Uncovering proteome variations and concomitant quality changes of differently drying-treated rape (Brassica napus) bee pollen by label-free quantitative proteomics.

High moisture content of fresh bee pollen makes it difficult to preserve and thus makes drying a necessary process during the bee pollen production. Drying treatment will affect its quality and the effects of sun drying, hot-air drying and freeze drying on the proteome of rape (Brassica napus) bee pollen have been evaluated using label-free quantitative proteomics by liquid chromatography-tandem mass spectrometer (LC-MS/MS). A total of 8377 proteins are identified, among which the most abundant differential proteins were found in freeze drying-treated samples. Also freeze-drying treatment maximizes the content of antioxidant, antibacterial and anemic bioactive pollen protein. Besides, rape bee pollen is found to adjust its metabolism to protect itself during the drying process. These results can be favorable to evaluate the effects of drying treatment on the nutrition and function of processed rape bee pollen and insight into how rape bee pollen proteins respond to dehydration.

Development of a sample preparation method for micro-proteomics analysis of the formaldehyde-fixed paraffin-embedded liver tissue samples.

Liver micro-proteomics based on the routinely used formaldehyde-fixed paraffin-embedded (FFPE) samples is valuable for innovative research, but the technical approach for sample preparation is often challenging. In this study, we aimed to develop a method for sample preparation for micro-proteomics on using the FFPE liver samples. We collected 2000 individual cells per batch from FFPE liver slices with laser capture microdissection and used them as test samples. We used the microscale fresh-frozen liver samples or HepG2 cells as control samples. For the FFPE samples, we first established a procedure for protein extraction. 2 h incubation at 95 °C in alkaline amine buffer supplemented with 4% sodium dodecyl sulfate allows improved production, efficiency, and quality of protein extraction. Then, we developed a dedicated protocol HDMSP for the micro-concentrated (<0.05 µg/µL) protein preparation for mass spectrometry (MS) based analysis, in which 2 µg/µL carboxyl magnetic beads and 70% acetonitrile are used to induce protein precipitation. For the 0.01 µg/µL protein control samples, protein recovery rate (PRR) by HDMSP is 72.1%, while the PRR is 5.9% if using a standard method solid phase-enhanced sample preparation. For the FFPE samples, the HDMSP PRR is 88.8%, and the subsequent MS analysis demonstrates increased depth, robustness, and quantitation accuracy for HDMSP relative to the control of in-gel digestion. Moreover, the physicochemical properties and subcellular location of the FFPE liver micro-proteome are comparable to those of the fresh-frozen control samples processed with filter-aided sample preparation (FASP). HDMSP is also comparable to FASP in terms of reproducibility and physicochemical properties in liver subcellular proteomes, and meanwhile reduces the sample preparation time by 15.9% and the experimental cost by 30.8%. Overall, the new method is simple and highly effective for preparing the microscale FFPE liver protein samples for MS analysis. This study provides a useful solution for FFPE liver micro-proteomics.

Investigating comprehensive effects of depuration salinity and duration on posterior anhydrous living-preservation of Pacific oyster (Crassostrea gigas).

Depuration and anhydrous living-preservation are two important and consecutive steps during the circulation of Pacific oyster (Crassostrea gigas), and two main factors in depuration, salinity and duration, are pivotal to posterior anhydrous living-preservation of C. gigas. In this work, the comprehensive effects of depuration salinity (26-38 g/L) and duration (0-72 h) on anhydrous living-preservation at 4 °C for 7 days were investigated in regard of mortality, biochemical indexes (fatty acids profile analysis, glycogen) and proteome changes as well. The results showed that the mortality of C. gigas increased obviously with 72 h depuration and especially with 20 % salinity fluctuation, concomitantly accompanying metabolism disorder. Furthermore, alterations in salinity and duration resulted in 381 different expression proteins (DEPs), which were gotten more involved in the pathways related to amino acid metabolism. Taken together, it was suggested that duration < 48 h and salinity fluctuations lower than 10 % were the preferred conditions for anhydrous living-preservation.

Insight into differences in whey proteome from human and eight dairy animal species for formula humanization.

