Explore the vast range of titles available.

In recent years, immune repertoire profiling with high-throughput sequencing (HTS) has advanced our understanding of adaptive immunity. However, fast progress in the field applied mostly to human and mouse research, with only few studies devoted to other model vertebrates. We present the first in-depth characterization of the T-cell receptor (TCR) repertoire in a non-model mammal (bank vole, Myodes glareolus ), widely used in ecological and evolutionary research. We used RNA from spleens, 5′RACE and HTS to describe V and J segments of TCRβ, qualitatively characterize preferential V–J segment usage and CDR3 length distribution. Overall orthology to murine genes was preserved, with 11 J and 37 V genes found in voles (although 3 V genes lacked a close orthologue). Further, we implemented unique molecular identifiers for quantitative analysis of CDR3 repertoire with stringent error correction. A conservative, lower bound estimation of the TCRβ repertoire was similar to that found for mice (1.7–2.3 × 10 5 clonotypes). We hope that by providing an easy-to-follow molecular protocol and on-line bioinformatics tools that do not require reference sequences (AmpliTCR and AmpliCDR3), we will encourage HTS immune repertoire profiling in other non-model vertebrates, thus opening new research avenues in e.g. comparative immunology, ecology and evolutionary biology.

Several challenges remain in data-independent acquisition (DIA) data analysis, such as to confidently identify peptides, define integration boundaries, remove interferences, and control false discovery rates. In practice, a visual inspection of the signals is still required, which is impractical with large datasets. We present Avant-garde as a tool to refine DIA (and parallel reaction monitoring) data. Avant-garde uses a novel data-driven scoring strategy: signals are refined by learning from the dataset itself, using all measurements in all samples to achieve the best optimization. We evaluate the performance of Avant-garde using benchmark DIA datasets and show that it can determine the quantitative suitability of a peptide peak, and reach the same levels of selectivity, accuracy, and reproducibility as manual validation. Avant-garde is complementary to existing DIA analysis engines and aims to establish a strong foundation for subsequent analysis of quantitative mass spectrometry data. A computational tool, Avant-garde, automates refinement of data-independent acquisition mass spectrometry-based quantitative proteomics data.

The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.

Background Recent work suggests that gene duplications may play an important role in the evolution of immunity genes. Passerine birds, and in particular Sylvioidea warblers, have highly duplicated major histocompatibility complex (MHC) genes, which are key in immunity, compared to other vertebrates. However, reasons for this high MHC gene copy number are yet unclear. High-throughput sequencing (HTS) allows MHC genotyping even in individuals with extremely duplicated genes. This HTS data can reveal evidence of selection, which may help to unravel the putative functions of different gene copies, i.e. neofunctionalization. We performed exhaustive genotyping of MHC class I in a Sylvioidea warbler, the sedge warbler, Acrocephalus schoenobaenus , using the Illumina MiSeq technique on individuals from a wild study population. Results The MHC diversity in 863 genotyped individuals by far exceeds that of any other bird species described to date. A single individual could carry up to 65 different alleles, a large proportion of which are expressed (transcribed). The MHC alleles were of three different lengths differing in evidence of selection, diversity and divergence within our study population. Alleles without any deletions and alleles containing a 6 bp deletion showed characteristics of classical MHC genes, with evidence of multiple sites subject to positive selection and high sequence divergence. In contrast, alleles containing a 3 bp deletion had no sites subject to positive selection and had low divergence. Conclusions Our results suggest that sedge warbler MHC alleles that either have no deletion, or contain a 6 bp deletion, encode classical antigen presenting MHC molecules. In contrast, MHC alleles containing a 3 bp deletion may encode molecules with a different function. This study demonstrates that highly duplicated MHC genes can be characterised with HTS and that selection patterns can be useful for revealing neofunctionalization. Importantly, our results highlight the need to consider the putative function of different MHC genes in future studies of MHC in relation to disease resistance and fitness.

