Indexed retention times (iRT), MS1 (the first level of mass analysis) or survey scan charge state distributions, and sequence ion intensities of MSMS (tandom mass spectrometry) spectra were predicted ...from peptide sequence by use of long-short term memory (LSTM) recurrent neural networks models. Data points on the order of 105 were used to train the iRT and charge state distribution models. An HCD sequence ion prediction model was trained with 2×106 experimental spectra. The models with a simple deep learning architecture can predict those three key LC-MS/MS (Liquid chromatography-tandem mass spectrometry) properties with superior accuracies.
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Highlights
•Deep learning models for prediction of LC-MS/MS properties.
Deep learning models for prediction of three key LC-MS/MS properties from peptide sequences were developed. The LC-MS/MS properties or behaviors are indexed retention times (iRT), MS1 or survey scan charge state distributions, and sequence ion intensities of HCD spectra. A common core deep supervised learning architecture, bidirectional long-short term memory (LSTM) recurrent neural networks was used to construct the three prediction models. Two featurization schemes were proposed and demonstrated to allow for efficient encoding of modifications. The iRT and charge state distribution models were trained with on order of 105 data points each. An HCD sequence ion prediction model was trained with 2 × 106 experimental spectra. The iRT prediction model and HCD sequence ion prediction model provide improved accuracies over the start-of-the-art models available in literature. The MS1 charge state distribution prediction model offers excellent performance. The prediction models can be used to enhance peptide identification and quantification in data-dependent acquisition and data-independent acquisition (DIA) experiments as well as to assist MRM (multiple reaction monitoring) and PRM (parallel reaction monitoring) experiment design.
Purpose Alterations in DNA damage response and repair (DDR) genes are associated with increased mutation load and improved clinical outcomes in platinum-treated metastatic urothelial carcinoma. We ...examined the relationship between DDR alterations and response to PD-1/PD-L1 blockade. Methods Detailed demographic, treatment response, and long-term outcome data were collected on patients with metastatic urothelial carcinoma treated with atezolizumab or nivolumab who had targeted exon sequencing performed on pre-immunotherapy tumor specimens. Presence of DDR alterations was correlated with best objective response per Response Evaluation Criteria in Solid Tumors (RECIST) and progression-free and overall survival. Results Sixty patients with urothelial cancer enrolled in prospective trials of anti-PD-1/PD-L1 antibodies met inclusion criteria. Any DDR and known or likely deleterious DDR mutations were identified in 28 (47%) and 15 (25%) patients, respectively. The presence of any DDR alteration was associated with a higher response rate (67.9% v 18.8%; P < .001). A higher response rate was observed in patients whose tumors harbored known or likely deleterious DDR alterations (80%) compared with DDR alterations of unknown significance (54%) and in those whose tumors were wild-type for DDR genes (19%; P < .001). The correlation remained significant in multivariable analysis that included presence of visceral metastases. DDR alterations also were associated with longer progression-free and overall survival. Conclusion DDR alterations are independently associated with response to PD-1/PD-L1 blockade in patients with metastatic urothelial carcinoma. These observations warrant additional study, including prospective validation and exploration of the interaction between tumor DDR alteration and other tumor/host biomarkers of immunotherapy response.
Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein ...interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S1104. Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y998 or in the S1039 region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation.
Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a ...nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H
2O
2. Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.
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► Proteomic approach monitors classical protein-tyrosine phosphatase (PTP) expression ► Modified method measures the fraction of PTPs that are oxidized ► Different cancer cell lines contain unique PTP oxidation profiles ► Method can be combined with phosphotyrosine proteomics to suggest PTP substrates
A method for quantifying the expression of reduced and oxidized protein tyrosine phosphatases (PTPs) shows that healthy and cancer cells have distinct PTP proteomes and reveals the extensive posttranslational regulation of these enzymes.