Human breastmilk fulfills the nutritional needs of infants and therefore is the best template for formula. In this study, whey proteins were investigated among human and eight dairy animal species using label-free proteomics approach. Totally, 965 proteins from milk whey were identified and large variations were observed between human and animals. Several proteins, including ß-galactosidase, fatty acid synthase, osteopontin, lactoferrin, mannose receptor, and complement C4-A, which are associated with digestion and immune response, exhibited significantly higher levels in human milk whey. Conversely, specific animal milk whey demonstrated elevated abundance of lipocalin 2, lysozyme, and glycosylation-dependent cell adhesion molecule 1. These differential proteins are enriched in complement and coagulation cascades, lysosome, and phagosome pathways. The findings shed light on the variations in the whey proteome composition between human and animal milk, which can contribute to optimizing formula humanization.

Plasma exosomes proteome profiling discovers protein markers associated with the therapeutic effect of Chaihu-Longgu-Muli decoction on temporal lobe epilepsy.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine (TCM) uses Chaihu-Longgu-Muli decoction (CLMD) to alleviate disease, clear away heat, calm the mind, and temper excitation. It has been widely used for the therapy of neuropsychiatric disorders including epilepsy, dementia, anxiety, insomnia, and depression for several centuries in China. AIM OF THE STUDY: This study aims to analyze differentially expressed proteins (DEPs) in the plasma exosomes of patients with temporal lobe epilepsy (TLE) and after the Chaihu-Longgu-Muli Decoction (CLMD) therapy and to explore the biomarkers of TLE and the potential targets of CLMD in treating TLE. MATERIALS AND METHODS: The plasma exosomes of normal people and patients with TLE before the treatment of oxcarbazepine (OXC) and combined treatment of OXC and CLMD (OXC.CLMD) were harvested. The exosomes were separated from plasma through ultracentrifugation and then identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. The DEPs were analyzed by proteomics and then subjected to gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The protein level of key genes was detected using Western blot. A lithium chloride-pilocarpine-induced epilepsy rat model was established and treated with OXC alone, OXC. CLMD, and CLMD alone (low dose and high dose). Neuronal injury in the hippocampal dentate gyrus and ribosomal protein L6 (RPL6) expression in the brain tissues were detected using H&E staining, Nissl staining, and Western blot. RESULTS: The proteomic analysis showed several DEPs were present among plasma exosomes in the four groups; DEPs were enriched in epilepsy-related function and pathway. Four key proteins were screened, including RPL6, Nucleolin (NCL), Apolipoprotein A1 (APOA1), and Lactate Dehydrogenase A (LDHA). Among them, RPL6, NCL, and LDHA protein levels were downregulated and APOA1 protein level was upregulated in the plasma exosomes of TLE patients. After OXC and OXC. CLMD treatment, the protein level of RPL6, NCL, and LDHA was increased, and the APOA1 protein level was decreased. Moreover, the RPL6 protein level was further elevated after OXC. CLMD treatment than that after OXC treatment. In the TLE rat model, neuronal degeneration and necrosis in the hippocampal dentate gyrus increased and RPL6 expression level decreased. After the treatment with OXC, OXC. CLMD, and CLMD alone, the degeneration and necrosis of neurons decreased, and the RPL6 expression level was increased; RPL6 upregulation was remarkably obvious after CLMD treatment. CONCLUSIONS: RPL6, NCL, LDHA and APOA1 are the DEPs in the plasma exosomes of patients with TLE before and after therapy. RPL6 might be a potential biomarker of CLMD in treating TLE.

Quantitative serum proteome analysis using tandem mass tags in dogs with epilepsy.

