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The resurgence of yellow fever in South America has prompted vaccination against the etiologic agent, yellow fever virus (YFV). Current vaccines are based on a live-attenuated YF-17D virus derived from a virulent African isolate. The capacity of these vaccines to induce neutralizing antibodies against the vaccine strain is used as a surrogate for protection. However, the sensitivity of genetically distinct South American strains to vaccine-induced antibodies is unknown. We show that antiviral potency of the polyclonal antibody response in vaccinees is attenuated against an emergent Brazilian strain. This reduction was attributable to amino acid changes at two sites in central domain II of the glycoprotein E, including multiple changes at the domain I-domain II hinge, which are unique to and shared among most South American YFV strains. Our findings call for a reevaluation of current approaches to YFV immunological surveillance in South America and suggest approaches for updating vaccines.

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Paradoxically, oncogenes that drive cell cycle progression may also trigger pathways leading to senescence, thereby inhibiting the growth of tumorigenic cells. Knowledge of how these pathways operate, and how tumor cells may evade these pathways, is important for understanding tumorigenesis. The Y1 cell line, which harbors an amplification of the proto-oncogene Ras, rapidly senesces in response to the mitogen fibroblast growth factor-2 (FGF-2). To gain a more complete picture of how FGF-2 promotes senescence, we employed a multi-omics approach to analyze histone modifications, mRNA and protein expression, and protein phosphorylation in Y1 cells treated with FGF-2. Compared to control cells treated with serum alone, FGF-2 caused a delayed accumulation of acetylation on histone H4 and higher levels of H3K27me3. Sequencing analysis revealed decreased expression of cell cycle-related genes with concomitant loss of H3K27ac. At the same time, FGF-2 promoted the expression of p21, various cytokines, and MAPK-related genes. Nuclear envelope proteins, particularly lamin B1, displayed increased phosphorylation in response to FGF-2. Proteome analysis suggested alterations in cellular metabolism, as evident by modulated expression of enzymes involved in purine biosynthesis, tRNA aminoacylation, and the TCA cycle. We propose that Y1 cells senesce due to an inability to progress through the cell cycle, which may stem from DNA damage or TGFb signaling. Altogether, the phenotype of Y1 cells is consistent with rapidly established oncogene-induced senescence, demonstrating the synergy between growth factors and oncogenes in driving senescence and bringing additional insight into this tumor suppressor mechanism.

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Intestinal ischemia reperfusion injury (iIRI) is a severe clinical condition presenting high morbidity and mortality worldwide. Some of the systemic consequences of IRI can be prevented by applying ischemic preconditioning (IPC), a series of short ischemia/reperfusion events preceding the major ischemia. Although neutrophils are key players in the pathophysiology of ischemic injuries, neither the dysregulation presented by these cells in iIRI nor the protective effect of iIPC have their regulation mechanisms fully understood. Protein phosphorylation, as well as the regulation of the respective phosphatases and kinases are responsible for regulating a large number of cellular functions in the inflammatory response. Moreover, in previous work we found hydrolases and transferases to be modulated in iIR and iIPC, suggesting the possible involvement of phosphatases and kinases in the process. Therefore, in the present study, we analyzed the phosphoproteome of neutrophils from rats submitted to mesenteric ischemia and reperfusion, either submitted or not to IPC, compared to quiescent controls and sham laparotomy. Proteomic analysis was performed by multi-step enrichment of phosphopeptides, isobaric labeling, and LC-MS/MS analysis. Bioinformatics was used to determine phosphosite and phosphopeptide abundance and clustering, as well as kinases and phosphatases sites and domains. We found that most of the phosphorylation-regulated proteins are involved in apoptosis and migration, and most of the regulatory kinases belong to CAMK and CMGC families. An interesting finding revealed groups of proteins that are modulated by iIR, but such modulation can be prevented by iIPC. Among the regulated proteins related to the iIPC protective effect, Vamp8 and Inpp5d/Ship are discussed as possible candidates for control of the iIR damage.

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Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

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DNA and histone proteins define the structure and composition of chromatin. Histone posttranslational modifications (PTMs) are covalent chemical groups capable of modeling chromatin accessibility, mostly due to their ability in recruiting enzymes responsible for DNA readout and remodeling. Mass spectrometry (MS)-based proteomics is the methodology of choice for large-scale identification and quantification of protein PTMs, including histones. High sensitivity proteomics requires online MS coupling with relatively low throughput and poorly robust nano-liquid chromatography (nanoLC) and, for histone proteins, a 2-d sample preparation that includes histone purification, derivatization, and digestion. We present a new protocol that achieves quantitative data on about 200 histone PTMs from tissue or cell lines in 7 h from start to finish. This protocol includes 4 h of histone extraction, 3 h of derivatization and digestion, and only 1 min of MS analysis via direct injection (DI-MS). We demonstrate that this sample preparation can be parallelized for 384 samples by using multichannel pipettes and 96-well plates. We also engineered the sequence of a synthetic "histone-like" peptide to spike into the sample, of which derivatization and digestion benchmarks the quality of the sample preparation. We ensure that DI-MS does not introduce biases in histone peptide ionization as compared to nanoLC-MS/MS by producing and analyzing a library of synthetically modified histone peptides mixed in equal molarity. Finally, we introduce EpiProfileLite for comprehensive analysis of this new data type. Altogether, our workflow is suitable for high-throughput screening of >1000 samples per day using a single mass spectrometer.

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Intestinal ischemia and reperfusion injury is a model system of possible consequences of severe trauma and surgery, which might result into tissue dysfunction and organ failure. Neutrophils contribute to the injuries preceded by ischemia and reperfusion. However, the mechanisms by which intestinal ischemia and reperfusion stimulate and activate circulating neutrophils is still not clear. In this work, we used proteomics approach to explore the underlying regulated mechanisms in Wistar rat neutrophils after ischemia and reperfusion. We isolated neutrophils from three different biological groups; control, sham laparotomy, and intestinal ischemia/reperfusion. In the workflow, we included iTRAQ-labeling quantification and peptide fractionation using HILIC prior to LC-MS/MS analysis. From proteomic analysis, we identified 2,045 proteins in total that were grouped into five different clusters based on their regulation trend between the experimental groups. A total of 417 proteins were found as significantly regulated in at least one of the analyzed conditions. Interestingly, the enzyme prediction analysis revealed that ischemia/reperfusion significantly reduced the relative abundance of most of the antioxidant and pro-survival molecules to cause more tissue damage and ROS production whereas some of the significantly up regulated enzymes were involved in cytoskeletal rearrangement, adhesion and migration. Clusters based KEGG pathways analysis revealed high motility, phagocytosis, directional migration, and activation of the cytoskeletal machinery in neutrophils after ischemia and reperfusion. Increased ROS production and decreased phagocytosis were experimentally validated by microscopy assays. Taken together, our findings provide a characterization of the rat neutrophil response to intestinal ischemia and reperfusion and the possible mechanisms involved in the tissue injury by neutrophils after intestinal ischemia and reperfusion.

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PURPOSE: In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma EXPERIMENTAL DESIGN: To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils are isolated from control and surgical trauma rats in this study. Extracted proteins are analyzed using nano liquid chromatography coupled with tandem mass spectrometry. RESULTS: A total of 2924 rat neutrophil proteins are identified in our analysis, of which 393 are found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma, we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations are then confirmed by in silico protein-protein interaction analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage.

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