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This study aims at identifying molecular biomarkers differentiating responders and non-responders to treatment with Tumor Necrosis Factor inhibitors (TNFi) among patients with axial spondyloarthritis (axSpA). Whole blood mRNA and plasma proteins were measured in a cohort of biologic-naïve axSpA patients (n = 35), pre and post (14 weeks) TNFi treatment with adalimumab. Differential expression analysis was used to identify the most enriched pathways and in predictive models to distinguish responses to TNFi. A treatment-associated signature suggests a reduction in inflammatory activity. We found transcripts and proteins robustly differentially expressed between baseline and week 14 in responders. C-reactive protein (CRP) and Haptoglobin (HP) proteins showed strong and early decrease in the plasma of axSpA patients, while a cluster of apolipoproteins (APOD, APOA2, APOA1) showed increased expression at week 14. Responders to TNFi treatment present higher levels of markers of innate immunity at baseline, and lower levels of adaptive immunity markers, particularly B-cells. A logistic regression model incorporating ASDAS-CRP, gender, and AFF3, the top differentially expressed gene at baseline, enabled an accurate prediction of response to adalimumab in our cohort (AUC = 0.97). In conclusion, innate and adaptive immune cell type composition at baseline may be a major contributor to response to adalimumab in axSpA patients. A model including clinical and gene expression variables should also be considered. [ABSTRACT FROM AUTHOR]

In cosmetics, there is some interest to solubilized polar antioxidant molecules in oil. This is a matrix that can indeed protect these fragile molecules against oxidation and that makes possible the polar antioxidants transfer through the epidermis in order to be delivered within the dermis. Varying the oil formulation allows the achievement of a high content of antioxidants in a natural vegetable oil using an oleo-eco extraction process. However, it was necessary to characterize this content using quantitative and efficient methods such as High-Performance Thin-Layer Chromatography technique. The aim of this work was to develop a method of antioxidant’s characterization with this technique without a preliminary re-extraction of the antioxidants.

Creatine transporter deficiency (CTD), a leading cause of intellectual disability is a result of the mutation in the gene encoding the creatine transporter SLC6A8, which prevents creatine uptake into the brain, causing mental retardation, expressive speech and language delay, autistic-like behavior and epilepsy. Preclinical in vitro and in vivo data indicate that dodecyl creatine ester (DCE) which increases the creatine brain content, might be a therapeutic option for CTD patients. To gain a better understanding of the pathophysiology and DCE treatment efficacy in CTD, this study focuses on the identification of biomarkers related to cognitive improvement in a Slc6a8 knockout mouse model (Slc6a8−/y) engineered to mimic the clinical features of CTD patients which have low brain creatine content. Shotgun proteomics analysis of 4,035 proteins in four different brain regions; the cerebellum, cortex, hippocampus (associated with cognitive functions) and brain stem, and muscle as a control, was performed in 24 mice. Comparison of the protein abundance in the four brain regions between DCE-treated intranasally Slc6a8−/y mice and wild type and DCE-treated Slc6a8−/y and vehicle group identified 14 biomarkers, shedding light on the mechanism of action of DCE. Integrative bioinformatics and statistical modeling identified key proteins in CTD, including KIF1A and PLCB1. The abundance of these proteins in the four brain regions was significantly correlated with both the object recognition and the Y-maze tests. Our findings suggest a major role for PLCB1, KIF1A, and associated molecules in the pathogenesis of CTD. [ABSTRACT FROM AUTHOR]

The vast majority of marine microorganisms and their functions are yet to be explored. The considerable diversity they encompass is an endless source of knowledge and wealth that can be valued on an industrial scale, emphasizing the need to develop rapid and efficient identification and characterization techniques. In this study, we identified 26 microbial isolates from coastal water of the NW Mediterranean Sea, using phylopeptidomics, a cutting-edge tandem mass spectrometry proteotyping technique. Taxonomical identification at the species level was successfully conducted for all isolates. The presence of strains belonging to the newly described Balneolaeota phylum, yet uncharacterized at the proteomics scale, was noted. The very first proteomics-based investigation of a representative of the Balneolaeota phylum, Balneola vulgaris, is proposed, demonstrating the use of our proteotyping workflow for the rapid identification and in-depth molecular characterization, in a single MS/MS analytical run. Tandem mass spectrometry proteotyping is a valuable asset for culturomic programs as the methodology is able to quickly classify the most atypical isolates. [ABSTRACT FROM AUTHOR]