This study included four groups of dogs (group A: healthy controls, group B: idiopathic epilepsy receiving antiepileptic medication (AEM), group C: idiopathic epilepsy without AEM, group D: structural epilepsy). Comparative quantitative proteomic analysis of serum samples among the groups was the main target of the study. Samples were analyzed by a quantitative Tandem-Mass-Tags approach on the Q-Exactive-Plus Hybrid Quadrupole-Orbitrap mass-spectrometer. Identification and relative quantification were performed in Proteome Discoverer. Data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD041129. Eighty-one proteins with different relative adundance were identified in the four groups and 25 were master proteins (p < 0.05). Clusterin (CLU), and apolipoprotein A1 (APOA1) had higher abundance in the three groups of dogs (groups B, C, D) compared to controls. Amine oxidase (AOC3) was higher in abundance in group B compared to groups C and D, and lower in group A. Adiponectin (ADIPOQ) had higher abundance in groups C compared to group A. ADIPOQ and fibronectin (FN1) had higher abundance in group B compared to group C and D. Peroxidase activity assay was used to quantify HP abundance change, validating and correlating with proteomic analysis (r = 0.8796). SIGNIFICANCE: The proteomic analysis of serum samples from epileptic dogs indicated potential markers of epilepsy (CLU), proteins that may contribute to nerve tissue regeneration (APOA1), and contributing factors to epileptogenesis (AOC3). AEM could alter extracellular matrix proteins (FN1). Illness (epilepsy) severity could influence ADIPOQ abundance.

Unraveling proteome changes of Sunit lamb meat in different feeding regimes and its relationship to flavor analyzed by TMT-labeled quantitative proteomic.

In order to explore the molecular mechanism of the effect of feeding regimes on lamb flavor, biceps femoris muscle samples from pasture-fed groups (PF) and concentrate-fed groups (CF) were chosen, and tandem mass tag (TMT) labeling combined with mass spectrometry (MS) was performed to find associations between flavor indicators and proteome profiles. The content and composition of amino acids and volatile flavor substances were better in the PF compared to the CF, with higher levels of some beneficial flavor components such as Arg, Pro Pentanal, Heptanal, Octanal, 1-octen-3-ol and 2,3-Octanedione. About 82 differentially abundant proteins (DAPs) were identified. The Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis indicated that the pathways that may be associated with lamb flavor are focused on amino acid anabolism. These results provide a basis for further understanding of the molecular mechanisms of proteins in meat flavor regulation.

Quantitative proteomics based bioactive proteins discovery and quality control of medicinal leeches.

ETHNOPHARMACOLOGICAL RELEVANCE: Leech, a classical traditional Chinese medicine for promoting blood circulation and removing blood stasis, is mainly used in the clinical treatment of cardiovascular and cerebrovascular diseases. The discovery of activity proteins or peptides in the dead and dried medicinal leech is an important task with great challenges. AIM OF THE STUDY: The aim of this study was to provide a basic proteome profile and help further discover active proteins and quality control for medicinal leeches, which would also provide insight into the research of animal medicines. MATERIALS AND METHODS: Seventeen batches of dried medicinal leeches covering three species were collected from medicinal markets, which were authenticated by DNA barcoding. Then the proteome of different species leeches was profiled to reveal the significantly different proteins using label-free proteomics. The characteristic peptides were screened out based on biological pathways analysis, which were further absolutely quantified using the developed stable isotope-labeled based parallel reaction monitoring method. RESULTS: Seventeen batches of leech materials were Whitmania pigra Whitman (WP), Whitmania laevis Whitman (WL) and Poecilobdella manillensis Lesson (PM), respectively. A total of 1,035 proteins (452 in WP, 425 in WL and 158 in PM) were identified. Among them, 90 overlapping proteins were mainly concentrated in diverse metabolic pathways and primarily localized in the cytoplasm and mitochondrial inner membrane, which mainly related to ATP binding, catalytic activity and structural molecular activity. In total of 51 uniquely expressed proteins (21 in WP, 23 in WL and 7 in PM), associated with multiple key signaling pathways, including Rap1, cGMP-PKG, PI3K-Akt, Wnt and HIF-1, etc., relevant to treating cardiovascular diseases, diabetes, cancer and even a variety of neurodegenerative diseases. Three proteins with potential bioactivities, including Neurohemerythrin, Hirudin and Eglin C, were selected as the quality makers and then quantified based on the characteristic peptides. CONCLUSIONS: This work profiled the proteome of three species of leeches, and addressed potential active proteins of the medicinal leech, which would help to provide the potential molecular mechanisms involved in disease treatment. The proteomics-based approach developed in this work is not only useful for the discovery of proteins with potential bioactivities but also helpful for the bioactivity relevant quality control of animal medicines.

