, adduct form and charge states. RESULTS: A distribution of monomeric, dimeric and tetrameric TAGGGT structures were formed in solution and separated in the gas phase based on their mobility and m/z value (e.g., [M + 2H]+2 , [2M + 3H]+3 , [M - 2H]-2 , [2M - 3H]-3 , [4M + 4H]+4 , [4M + 3H + NH4 ]+4 , [4M + 2H + 2NH4 ]+4 and [4M + H + 3NH4 ]+4 ). The high mobility resolution of the TIMS-MS analyzer permitted the observation of multiple CCS bands per molecular ion form. Comparison with theoretical candidate structures suggests that monomeric TAGGGT species are stabilized by A-T and G+ -G interactions, with the size of the conformer influenced by the proton location. In the case of the TAGGGT quadruplex, the protonated species displayed a broad CCS distribution, while six discrete conformers were stabilized by the presence of ammonium ions (n = 1-3). CONCLUSIONS: This is the first observation of multiple conformations of TAGGGT complexes (n = 1, 2 and 4) in 10 mM ammonium acetate. Candidate structures with intramolecular interactions of the form of G+ -G and traditional A-T base pairing agreed with the experimental trends. Our results demonstrate the structural diversity of TAGGGT monomers, dimers and tetramers in the gas phase beyond the previously reported solution structure, using 10 mM ammonium acetate to replicate biological conditions.
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DNA de Cadeia Simples/química , Quadruplex G , Dimerização , Espectrometria de Mobilidade Iônica , Simulação de Dinâmica Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Comprehensive characterization of proteomes comprising the same proteins with distinct post-translational modifications (PTMs) is a staggering challenge. Many such proteoforms are isomers (localization variants) that require separation followed by top-down or middle-down mass spectrometric analyses, but condensed-phase separations are ineffective in those size ranges. The variants for "middle-down" peptides were resolved by differential ion mobility spectrometry (FAIMS), relying on the mobility increment at high electric fields, but not previously by linear IMS on the basis of absolute mobility. We now use complete histone tails with diverse PTMs on alternative sites to demonstrate that high-resolution linear IMS, here trapped IMS (TIMS), broadly resolves the variants of â¼50 residues in full or into binary mixtures quantifiable by tandem MS, largely thanks to orthogonal separations across charge states. Separations using traveling-wave (TWIMS) and/or involving various time scales and electrospray ionization source conditions are similar (with lower resolution for TWIMS), showing the transferability of results across linear IMS instruments. The linear IMS and FAIMS dimensions are substantially orthogonal, suggesting FAIMS/IMS/MS as a powerful platform for proteoform analyses.
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Histonas/isolamento & purificação , Peptídeos/isolamento & purificação , Proteoma/isolamento & purificação , Histonas/química , Histonas/metabolismo , Espectrometria de Massas , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/química , Proteoma/metabolismoRESUMO
In this work, nonlinear, stepping analytical mobility scan functions are implemented to increase the analytical separation and duty cycle during tandem Trapped Ion Mobility Spectrometry and FT-ICR MS operation. The differences between linear and stepping scan functions are described based on length of analysis, mobility scan rate, signal-to-noise, and mobility resolving power. Results showed that for the linear mobility scan function only a small fraction of the scan is sampled, resulting in the lowest duty cycle 0.5% and longest experiment times. Implementing nonlinear targeted scan functions for analysis of known mobilities resulted in increased duty cycle (0.85%) and resolving powers (R up to 300) with a 6-fold reduction in time from 30 to 5 min. For broad range characterization, a nonlinear mobility stepping scan function provided the best sensitivity, resolving power, duty cycle (4%), and points per peak. The applicability of nonlinear mobility scan functions for the analysis of complex mixtures is illustrated for the case of a direct infusion of a MCF-7 breast cancer cell digest, where isobaric peptides (e.g., DFTPAELR and TTILQSTGK) were separated in the mobility domain (RIMS: 110) and identified based on their CCS, accurate mass (RMS: 550k), and tandem MS using IRMPD in the ICR cell.