Lassa virus (LASV), an Old World arenavirus, is responsible for hemorrhagic fevers in western Africa. The privileged tropism of LASV for endothelial cells combined with a dysregulated inflammatory response are the main cause of the increase in vascular permeability observed during the disease. Mopeia virus (MOPV) is another arenavirus closely related to LASV but nonpathogenic for non-human primates (NHPs) and has never been described in humans. MOPV is more immunogenic than LASV in NHPs and in vitro in human immune cell models, with more intense type I IFN and adaptive cellular responses. Here, we compared the transcriptomic and proteomic responses of human umbilical vein endothelial cells (HUVECs) to infection with the two viruses to further decipher the mechanisms involved in their differences in immunogenicity and pathogenicity. Both viruses replicated durably and efficiently in HUVECs, but the responses they induced were strikingly different. Modest activation was observed at an early stage of LASV infection and then rapidly shut down. By contrast, MOPV induced a late but more intense response, characterized by the expression of genes and proteins mainly associated with the type I IFN response and antigen processing/presentation. Such a response is consistent with the higher immunogenicity of MOPV relative to LASV, whereas the lack of an innate response induced in HUVECs by LASV is consistent with its uncontrolled systemic dissemination through the vascular endothelium. [ABSTRACT FROM AUTHOR]

COVID-19 is the most disturbing pandemic of the past hundred years. Its causative agent, the SARS-CoV-2 virus, has been the subject of an unprecedented investigation to characterize its molecular structure and intimate functioning. While markers for its detection have been proposed and several diagnostic methodologies developed, its propensity to evolve and evade diagnostic tools and the immune response is of great concern. The recent spread of new variants with increased infectivity requires even more attention. Here, we document how shotgun proteomics can be useful for rapidly monitoring the evolution of the SARS-CoV-2 virus. We evaluated the heterogeneity of purified SARS-CoV-2 virus obtained after culturing in the Vero E6 cell line. We found that cell culture induces significant changes that are translated at the protein level, such changes being detectable by tandem mass spectrometry. Production of viral particles requires careful quality control which can be easily performed by shotgun proteomics. Although considered relatively stable so far, the SARS-CoV-2 genome turns out to be prone to frequent variations. Therefore, the sequencing of SARS-CoV-2 variants from patients reporting only the consensus genome after its amplification would deserve more attention and could benefit from more in-depth analysis of low level but crystal-clear signals, as well as complementary and rapid analysis by shotgun proteomics. [ABSTRACT FROM AUTHOR]

A vaccine to fight Lassa fever: Thousands of people in Western Africa succumb to Lassa fever each year. Lassa virus infections can in some cases respond to antiviral treatment, but there is no vaccine. Mateo et al. tested three different viral-vectored Lassa virus vaccines in a nonhuman primate model. They compared transcriptomic and proteomic signatures as well as B cell and T cell responses after vaccination. One vaccine using a measles virus vector with Lassa virus glycoprotein and nucleoprotein provided almost sterilizing protection upon a lethal Lassa virus challenge. This vaccine will soon be tested in humans and has the potential to make a major impact on the incidence of Lassa fever. Lassa fever is a major threat in Western Africa. The large number of people living at risk for this disease calls for the development of a vaccine against Lassa virus (LASV). We generated live-attenuated LASV vaccines based on measles virus and Mopeia virus platforms and expressing different LASV antigens, with the aim to develop a vaccine able to protect after a single shot. We compared the efficacy of these vaccines against LASV in cynomolgus monkeys. The vaccines were well tolerated and protected the animals from LASV infection and disease after a single immunization but with varying efficacy. Analysis of the immune responses showed that complete protection was associated with robust secondary T cell and antibody responses against LASV. Transcriptomic and proteomic analyses showed an early activation of innate immunity and T cell priming after immunization with the most effective vaccines, with changes detectable as early as 2 days after immunization. The most efficacious vaccine candidate, a measles vector simultaneously expressing LASV glycoprotein and nucleoprotein, has been selected for further clinical evaluation. [ABSTRACT FROM AUTHOR]