Mitochondrial phosphoproteomes are functionally specialized across tissues.

Mitochondria are essential organelles whose dysfunction causes human pathologies that often manifest in a tissue-specific manner. Accordingly, mitochondrial fitness depends on versatile proteomes specialized to meet diverse tissue-specific requirements. Increasing evidence suggests that phosphorylation may play an important role in regulating tissue-specific mitochondrial functions and pathophysiology. Building on recent advances in mass spectrometry (MS)-based proteomics, we here quantitatively profile mitochondrial tissue proteomes along with their matching phosphoproteomes. We isolated mitochondria from mouse heart, skeletal muscle, brown adipose tissue, kidney, liver, brain, and spleen by differential centrifugation followed by separation on Percoll gradients and performed high-resolution MS analysis of the proteomes and phosphoproteomes. This in-depth map substantially quantifies known and predicted mitochondrial proteins and provides a resource of core and tissue-specific mitochondrial proteins (mitophos.de). Predicting kinase substrate associations for different mitochondrial compartments indicates tissue-specific regulation at the phosphoproteome level. Illustrating the functional value of our resource, we reproduce mitochondrial phosphorylation events on dynamin-related protein 1 responsible for its mitochondrial recruitment and fission initiation and describe phosphorylation clusters on MIGA2 linked to mitochondrial fusion.

Changes of the salivary and serum proteome in canine hypothyroidism.

In this study, changes in salivary and serum proteome of dogs with hypothyroidism were studied using tandem mass tags (TMT) labelling and liquid chromatography-mass spectrometry (LC-MS/MS). Saliva and serum proteome from 10 dogs with hypothyroidism were compared with 10 healthy dogs. In saliva, a total of seven proteins showed significant changes between the two groups, being six downregulated and one upregulated, meanwhile, in serum, a total of six proteins showed significant changes, being five downregulated and one upregulated. The altered proteins reflected metabolic and immunologic changes, as well as, skin and coagulation alterations, and these proteins were not affected by gender. One of the proteins that were downregulated in saliva, lactate dehydrognease (LDH), was measured by a spectrophotometric assay in saliva samples from 42 dogs with hypothyroidism, 42 dogs with non-thyroid diseases and 46 healthy dogs. The activity of LDH was lower in the saliva of hypothyroid dogs when compared to non-thyroid diseased dogs and healthy controls. This study indicates that canine hypothyroidism can produce changes in the proteome of saliva and serum. These two sample types showed different variations in their proteins reflecting physiopathological changes that occur in this disease, mainly related to the immune system, metabolism, skin and coagulation. In addition, some of the proteins identified in this study, specially LDH in saliva, should be further explored as potential biomarkers of canine hypothyroidism.

Defining Membrane Protein Localization by Isopycnic Density Gradients.

Bacterial membrane proteins account for around one-third of the proteome in many species and can represent much more than half of the mass of the membranes. Classic techniques in cell biology can be applied to characterize bacterial membranes and their membrane protein constituents, and here we describe a protocol for the purification of outer membranes and inner membranes from Escherichia coli. This allows for compositional analysis of the membranes as well as functional analyses. The procedure can be applied with minor modifications to other bacterial species including those carrying capsular polysaccharide attached to the outer membrane.

Integrative proteome and metabolome analyses reveal molecular basis underlying growth and nutrient composition in the Pacific oyster, Crassostrea gigas.

