Ten questions to AI regarding the present and future of proteomics

The role of a scientist is at first not so different from a philosopher. They both need to question common thinking and evaluate whether reality is not as we always thought. Based on this, we need to design hypotheses, experiments, and analyses to prove our alternative vision. Artificial Intelligence (AI) is rapidly moving from an “assistant” into a proper “colleague” for literature mining, data analysis and interpretation, and literally having (almost) real scientific conversations. However, being AI based on existing information, if we rely on it excessively will we still be able to question the status quo? In this article, we are particularly interested in discussing the future of proteomics and mass spectrometry with our new electronic collaborator. We leave to the reader the judgement whether the answers we received are satisfactory or superficial. What we were mostly interested in was laying down what we think are critical questions that the proteomics community should occasionally ask to itself. Proteomics has been around for more than 30 years, but it is still missing a few critical steps to fully address its promises as being the new genomics for clinical diagnostics and fundamental science, while becoming a user-friendly tool for every lab. Will we get there with the help of AI? And will these answers change in a short period, as AI continues to advance

Nuclear Factor I/B is an Oncogene in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive cancer often diagnosed after it has metastasized. Despite the need to better understand this disease, SCLC remains poorly characterized at the molecular and genomic levels. Using a genetically engineered mouse model of SCLC driven by conditional deletion of Trp53 and Rb1 in the lung, we identified several frequent, high-magnitude focal DNA copy number alterations in SCLC. We uncovered amplification of a novel, oncogenic transcription factor, Nuclear factor I/B (Nfib), in the mouse SCLC model and in human SCLC. Functional studies indicate that NFIB regulates cell viability and proliferation during transformation.National Cancer Institute (U.S.) (grant P30-CA14051)David H. Koch Institute for Integrative Cancer Research at MIT (Ludwig Center for Molecular Oncology)Howard Hughes Medical InstituteAlfred P. Sloan Foundation (Research Fellowship)International Association for the Study of Lung Cance

Geração e caracterização molecular de anticorpos contra canais para sódio voltagem dependentes

Exportado OPUSMade available in DSpace on 2019-08-10T10:13:28Z (GMT). No. of bitstreams: 1 dissertac_a_o_stephanie_stransky_lauar.pdf: 7108404 bytes, checksum: 8f212eb27649ecca003c409ab602ab85 (MD5) Previous issue date: 5Canais para sódio voltagem dependentes (Nav) são canais iônicos responsáveis pela iniciação e propagação de potenciais de ação em células eletricamente excitáveis. Estes canais são expressos em células metastáticas de diversos tipos de câncer, e mutações associadas a eles estão relacionados à síndromes epilépticas, miopatias esqueléticas, arritmias cardíacas, neuropatias entre outras. Devido ao importante papel dos canais para Na+ em diferentes patologias, o objetivo deste estudo foi a produção de anticorpos contra uma sequência extracelular conservada das subfamílias Nav. A sequência de 15 aminoácidos (peptídeo alpha) correspondente ao 11º segmento extracelular localizado no domínio IV do canal Nav1.1, foi sintetizada utilizando o método FMOC. Camundongos Balb/C e coelhos New Zealand foram imunizados com o peptídeo sintético conjugado à ovalbumina. Anticorpos anti-peptídeo alpha foram testados quanto à capacidade de reconhecer tanto o próprio peptídeo alpha, através de ensaios de ELISA e SPOT, mas também de reconhecer o próprio canal para Na+, tanto em células HEK expressando o canal Nav1.3 e também em extrato de córtex de cérebro de camundongos. Os resultados demonstraram que o peptídeo sintetizado é capaz de induzir a produção de anticorpos sítio específicos. Além disso, nossa metodologia foi eficaz no mapeamento de um sítio extracelular do canal para Na+ e para a identificação de resíduos de aminoácidos críticos para a interação com o anticorpo, proporcionando novas perspectivas para o estudo da modulação de canais para Na+ e seus efeitos fisiopatológicos associadosVoltage-gated sodium channels (Nav) are ion channels responsible for the initiation and propagation of action potentials in electrically excitable cells. These channels are expressed in metastatic cells of several types of cancer, and mutations associated with them are related to epileptic syndromes, skeletal myopathy, cardiac arrhythmias, neuropathies and others. Due to the important role of Na+ channels in different pathologies, the aim of this study was the production of antibodies against a conserved extracellular sequence of the Nav subfamilies. The sequence of 15 amino acids (alpha peptide) corresponding to the 11º extracellular segment located in domain IV of Nav1.1 channel, was synthesized using the FMOC method. Balb/C mice and New Zealand rabbits were immunized with the synthetic peptide conjugated to ovalbumin. Alpha peptide antibodies were tested for their ability to recognize alpha peptide in an ELISA and SPOT assay, and also for their ability to recognize the Na+ channel in HEK cells expressing Nav1.3 channels and mouse brain cortex extract. The results showed that the synthesized peptide was capable of inducing the production of site specific antibodies. Moreover, our method was effective in mapping a Na+ channel extracellular site and in identification of critical amino acids residues for the interaction with the antibody, providing new prospects for studying modulation of sodium channel and their associated pathophysiological effect