White-rot fungi employ secreted carbohydrate-active enzymes (CAZymes) along with reactive oxygen species (ROS), like hydrogen peroxide (H2O2), to degrade lignocellulose in wood. H2O2 serves as a co-substrate for key oxidoreductases during the initial decay phase. While the degradation of lignocellulose by CAZymes is well documented, the impact of ROS on the oxidation of the secreted proteins remains unclear, and the identity of the oxidized proteins is unknown. Methionine (Met) can be oxidized to Met sulfoxide (MetO) or Met sulfone (MetO2) with potential deleterious, antioxidant, or regulatory effects. Other residues, like proline (Pro), can undergo carbonylation. Using the white-rot Pycnoporus cinnabarinus grown on aspen wood, we analyzed the Met content of the secreted proteins and their susceptibility to oxidation combining H218O2 with deep shotgun proteomics. Strikingly, their overall Met content was significantly lower (1.4%) compared to intracellular proteins (2.1%), a feature conserved in fungi but not in metazoans or plants. We evidenced that a catalase, widespread in white-rot fungi, protects the secreted proteins from oxidation. Our redox proteomics approach allowed the identification of 49 oxidizable Met and 40 oxidizable Pro residues within few secreted proteins, mostly CAZymes. Interestingly, many of them had several oxidized residues localized in hotspots. Some Met, including those in GH7 cellobiohydrolases, were oxidized up to 47%, with a substantial percentage of sulfone (13%). These Met are conserved in fungal homologs, suggesting important functional roles. Our findings reveal that white-rot fungi safeguard their secreted proteins by minimizing their Met content and by scavenging ROS and pinpoint redox-active residues in CAZymes.IMPORTANCEThe study of lignocellulose degradation by fungi is critical for understanding the ecological and industrial implications of wood decay. While carbohydrate-active enzymes (CAZymes) play a well-established role in lignocellulose degradation, the impact of hydrogen peroxide (H2O2) on secreted proteins remains unclear. This study aims at evaluating the effect of H2O2 on secreted proteins, focusing on the oxidation of methionine (Met). Using the model white-rot fungi Pycnoporus cinnabarinus grown on aspen wood, we showed that fungi protect their secreted proteins from oxidation by reducing their Met content and utilizing a secreted catalase to scavenge exogenous H2O2. The research identified key oxidizable Met within secreted CAZymes. Importantly, some Met, like those of GH7 cellobiohydrolases, undergone substantial oxidation levels suggesting important roles in lignocellulose degradation. These findings highlight the adaptive mechanisms employed by white-rot fungi to safeguard their secreted proteins during wood decay and emphasize the importance of these processes in lignocellulose breakdown. [ABSTRACT FROM AUTHOR]

The formation of a protein corona around nanoparticles can influence their toxicity, triggering cellular responses that may be totally different from those elicited by pristine nanoparticles. The main objective of this study was to investigate whether the species origin of the serum proteins forming the corona influences the in vitro toxicity assessment of silica nanoparticles. Coronas were preformed around nanoparticles before cell exposures by incubation in fetal bovine (FBS) or human (HS) serum. The compositions of these protein coronas were assessed by nano-LC MS/MS. The effects of these protein-coated nanoparticles on HepG2 cells were monitored using real-time cell impedance technology. The nanoparticle coronas formed in human or fetal bovine serum comprised many homologous proteins. Using human compared with fetal bovine serum, nanoparticle toxicity in HepG2 cells decreased by 4-fold and 1.5-fold, when used at 50 and 10μg/mL, respectively. It is likely that “markers of self” are present in the serum and are recognized by human cell receptors. Preforming a corona with human serum seems to be more appropriate for in vitro toxicity testing of potential nanocarriers using human cells. In vitro cytotoxicity assays must reflect in vivo conditions as closely as possible to provide solid and useful results. [ABSTRACT FROM AUTHOR]

Upon contact with biological fluids, nanoparticles (NPs) are readily coated by cellular compounds, particularly proteins, which are determining factors for the localization and toxicity of NPs in the organism. Here, we improved a methodological approach to identify proteins that adsorb on silica NPs with high affinity. Using large-scale proteomics and mixtures of soluble proteins prepared either from yeast cells or from alveolar human cells, we observed that proteins with large unstructured region(s) are more prone to bind on silica NPs. These disordered regions provide flexibility to proteins, a property that promotes their adsorption. The statistical analyses also pointed to a marked overrepresentation of RNA-binding proteins (RBPs) and of translation initiation factors among the adsorbed proteins. We propose that silica surfaces, which are mainly composed of Si–O−and Si–OH groups, mimic ribose-phosphate molecules (rich in –O−and –OH) and trap the proteins able to interact with ribose-phosphate containing molecules. Finally, using anin vitroassay, we showed that the sequestration of translation initiation factors by silica NPs results in an inhibition of thein vitrotranslational activity. This result demonstrates that characterizing the protein corona of various NPs would be a relevant approach to predict their potential toxicological effects. [ABSTRACT FROM AUTHOR]