In order to comprehend the molecular basis of growth, nutrient composition, and color pigmentation in oysters, comparative proteome and metabolome analyses of two selectively bred oyster strains with contrasting growth rate and shell color were used in this study. A total of 289 proteins and 224 metabolites were identified differentially expressed between the two strains. We identified a series of specifically enriched functional clusters implicated in protein biosynthesis (RPL4, MRPS7, and CARS), fatty acid metabolism (ACSL5, PEX3, ACOXI, CPTIA, FABP6, and HSD17B12), energy metabolism (FH, PPP1R7, CLAM2, and RGN), cell proliferation (MYB, NFYC, DOHH, TOP2a, SMARCA5, and SMARCC2), material transport (ABCB1, ABCB8, VPS16, and VPS33a), and pigmentation (RDH7, RDH13, Retsat, COX15, and Cyp3a9). Integrated proteome and metabolome analyses indicate that fast-growing strain utilize energy-efficient mechanisms of ATP generation while promoting protein and polyunsaturated fatty acid synthesis, activating the cell cycle to increase cell proliferation and thus promoting their biomass increase. These results uncovered molecular mechanisms underlying growth regulation, nutrition quality, and pigmentation and provided candidate biomarkers for molecular breeding in oysters. SIGNIFICANCE: Rapid growth has always been the primary breeding objective to increase the production profits of Pacific oyster (Crassostrea gigas), while favorable nutritional quality and beautiful color add commercial value. In recent years, proteomic and metabolomic techniques have been widely used in marine organisms, although these techniques are seldom utilized to study oyster growth and development. In this study, two C. gigas strains with contrasted phenotypes in growth and shell color provided an ideal model for unraveling the molecular basis of growth and nutrient composition through a comparison of the proteome and metabolome. Since proteins and metabolites are the critical undertakers and the end products of cellular regulatory processes, identifying the differentially expressed proteins and metabolites would allow for discovering biomarkers and pathways that were implicated in cell growth, proliferation, and other critical functions. This work provides valuable resources in assistance with molecular breeding of oyster strains with superior production traits of fast-growth and high-quality nutrient value.

Histologic and proteomic profile of two methods to decellularize human dental pulp tissue.

OBJECTIVE: The present study compared the structural and proteomic architecture of extracellular matrices (ECM) of decellularized human dental pulp using two previously described protocols. DESIGN: Pulp tissue from 150 molars was extracted and three treatments took place, based on the Matoug-Elwerfelli Group (MG) and the Song Group (SG) protocols and an untreated pulp group (CG), to examine histoarchitecture and the effectiveness of the decellularization process, using histological analysis (n = 12) and scanning electron microscopy (SEM) (n = 3). Protein extraction took place using 100 mg dry weight of pulp, in triplicates for each group, and the shotgun proteome analysis was performed by nanoUPLC-MSE. Proteins were identified using the revised human UNIPROT database attached to the PLGS search engine. RESULTS: Histological analysis and SEM demonstrated that ECM in MG was more preserved. Proteome analysis showed that the decellularized process in MG maintained approximately 69.56% of proteins identified in untreated pulp tissue while SG maintained 28.26%. CONCLUSIONS: ECM appears to be suitable as a potential biological scaffold for pulp revascularization and regeneration procedures, especially those processed according to the Matoug-Elwerfelli protocol. This finding can collaborate to enhance clinical solutions for young permanent teeth that have suffered necrosis.

The GENTIL Method for Isolation of Human Adult Cardiomyocytes from Cryopreserved Tissue for Proteomic Analyses.

Heart failure is a serious clinical and economic health care problem, and its clinical progression is linked to pathological cardiac remodeling. Due to the heterogeneity of heart failure, lack of animal models to accurately represent advanced heart failure, and limited access to fresh human cardiac tissue, little is known regarding cell-type-specific mechanisms and context-specific functions of cardiomyocytes during disease development processes. While mass spectrometry has been increasingly applied to unravel changes in the proteome associated with cardiovascular physiology and disease, most studies have used homogenized tissue. Therefore, new studies using isolated cardiomyocytes are necessary to gain a better understanding of the intricate cell-type-specific molecular mechanisms underlying the pathophysiology of heart failure. This chapter describes the GENTIL method, which incorporates recent technological developments in sample handling, for isolation of cardiomyocytes from cryopreserved human cardiac tissues for use in proteomic analyses.

N-Terminomics/TAILS of Human Tumor Biopsies and Cancer Cell Lines.