Single-cell proteomics confidently quantifies cellular heterogeneity, however quantification of post-translational modifications, such as those deposited on histone proteins, remains elusive. Here, we develop a robust mass spectrometry-based method for the unbiased analysis of single-cell histone post-translational modifications (sc-hPTM). sc-hPTM identifies both single- and combinatorial histone post-translational modifications (67 peptidoforms in total), which includes nearly all frequently studied histone post-translational modifications with comparable reproducibility to traditional bulk experiments. As a proof of concept, we treat cells with sodium butyrate, a histone deacetylase inhibitor, and demonstrate that our method can i) distinguish between treated and untreated cells, ii) identify sub-populations of cells with heterogeneous response to the treatment, and iii) reveal differential co-regulation of histone post-translational modifications in the context of drug treatment. The sc-hPTM method enables comprehensive investigation of chromatin heterogeneity at single-cell resolution and provides a further understanding of the histone code. Chromatin structure is regulated by chemical modifications of histone proteins, but measuring these at single-cell resolution has been challenging. Here, the authors develop a mass spectrometry-based method to profile histone modifications in individual cells, revealing chromatin heterogeneity and differential co-regulation.

Mass spectrometry with data-independent acquisition (DIA) is a promising method to improve the comprehensiveness and reproducibility of targeted and discovery proteomics, in theory by systematically measuring all peptide precursors in a biological sample. However, the analytical challenges involved in discriminating between peptides with similar sequences in convoluted spectra have limited its applicability in important cases, such as the detection of single-nucleotide polymorphisms (SNPs) and alternative site localizations in phosphoproteomics data. We report Specter (https://github.com/rpeckner-broad/Specter), an open-source software tool that uses linear algebra to deconvolute DIA mixture spectra directly through comparison to a spectral library, thus circumventing the problems associated with typical fragment-correlation-based approaches. We validate the sensitivity of Specter and its performance relative to that of other methods, and show that Specter is able to successfully analyze cases involving highly similar peptides that are typically challenging for DIA analysis methods.

ObjectivesThe objective of the present study was to explain why two siblings carrying both the same homozygous pathogenic mutation for the autoinflammatory disease hyper IgD syndrome, show opposite phenotypes, that is, the first being asymptomatic, the second presenting all classical characteristics of the disease.MethodsWhere single omics (mainly exome) analysis fails to identify culprit genes/mutations in human complex diseases, multiomics analyses may provide solutions, although this has been seldom used in a clinical setting. Here we combine exome, transcriptome and proteome analyses to decipher at a molecular level, the phenotypic differences between the two siblings.ResultsThis multiomics approach led to the identification of a single gene—STAT1—which harboured a rare missense variant and showed a significant overexpression of both mRNA and protein in the symptomatic versus the asymptomatic sister. This variant was shown to be of gain of function nature, involved in an increased activation of the Janus kinase/signal transducer and activator of transcription signalling (JAK/STAT) pathway, known to play a critical role in inflammatory diseases and for which specific biotherapies presently exist. Pathway analyses based on information from differentially expressed transcripts and proteins confirmed the central role of STAT1 in the proposed regulatory network leading to an increased inflammatory phenotype in the symptomatic sibling.ConclusionsThis study demonstrates the power of a multiomics approach to uncover potential clinically actionable targets for a personalised therapy. In more general terms, we provide a proteogenomics analysis pipeline that takes advantage of subject-specific genomic and transcriptomic information to improve protein identification and hence advance individualised medicine.