Background: The toxicity of manufactured fumed silica nanoparticles (NPs) remains poorly investigated compared to that of crystalline silica NPs, which have been associated with lung diseases after inhalation. Amorphous silica NPs are a raw material for manufactured nanocomposites, such as cosmetics, foods, and drugs, raising concerns about their potential toxicity. Results: The size of the NPs was determined by dynamic light scattering and their shape was visualized by atomic force microscopy (10 ± 4 nm). The pertinent toxicological concentration and dynamic ranges were determined using viability tests and cellular impedance. We combined transcriptomics and proteomics to assess the cellular and molecular effects of fumed silica in A549 human alveolar epithelial cells. The "no observed transcriptomic adverse effect level" (NOTEL) was set to 1.0 µg/cm², and the "lowest observed adverse transcriptional effect level" (LOTEL) was set at 1.5 µg/cm². We carried out genome-wide expression profiles with microarrays and identified, by shotgun proteomics, the exoproteome changes in lung cells after exposure to NP doses (0.1, 1.0, 1.5, 3.0, and 6.0 µg/cm²)at two time points (24 h and 72 h). The data revealed a hierarchical, dose-dependent cellular response to silica NPs. At 1.5 µg/cm², the Rho signaling cascade, actin cytoskeleton remodeling, and clathrin-mediated endocytosis were induced. At 3.0 µg/cm², many inflammatory mediators were upregulated and the coagulation system pathway was triggered. Lastly, at 6.0 µg/cm², oxidative stress was initiated. The proteins identified in the extracellular compartment were consistent with these findings. Conclusions: The alliance of two high-throughput technologies allowed the quantitative assessment of the cellular effects and molecular consequences of exposure of lung cells to low doses of NPs. These results were obtained using a pathway-driven analysis instead of isolated genes. As in photography, toxicogenomics allows, at the same time, the visualization of a wide spectrum of biological responses and a "zoom in" to the details with a great depth of field. This study illustrates how such an approach based on human cell culture models is a valuable predictive screening tool to evaluate the toxicity of many potentially harmful emerging substances, alone or in mixtures, in the framework of future regulatory reinforcements. [ABSTRACT FROM AUTHOR]

To identify nucleo-cytoplasmic shuttle proteins that relocate to the nucleus upon UV stress, we selected 18 targets on the basis of their conservation amongst eukaryotes and their relatively poor functional description. Their relocation was assayed using quantitative nuclear relocation assay (QNR). We focused on Pat1, a component of the cytoplasmic foci called processing bodies (p-bodies), because it had the strongest response to the stress. We verified that Pat1 accumulates in the nucleus after GFP tagging and fluorescence microscopy. Using tandem affinity purification coupled to a mass spectrometry shotgun detection and quantitation approach, we explored the dynamics of Pat1 protein-protein interaction network after UV stress. We have shown that Pat1 co-purifies with Dhh1 specifically upon UV stress. We observed that the nuclear accumulation of Pat1 upon UV stress is abolished in a dhh1∆ strain. These data provide the first evidence that Dhh1 is required for Pat1 nuclear relocation after UV stress. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Thyroglobulin (Tg) is secreted by thyroid epithelial cells. It is essential for thyroid hormonogenesis and iodine storage. Although studied for many years, only indirect and partial surveys of its post-translational modifications were reported. Here, we present a direct proteomic approach, used to study the degree of iodination of mouse Tg without any preliminary purification. A comprehensive coverage of Tg was obtained using a combination of different proteases, MS/MS fragmentation procedures with inclusion lists and a hybrid mass high-resolution LTQ-Orbitrap XL mass spectrometer. Although only 16 iodinated sites are currently known for human Tg, we uncovered 37 iodinated tyrosine residues, most of them being mono- or diiodinated. We report the specific isotopic pattern of thyroxine modification, not recognized as a normal peptide pattern. Four hormonogenic sites were detected. Two donor sites were identified through the detection of a pyruvic acid residue in place of the initial tyrosine. Evidence for polypeptide cleavages sites due to the action of cathepsins and dipeptidyl proteases in the thyroid were also detected. This work shows that semi-quantitation of Tg iodination states is feasible for human biopsies and should be of significant medical interest for further characterization of human thyroid pathologies. [ABSTRACT FROM AUTHOR]