Proteases serve essential roles in numerous biological processes and signaling cascades by cleaving their substrates in a restricted manner or via degradation. It is important to determine which proteins are protease substrates and where their cleavage sites are located to characterize the impact of proteolysis on the molecular mechanisms of their substrates. N-terminomics is a branch of proteomics that enriches the N-terminal sequence of proteins. A proteome-wide collection of these sequences has been broadly applied to comprehend proteolytic cascades and for genome annotation. Terminal Amine Isotopic Labeling of Substrates (TAILS) is a combined N-terminomics and proteomics technique that has been applied for protein N-terminal characterization and quantification of natural and neo-N-termini of proteins using liquid chromatography and tandem mass spectrometry (LC-MS/MS). TAILS uses negative selection to enrich both original mature protein N-termini and neo-N-termini produced from proteolysis in a proteome labeled with isotopic tags. This approach has been applied to the investigation of protease function and substrate identification in cell culture systems, animal disease models, and, most recently, clinical samples such as blood and tumor tissues from cancer patients.

MultiPro: DDA-PASEF and diaPASEF acquired cell line proteomic datasets with deliberate batch effects.

Mass spectrometry-based proteomics plays a critical role in current biological and clinical research. Technical issues like data integration, missing value imputation, batch effect correction and the exploration of inter-connections amongst these technical issues, can produce errors but are not well studied. Although proteomic technologies have improved significantly in recent years, this alone cannot resolve these issues. What is needed are better algorithms and data processing knowledge. But to obtain these, we need appropriate proteomics datasets for exploration, investigation, and benchmarking. To meet this need, we developed MultiPro (Multi-purpose Proteome Resource), a resource comprising four comprehensive large-scale proteomics datasets with deliberate batch effects using the latest parallel accumulation-serial fragmentation in both Data-Dependent Acquisition (DDA) and Data Independent Acquisition (DIA) modes. Each dataset contains a balanced two-class design based on well-characterized and widely studied cell lines (A549 vs K562 or HCC1806 vs HS578T) with 48 or 36 biological and technical replicates altogether, allowing for investigation of a multitude of technical issues. These datasets allow for investigation of inter-connections between class and batch factors, or to develop approaches to compare and integrate data from DDA and DIA platforms.

Proteomic analysis reveals changes in the proteome of human THP-1 macrophages infected with Paracoccidioides brasiliensis.

Paracoccidioides spp. is the etiologic agent of Paracoccidioidomycosis (PCM), a systemic disease with wide distribution in Latin America. Macrophages are very important cells during the response to infection by P. brasiliensis. In this study, we performed a proteomic analysis to evaluate the consequences of P. brasiliensis yeast cells on the human THP-1 macrophage proteome. We have identified 443 and 2247 upregulated or downregulated proteins, respectively, in macrophages co-cultured with yeast cells of P. brasiliensis in comparison to control macrophages unexposed to the fungus. Proteomic analysis revealed that interaction with P. brasiliensis caused metabolic changes in macrophages that drastically affected energy production pathways. In addition, these macrophages presented regulated many factors related to epigenetic modifications and gene transcription as well as a decrease of many proteins associated to the immune system activity. This is the first human macrophage proteome derived from interactions with P. brasiliensis, which contributes to elucidating the changes that occur during the host response to this fungus. Furthermore, it highlights proteins that may be targets for the development of new therapeutic approaches to PCM.

An interdisciplinary therapy for lifestyle change is effective in improving psychological and inflammatory parameters in women with grade I obesity.

Obesity and depression, disorders associated with inflammation, have high incidences in women. Understanding the derangements present in the initial phase of obesity may point to factors that could help avoiding disease aggravation. The present study aimed at investigating the effects of a 6-months interdisciplinary therapy for weight loss in women with grade I obesity. Before and after the therapy, 37 middle-aged women donated blood and responded to questionnaires for depression and anxiety symptoms. Inflammatory parameters were evaluated in serum and a preliminary screening of the plasma proteome was performed. The therapy decreased anthropometric, psychological scores, and serum levels of inflammatory parameters. Depression and anxiety scores correlated positively with some inflammatory parameters. The proteomic analysis showed changes in proteins related to cholesterol metabolism and inflammatory response. Interdisciplinary therapy improves anthropometric and inflammatory statuses and ameliorating psychological symptoms. The decrease of MCP-1 levels after interdisciplinary therapy has not been reported so far, at the best of our knowledge. The present demonstration of positive associations of inflammatory markers and psychological scores indicate that these mediators may be useful to monitor psychological status in obesity. The present proteome data, although preliminary, pointed to plasma alterations indicative of improvement of inflammation after interdisciplinary therapy.