Mass Spectrometry to Study Chromatin Compaction

Chromatin accessibility is a major regulator of gene expression. Histone writers/erasers have a critical role in chromatin compaction, as they “flag” chromatin regions by catalyzing/removing covalent post-translational modifications on histone proteins. Anomalous chromatin decondensation is a common phenomenon in cells experiencing aging and viral infection. Moreover, about 50% of cancers have mutations in enzymes regulating chromatin state. Numerous genomics methods have evolved to characterize chromatin state, but the analysis of (in)accessible chromatin from the protein perspective is not yet in the spotlight. We present an overview of the most used approaches to generate data on chromatin accessibility and then focus on emerging methods that utilize mass spectrometry to quantify the accessibility of histones and the rest of the chromatin bound proteome. Mass spectrometry is currently the method of choice to quantify entire proteomes in an unbiased large-scale manner; accessibility on chromatin of proteins and protein modifications adds an extra quantitative layer to proteomics dataset that assist more informed data-driven hypotheses in chromatin biology. We speculate that this emerging new set of methods will enhance predictive strength on which proteins and histone modifications are critical in gene regulation, and which proteins occupy different chromatin states in health and disease

Additional file 3 of Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model

Additional file 3: Table S1. Full metabolite profiling of the cell culture supernatan

Additional file 2 of Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model

Additional file 2. Protein Coding Time-course analysi

Additional file 5 of Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model

Additional file 5: Table 3. Proteome of HepG2/C3A spheroids and flat cell

Additional file 4 of Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model

Additional file 4: Table S2. Gene expression clusterin

Additional file 1 of Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model

Additional file 1: Fig. S1. Spheroids express proteins characteristic of the human liver. (A) Size distribution of liver spheroids. For the proteome analysis, flat cells and spheroids were collected, processed and the peptides analyzed by mass spectrometry. Bar graphs show the relative abundance of (B) metallothioneins and (C) liver biomarkers. Data are represented as means ± SEM. Fig. S2. NaBut treatment induce histone hyperacetylation in flat cells. HepG2/C3A cells were treated with 20 mM NaBut and were kept in culture for 3 days. After treatment, histones were extracted and analyzed by mass spectrometry. (A) Total levels of histone peptides containing 1, 2 or 3 methylations (me1, me2, me3, respectively) or containing acetylations (ac). (B) Total levels of histone H4 peptides containing acetylations (1ac, 2ac, 3ac, 4ac). Unmod, unmodified peptide. (C) Relative abundance of histone H3 acetylated peptides. Data are represented as means ± SD. Nt, non-treated. (D) Volcano plot representing NaBut vs Nt fold change after 3 days of treatment. Fig. S3. Liver spheroids have a slow replication rate and can recover from treatment. (A) Adenylate kinase was measured following NaBut treatment. The culture supernatant from flat cells and spheroids were collected and analyzed by luminescence. (B) Relative abundance of DNMT1 in flat cells and spheroids. (C) Labeling incorporation in flat cells and spheroids. Data are represented as means ± SEM. * p < 0.05, ** p < 0.005, and *** p < 0.0005 when compared with flat cells

Acidic Phospholipase A2-Peptide Derivative Modulates Oxidative Status and Microstructural Reorganization of Scar Tissue after Cutaneous Injury

From in vitro and in vivo models, the proliferative and healing potential of an acidic phospholipase A2 (LAPLA2) from Lachesis muta venom was investigated. The LAPLA2 proliferative activity was evaluated on fibroblasts and keratinocytes cultured, and the antioxidant and regenerative potential of LAPLA2 was analyzed in a murine model. The animal study consisted of four groups: C (negative control): 0.9% NaCl; SS (positive control): 1% silver sulfadiazine; L1 group: 0.5% LAPLA2; and L2 group: 0.25% LAPLA2. Wounds were topically treated daily for 12 days, and scar tissue samples were collected every 4 days. In vitro, LAPLA2 stimulated marked time-dependent cell proliferation. In vivo, it increased the antioxidant activity of superoxide dismutase (SOD) and catalase (CAT) and decreased malondialdehyde (MDA) and carbonyl protein (CP) levels in scar tissue treated with LAPLA2 at 0.5%. This peptide was effective in stimulating cellular proliferation, neoangiogenesis, type I and III collagen deposition, and maturation in a time-dependent-way, reducing the time required for wound closure. Our results indicated that LAPLA2 presented a remarkable potential in improving the oxidative status and microstructural reorganization of the scar tissue by stimulation of cellularity, angiogenesis, colagenogenesis, and wound contraction, suggesting that the peptide could be a potential candidate for a new healing drug