Drought is a major threat to coffee, compromising the quality and quantity of its production. We have analyzed the core proteome of 18 Coffea canephora cv. Conilon Clone 153 and C. arabica cv. Icatu plants and assessed their responses to moderate (MWD) and severe (SWD) water deficits. Label-free quantitative shotgun proteomics identified 3000 proteins in both genotypes, but less than 0.8% contributed to ca. 20% of proteome biomass. Proteomic changes were dependent on the severity of drought, being stronger under SWD and with an enrolment of different proteins, functions, and pathways than under MWD. The two genotypes displayed stress-responsive proteins under SWD, but only C. arabica showed a higher abundance of proteins involved in antioxidant detoxification activities. Overall, the impact of MWD was minor in the two genotypes, contrary to previous studies. In contrast, an extensive proteomic response was found under SWD, with C. arabica having a greater potential for acclimation/resilience than C. canephora. This is likely supported by a wider antioxidative response and an ability to repair photosynthetic structures, being crucial to develop new elite genotypes that assure coffee supply under water scarcity levels. [ABSTRACT FROM AUTHOR]

TiO2 nanoparticles (NPs) are one of the most produced NPs worldwide and are used in many consumer products. Their impact on human health, especially through inhalation, has been studied for more than two decades. TiO2 is known for its strong affinity towards phosphates, and consequently interaction with cellular phosphates may be one of the mechanisms driving its toxicity. In the present study, we used a phosphoproteomics approach to document the interaction of TiO2-NP with phosphoproteins from A549 human pulmonary alveolar epithelial cells. Cells were exposed to 21 nm anatase/rutile TiO2-NPs, then their phosphopeptides were extracted and analyzed using shotgun proteomics. By comparing the phosphoprotein content, phosphorylation status and phosphorylation sites of exposed cells with that of control cells, our results show that by affecting the phosphoproteome, TiO2-NPs affect cellular processes such as apoptosis, linked with cell cycle and the DNA damage response, TP53 being central to these pathways. Other pathways including inflammation and molecular transport are also affected. These molecular mechanisms of TiO2-NP toxicity have been reported previously, our study shows for the first time that they may derive from phosphoproteome modulation, which could be one of their upstream regulators. [ABSTRACT FROM AUTHOR]

Estuaries are important areas highly vulnerable to anthropogenic pollutions. Therefore, the assessment of estuarine water quality is a major ecological issue. In this study, we sampled juveniles of the European flounder in the "pristine" Canche estuary, and caged them in Canche and in two polluted sites of the Seine estuary, Rouen and Fosse Nord. After one month, the metal and organic pollutants in these sites were assessed, and we evaluated several phenotypic indicators (condition index, RNA/DNA ratios and genotoxicity), and extracted the proteins in fish livers for analysis using a shotgun proteomics approach. The results showed strong modifications in the fish caged in both sites of the Seine estuary, as compared to those caged in Canche. In particular, many proteins involved in phase I and phase II detoxification reactions were accumulated in the liver of fish caged in the site showing the highest pollution, Rouen. In addition, we observed a general disruption of metabolism, in particular an increase in lipid synthesis and carbohydrate degradation in Rouen, and a decrease in the abundance of proteins associated to translational activity in Fosse Nord. At both sites, several stress proteins were decreased. The proteomic impact of the encagement by itself was also evaluated, by comparing the liver proteome of fish caged in Canche to that of fish stayed in natura during the same time. The results showed proteomic signatures of exposure to stressful conditions (particularly heat stress), most probably related to the micro-habitat in which the cages were placed. In conclusion, the caging technique is of great interest for ecotoxicological assessment of estuarine waters, but should consider that the results are representative of the micro-habitat around the cages, which does not necessarily represent the overall heterogeneity of the estuarine environment. Unlabelled Image • Platichthys flesus juveniles were caged in two differentially polluted sites of the Seine estuary. • The liver proteome of fish phenotypes were determined. • Proteins implied in xenobiotics detoxification were positively deregulated in the most polluted area. • Control fish (caged in Canche) overexpressed many stress proteins. • Caging is an interesting technique to evaluate estuarine water quality at a micro-scale. [ABSTRACT FROM AUTHOR]