N6-Methyladenosine (m6A) accounts for approximately 0.2% to 0.6% of all adenosine in mammalian mRNA, representing the most abundant internal mRNA modifications. m6A RNA immunoprecipitation followed ...by high-throughput sequencing (MeRIP-seq) is a powerful technique to map the m6A location transcriptome-wide. However, this method typically requires 300 μg of total RNA, which limits its application to patient tumors. In this study, we present a refined m6A MeRIP-seq protocol and analysis pipeline that can be applied to profile low-input RNA samples from patient tumors. We optimized the key parameters of m6A MeRIP-seq, including the starting amount of RNA, RNA fragmentation, antibody selection, MeRIP washing/elution conditions, methods for RNA library construction, and the bioinformatics analysis pipeline. With the optimized immunoprecipitation (IP) conditions and a postamplification rRNA depletion strategy, we were able to profile the m6A epitranscriptome using 500 ng of total RNA. We identified approximately 12,000 m6A peaks with a high signal-to-noise (S/N) ratio from 2 lung adenocarcinoma (ADC) patient tumors. Through integrative analysis of the transcriptome, m6A epitranscriptome, and proteome data in the same patient tumors, we identified dynamics at the m6A level that account for the discordance between mRNA and protein levels in these tumors. The refined m6A MeRIP-seq method is suitable for m6A epitranscriptome profiling in a limited amount of patient tumors, setting the ground for unraveling the dynamics of the m6A epitranscriptome and the underlying mechanisms in clinical settings.
Fire and biodiversity in the Anthropocene Kelly, Luke T; Giljohann, Katherine M; Duane, Andrea ...
Science (American Association for the Advancement of Science),
11/2020, Letnik:
370, Številka:
6519
Journal Article
Recenzirano
Odprti dostop
Fire has been a source of global biodiversity for millions of years. However, interactions with anthropogenic drivers such as climate change, land use, and invasive species are changing the nature of ...fire activity and its impacts. We review how such changes are threatening species with extinction and transforming terrestrial ecosystems. Conservation of Earth's biological diversity will be achieved only by recognizing and responding to the critical role of fire. In the Anthropocene, this requires that conservation planning explicitly includes the combined effects of human activities and fire regimes. Improved forecasts for biodiversity must also integrate the connections among people, fire, and ecosystems. Such integration provides an opportunity for new actions that could revolutionize how society sustains biodiversity in a time of changing fire activity.
Abstract
STUDY QUESTION
What is the recommended assessment and management of women with polycystic ovary syndrome (PCOS), based on the best available evidence, clinical expertise and consumer ...preference?
SUMMARY ANSWER
International evidence-based guidelines, including 166 recommendations and practice points, addressed prioritized questions to promote consistent, evidence-based care and improve the experience and health outcomes of women with PCOS.
WHAT IS KNOWN ALREADY
Previous guidelines either lacked rigorous evidence-based processes, did not engage consumer and international multidisciplinary perspectives, or were outdated. Diagnosis of PCOS remains controversial, and assessment and management are inconsistent. The needs of women with PCOS are not being adequately met and evidence practice gaps persist.
STUDY DESIGN, SIZE, DURATION
International evidence-based guideline development engaged professional societies and consumer organizations with multidisciplinary experts and women with PCOS directly involved at all stages. Appraisal of Guidelines for Research and Evaluation (AGREE) II-compliant processes were followed, with extensive evidence synthesis. The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) framework was applied across evidence quality, feasibility, acceptability, cost, implementation and ultimately recommendation strength.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Governance included a six continent international advisory and a project board, five guideline development groups, and consumer and translation committees. Extensive health professional and consumer engagement informed guideline scope and priorities. Engaged international society-nominated panels included pediatrics, endocrinology, gynecology, primary care, reproductive endocrinology, obstetrics, psychiatry, psychology, dietetics, exercise physiology, public health and other experts, alongside consumers, project management, evidence synthesis and translation experts. In total, 37 societies and organizations covering 71 countries engaged in the process. Twenty face-to-face meetings over 15 months addressed 60 prioritized clinical questions involving 40 systematic and 20 narrative reviews. Evidence-based recommendations were developed and approved via consensus voting within the five guideline panels, modified based on international feedback and peer review, with final recommendations approved across all panels.
MAIN RESULTS AND THE ROLE OF CHANCE
The evidence in the assessment and management of PCOS is generally of low to moderate quality. The guideline provides 31 evidence based recommendations, 59 clinical consensus recommendations and 76 clinical practice points all related to assessment and management of PCOS. Key changes in this guideline include: (i) considerable refinement of individual diagnostic criteria with a focus on improving accuracy of diagnosis; (ii) reducing unnecessary testing; (iii) increasing focus on education, lifestyle modification, emotional wellbeing and quality of life; and (iv) emphasizing evidence based medical therapy and cheaper and safer fertility management.
LIMITATIONS, REASONS FOR CAUTION
Overall evidence is generally low to moderate quality, requiring significantly greater research in this neglected, yet common condition, especially around refining specific diagnostic features in PCOS. Regional health system variation is acknowledged and a process for guideline and translation resource adaptation is provided.