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Despite often minute concentrations in vivo, d-amino acid containing peptides (DAACPs) are crucial to many life processes. Standard proteomics protocols fail to detect them as d/l substitutions do not affect the peptide parent and fragment masses. The differences in fragment yields are often limited, obstructing the investigations of important but low abundance epimers in isomeric mixtures. Separation of d/l-peptides using ion mobility spectrometry (IMS) was impeded by small collision cross section differences (commonly â¼1%). Here, broad baseline separation of DAACPs with up to â¼30 residues employing trapped IMS with resolving power up to â¼340, followed by time-of-flight mass spectrometry is demonstrated. The d/l-pairs coeluting in one charge state were resolved in another, and epimers merged as protonated species were resolved upon metalation, effectively turning the charge state and cationization mode into extra separation dimensions. Linear quantification down to 0.25% proved the utility of high resolution IMS-MS for real samples with large interisomeric dynamic range. Very close relative mobilities found for DAACP pairs using traveling-wave IMS (TWIMS) with different ion sources and faster IMS separations showed the transferability of results across IMS platforms. Fragmentation of epimers can enhance their identification and further improve detection and quantification limits, and we demonstrate the advantages of online mobility separated collision-induced dissociation (CID) followed by high resolution mass spectrometry (TIMS-CID-MS) for epimer analysis.
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Aminoácidos/química , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Peptídeos/química , Peptídeos/isolamento & purificação , Prótons , Estereoisomerismo , Fatores de TempoRESUMO
BACKGROUND: DNA methylation is an important epigenetic mark relevant to normal development and disease genesis. A common approach to characterizing genome-wide DNA methylation is using Next Generation Sequencing technology to sequence bisulfite treated DNA. The short sequence reads are mapped to the reference genome to determine the methylation statuses of Cs. However, despite intense effort, a much smaller proportion of the reads derived from bisulfite treated DNA (usually about 40-80%) can be mapped than regular short reads mapping (> 90%), and it is unclear what factors lead to this low mapping efficiency. RESULTS: To address this issue, we used the hairpin bisulfite sequencing technology to determine sequences of both DNA double strands simultaneously. This enabled the recovery of the original non-bisulfite-converted sequences. We used Bismark for bisulfite read mapping and Bowtie2 for recovered read mapping. We found that recovering the reads improved unique mapping efficiency by 9-10% compared to the bisulfite reads. Such improvement in mapping efficiency is related to sequence entropy. CONCLUSIONS: The hairpin recovery technique improves mapping efficiency, and sequence entropy relates to mapping efficiency.
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Mapeamento Cromossômico/métodos , Sequências Repetidas Invertidas/genética , Análise de Sequência de DNA/métodos , Sulfitos/farmacologia , Sequência de Bases , Metilação de DNA/efeitos dos fármacos , Genômica , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Background. Large-scale bisulfite treatment and short reads sequencing technology allow comprehensive estimation of methylation states of Cs in the genomes of different tissues, cell types, and developmental stages. Accurate characterization of DNA methylation is essential for understanding genotype phenotype association, gene and environment interaction, diseases, and cancer. Aligning bisulfite short reads to a reference genome has been a challenging task. We compared five bisulfite short read mapping tools, BSMAP, Bismark, BS-Seeker, BiSS, and BRAT-BW, representing two classes of mapping algorithms (hash table and suffix/prefix tries). We examined their mapping efficiency (i.e., the percentage of reads that can be mapped to the genomes), usability, running time, and effects of changing default parameter settings using both real and simulated reads. We also investigated how preprocessing data might affect mapping efficiency. Conclusion. Among the five programs compared, in terms of mapping efficiency, Bismark performs the best on the real data, followed by BiSS, BSMAP, and finally BRAT-BW and BS-Seeker with very similar performance. If CPU time is not a constraint, Bismark is a good choice of program for mapping bisulfite treated short reads. Data quality impacts a great deal mapping efficiency. Although increasing the number of mismatches allowed can increase mapping efficiency, it not only significantly slows down the program, but also runs the risk of having increased false positives. Therefore, users should carefully set the related parameters depending on the quality of their sequencing data.