The effects of Phycocyanobilin on experimental arthritis involve the reduction in nociception and synovial neutrophil infiltration, inhibition of cytokine production, and modulation of the neuronal proteome.

Introduction: The antinociceptive and pharmacological activities of C-Phycocyanin (C-PC) and Phycocyanobilin (PCB) in the context of inflammatory arthritis remain unexplored so far. In the present study, we aimed to assess the protective actions of these compounds in an experimental mice model that replicates key aspects of human rheumatoid arthritis. Methods: Antigen-induced arthritis (AIA) was established by intradermal injection of methylated bovine serum albumin in C57BL/6 mice, and one hour before the antigen challenge, either C-PC (2, 4, or 8 mg/kg) or PCB (0.1 or 1 mg/kg) were administered intraperitoneally. Proteome profiling was also conducted on glutamate-exposed SH-SY5Y neuronal cells to evaluate the PCB impact on this key signaling pathway associated with nociceptive neuronal sensitization. Results and discussion: C-PC and PCB notably ameliorated hypernociception, synovial neutrophil infiltration, myeloperoxidase activity, and the periarticular cytokine concentration of IFN-γ, TNF-α, IL-17A, and IL-4 dose-dependently in AIA mice. In addition, 1 mg/kg PCB downregulated the gene expression for T-bet, RORγ, and IFN-γ in the popliteal lymph nodes, accompanied by a significant reduction in the pathological arthritic index of AIA mice. Noteworthy, neuronal proteome analysis revealed that PCB modulated biological processes such as pain, inflammation, and glutamatergic transmission, all of which are involved in arthritic pathology. Conclusions: These findings demonstrate the remarkable efficacy of PCB in alleviating the nociception and inflammation in the AIA mice model and shed new light on mechanisms underlying the PCB modulation of the neuronal proteome. This research work opens a new avenue to explore the translational potential of PCB in developing a therapeutic strategy for inflammation and pain in rheumatoid arthritis.

Large-scale phosphoproteome analysis in wheat seedling leaves provides evidence for extensive phosphorylation of regulatory proteins during CWMV infection.

BACKGROUND: Chinese wheat mosaic virus (CWMV) often causes severe damage to wheat (Triticum aestivum L.) growth and yield. It is well known that a successful infection in plants depends on a complex interaction between the host plant and the pathogen. Post-translational modification (PTM) of proteins is considered to be one of the main processes that decides the outcome of the plant-pathogen arms race during this interaction. Although numerous studies have investigated PTM in various organisms, there has been no large-scale phosphoproteomic analysis of virus-infected wheat plants. We therefore aimed to investigate the CWMV infection-induced phosphoproteomics changes in wheat by high-resolution liquid chromatography-tandem mass spectroscopy (LC-MS/MS) using affinity-enriched peptides followed by comprehensive bioinformatics analysis. RESULTS: Through this study, a total of 4095 phosphorylation sites have been identified in 1968 proteins, and 11.6% of the phosphorylated proteins exhibited significant changes (PSPCs) in their phosphorylation levels upon CWMV infection. The result of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that most of the PSPCs were associated with photosynthesis, plant-pathogen interactions, and MAPK signaling pathways. The protein-protein interaction (PPI) network analysis result showed that these PSPCs were mainly participated in the regulation of biosynthesis and metabolism, protein kinase activities, and transcription factors. Furthermore, the phosphorylation levels of TaChi1 and TaP5CS, two plant immunity-related enzymes, were significantly changed upon CWMV infection, resulting in a significant decrease in CWMV accumulation in the infected plants. CONCLUSIONS: Our results indicate that phosphorylation modification of protein plays a critical role in wheat resistance to CWMV infection. Upon CWMV infection, wheat plants will regulate the levels of extra- and intra-cellular signals and modifications of enzyme activities via protein phosphorylation. This novel information about the strategies used by wheat to resist CWMV infection will help researchers to breed new CWMV-resistant cultivars and to better understand the arms race between wheat and CWMV.