WIDER IMPLICATIONS OF THE FINDINGS
The international guideline for the assessment and management of PCOS provides clinicians with clear advice on best practice based on the best available evidence, expert multidisciplinary input and consumer preferences. Research recommendations have been generated and a comprehensive multifaceted dissemination and translation program supports the guideline with an integrated evaluation program.
STUDY FUNDING/COMPETING INTEREST(S)
The guideline was primarily funded by the Australian National Health and Medical Research Council of Australia (NHMRC) supported by a partnership with ESHRE and the American Society for Reproductive Medicine. Guideline development group members did not receive payment. Travel expenses were covered by the sponsoring organizations. Disclosures of conflicts of interest were declared at the outset and updated throughout the guideline process, aligned with NHMRC guideline processes. Full details of conflicts declared across the guideline development groups are available at https://www.monash.edu/medicine/sphpm/mchri/pcos/guideline in the Register of disclosures of interest. Of named authors, Dr Costello has declared shares in Virtus Health and past sponsorship from Merck Serono for conference presentations. Prof. Laven declared grants from Ferring, Euroscreen and personal fees from Ferring, Euroscreen, Danone and Titus Healthcare. Prof. Norman has declared a minor shareholder interest in an IVF unit. The remaining authors have no conflicts of interest to declare. The guideline was peer reviewed by special interest groups across our partner and collaborating societies and consumer organizations, was independently assessed against AGREE-II criteria, and underwent methodological review. This guideline was approved by all members of the guideline development groups and was submitted for final approval by the NHMRC.
Data dependent acquisition (DDA) and data independent acquisition (DIA) are traditionally separate experimental paradigms in bottom-up proteomics. In this work, we developed a strategy combining the ...two experimental methods into a single LC-MS/MS run. We call the novel strategy data dependent–independent acquisition proteomics, or DDIA for short. Peptides identified from DDA scans by a conventional and robust DDA identification workflow provide useful information for interrogation of DIA scans. Deep learning based LC-MS/MS property prediction tools, developed previously, can be used repeatedly to produce spectral libraries facilitating DIA scan extraction. A complete DDIA data processing pipeline, including the modules for iRT vs RT calibration curve generation, DIA extraction classifier training, and false discovery rate control, has been developed. Compared to another spectral library-free method, DIA-Umpire, the DDIA method produced a similar number of peptide identifications, but nearly twice as many protein group identifications. The primary advantage of the DDIA method is that it requires minimal information for processing its data.
The sequence database searching method is widely used in proteomics for peptide identification. To control the false discovery rate (FDR) of the searching results, the target–decoy method generates ...and searches a decoy database together with the target database. A known problem is that the target protein sequence database may contain numerous repeated peptides. The structures of these repeats are not preserved by most existing decoy generation algorithms. Previous studies suggest that such discrepancy between the target and decoy databases may lead to an inaccurate FDR estimation. Based on the de Bruijn graph model, we propose a new repeat-preserving algorithm to generate decoy databases. We prove that this algorithm preserves the structures of the repeats in the target database to a great extent. The de Bruijn method has been compared with a few other commonly used methods and demonstrated superior FDR estimation accuracy and an improved number of peptide identification.
There is growing recognition that mammalian cells produce many thousands of large intergenic transcripts. However, the functional significance of these transcripts has been particularly ...controversial. Although there are some well-characterized examples, most (>95%) show little evidence of evolutionary conservation and have been suggested to represent transcriptional noise. Here we report a new approach to identifying large non-coding RNAs using chromatin-state maps to discover discrete transcriptional units intervening known protein-coding loci. Our approach identified ∼1,600 large multi-exonic RNAs across four mouse cell types. In sharp contrast to previous collections, these large intervening non-coding RNAs (lincRNAs) show strong purifying selection in their genomic loci, exonic sequences and promoter regions, with greater than 95% showing clear evolutionary conservation. We also developed a functional genomics approach that assigns putative functions to each lincRNA, demonstrating a diverse range of roles for lincRNAs in processes from embryonic stem cell pluripotency to cell proliferation. We obtained independent functional validation for the predictions for over 100 lincRNAs, using cell-based assays. In particular, we demonstrate that specific lincRNAs are transcriptionally regulated by key transcription factors in these processes such as p53, NF B, Sox2, Oct4 (also known as Pou5f1) and Nanog. Together, these results define a unique collection of functional lincRNAs that are highly conserved and implicated in diverse biological processes.
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