Background Over recent years, increasing evidence suggests a causal relationship between neurofibrillary tangles (NFTs) formation, the main histopathological hallmark of tauopathies, including Alzheimer’s disease (AD), and the ubiquitin–proteasome system (UPS) dysfunction detected in these patients. Nevertheless, the mechanisms underlying UPS failure and the factors involved remain poorly understood. Given that AD and tauopathies are associated with chronic neuroinflammation, here, we explore if ATP, one of the danger-associated molecules patterns (DAMPs) associated with neuroinflammation, impacts on AD-associated UPS dysfunction. Methods To evaluate if ATP may modulate the UPS via its selective P2X7 receptor, we combined in vitro and in vivo approaches using both pharmacological and genetic tools. We analyze postmortem samples from human AD patients and P301S mice, a mouse model that mimics pathology observed in AD patients, and those from the new transgenic mouse lines generated, such as P301S mice expressing the UPS reporter Ub G76V -YFP or P301S deficient of P2X7R. Results We describe for the first time that extracellular ATP-induced activation of the purinergic P2X7 receptor (P2X7R) downregulates the transcription of β5 and β1 proteasomal catalytic subunits via the PI3K/Akt/GSK3/Nfr2 pathway, leading to their deficient assembly into the 20S core proteasomal complex, resulting in a reduced proteasomal chymotrypsin-like and postglutamyl-like activities. Using UPS-reported mice (UbGFP mice), we identified neurons and microglial cells as the most sensitive cell linages to a P2X7R-mediated UPS regulation. In vivo pharmacological or genetic P2X7R blockade reverted the proteasomal impairment developed by P301S mice, which mimics that were detected in AD patients. Finally, the generation of P301S;UbGFP mice allowed us to identify those hippocampal cells more sensitive to UPS impairment and demonstrate that the pharmacological or genetic blockade of P2X7R promotes their survival. Conclusions Our work demonstrates the sustained and aberrant activation of P2X7R caused by Tau-induced neuroinflammation contributes to the UPS dysfunction and subsequent neuronal death associated with AD, especially in the hippocampus.

Alzheimer disease is a neurodegenerative disease characterized by the presence of senile plaques composed of amyloid-β (Aβ) peptide, neurofibrillary tangles, neuronal loss and neuroinflammation. Previous works have revealed that extracellular ATP, through its selective receptor P2X7 (P2X7R), is essential to neuroinflammation and neurotoxicity induced by Aβ. P2X7R is upregulated on microglial cells around the senile plaques. This upregulation progressively rises with age and is parallel with an accumulation of senile plaques and also correlates with the synaptic toxicity detected both in animal models reproducing AD and human patients of AD. Furthermore, the late onset of the first AD-associated symptoms suggests that aging associated-changes may be relevant to the disease progression. Thus, microglia motility and its capacity to respond to exogenous ATP stimulus decrease with aging. To evaluate whether the P2X7R age related-changes on microglia cells may be relevant to the AD progression, we generated a new transgenic mouse model crossing an Aβ peptide mouse model, J20 mice and the P2X7R reporter mice EGFP. Our results indicate that neuroinflammation induced by Aβ peptide causes changes in the P2X7R distribution pattern, increasing it s expression in microglial cells at advanced and late stages, when microgliosis occurs, but not in the early stages, in the absence of microgliosis. In addition, we found that P2X7R activation promotes microglial cells migration to senile plaques but decreases their phagocytic capacity. Moreover, we found a significant reduction of P2X7R transcription on neuronal cells at the early and advanced stages, but not at the late stages. Since previous studies have reported that either pharmacological inhibition or selective downregulation of P2X7R significantly improve behavioral alterations and reduce the incidence and size of senile plaques in the early and advanced stages of AD, the results presented here provide new evidence, indicating that this therapeutic approach could be also efficient in the late stages of the disease.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease characterized by a progressive cognitive decline associated with global brain damage. Initially, intracellular paired helical filaments composed by hyperphosphorylated tau and extracellular deposits of amyloid-β (Aβ) were postulated as the causing factors of the synaptic dysfunction, neuroinflammation, oxidative stress, and neuronal death, detected in AD patients. Therefore, the vast majority of clinical trials were focused on targeting Aβ and tau directly, but no effective treatment has been reported so far. Consequently, only palliative treatments are currently available for AD patients. Over recent years, several studies have suggested the involvement of the purinergic receptor P2X7 (P2X7R), a plasma membrane ionotropic ATP-gated receptor, in the AD brain pathology. In this line, altered expression levels and function of P2X7R were found both in AD patients and AD mouse models. Consequently, genetic depletion or pharmacological inhibition of P2X7R ameliorated the hallmarks and symptoms of different AD mouse models. In this review, we provide an overview of the current knowledge about the role of the P2X7R in AD.