High growth rates and body weight are important traits of young dairy goats that can shorten generation intervals, improve animal performance, and increase economic benefits. In the present study, ninety-nine, 6-month-old, female goats were fed with the same diet and kept under the same management condition. The ten goats with highest average daily gain (ADG, HADG, 135.27 ± 4.59 g/d) and ten goats with lowest ADG (LADG, 87.74 ± 3.13 g/d) were selected to identify the key serum metabolites associated with ADG, and to investigate the relationships of serum metabolome profiles with digestive tract microbiota. The results showed that a total of 125 serum metabolites were significantly different between HADG and LADG. Of these, 43 serum metabolites were significantly higher levels in HADG, including D-ornithine, l-glutamine, L-histidine, carnosine, LysoPC (16:1(9Z)/0:0), DCTP and hydroxylysine, while, 82 serum metabolites were significantly higher levels in LADG, including P-salicylic acid and deoxycholic acid 3-glucuronide. Pathway analysis indicated that these different metabolites were mainly involved in amino acid and lipid metabolism. Furthermore, Spearman's rank correlation analysis revealed that these differential serum metabolites were correlated with ADG and ADG-related bacteria. Notably, serum hydroxylysine and L-histidine could be used as biomarkers for distinguishing HADG and LADG goats, with an accuracy of >92.0%. SIGNIFICANCE: Our study confirms that individual microbiota and metabolic differences contribute to the variations of growth rate in young goats. Some serum metabolites may be useful in improving the growth performance of young goats, which provides directions for developing further nutritional regulation in the goat industry to achieve healthy feeding and efficiency enhancement.

Aging is accompanied by deterioration in physical condition, and creates high risks of diseases. Stem cell therapy exhibited promising potential in delaying aging. However, the unelucidated therapeutic mechanism limits future clinical application. Herein, to systematically understand the response to stem cell transfusion at the molecular level, we performed quantitative serum proteomic and peptidomics analyses in the 24-month-old aging mice model with or without mesenchymal stem cell (MSC) treatment. As a result, a total of 560 proteins and 2131 endogenous peptides were identified, among which, 6 proteins and 9 endogenous peptides derived from 6 precursor proteins were finally identified as therapeutic biomarkers after MSC transfusion on aging mice both by untargeted label-free quantification and targeted parallel reaction monitoring (PRM) quantification. Amazingly, the biological function of these differential proteins was mainly related to inflammation, which is not only the important hallmark of aging, but also the main cause of inducing aging. The reduction of these inflammatory protein content after MSC treatment further suggests the anti-inflammatory effect of MSC therapy reported elsewhere. Therefore, our study provides new evidence for the anti-inflammatory effect of MSC therapy for anti-aging and offers abundant data to support deeper investigations of the therapeutic mechanism of MSC in delaying aging.

Although the microgravity (μ-g) environment that astronauts encounter during spaceflight can cause severe acute bone loss, the molecular mechanism of this bone loss remains unclear. To investigate the gravity-response proteins involved in bone metabolism, it is important to comprehensively determine which proteins exhibit differential abundance associated with mechanical stimuli. However, comprehensive proteomic analysis using small bone samples is difficult because protein extraction in mineralized bone tissue is inefficient. Here, we established a high-sensitivity analysis system for mouse bone proteins using data-independent acquisition mass spectrometry. This system successfully detected 40 proteins in the femoral diaphysis showing differential abundance between mice raised in a μ-g environment, where the bone mass was reduced by gravity unloading, and mice raised in an artificial 1-gravity environment on the International Space Station. Additionally, 22 proteins, including noncollagenous bone matrix proteins, showed similar abundance between the two groups in the mandible, where bone mass was unaltered due to mastication stimuli, suggesting that these proteins are responsive to mechanical stimuli. One of these proteins, SPARCL1, is suggested to promote osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand. We expect these findings to lead to new insights into the mechanisms of bone metabolism induced by mechanical stimuli. SIGNIFICANCE: We aimed to investigate the gravity-response proteins involved in bone metabolism. To this end, we established a comprehensive analysis system for mouse bone proteins using data-independent acquisition mass spectrometry, which is particularly useful in comprehensively analyzing the bone proteome using small sample volumes. In addition, a comprehensive proteomic analysis of the femoral diaphysis and mandible, which exhibit different degrees of bone loss in mice raised on the International Space Station, identified proteins that respond to mechanical stimuli. SPARCL1, a mechanical stimulus-responsive protein, was consequently suggested to be involved in osteoclast differentiation associated with bone remodeling. Our findings represent an important step toward elucidating the molecular mechanism of bone metabolism induced by mechanical stimuli.

Cancer-associated fibroblasts (CAFs) are a key component of tumors. We aimed to profile the proteome of cancer cell lines representing three common cancer types (lung, colorectal and pancreatic) and a representative CAF cell line from each tumor type to gain insight into CAF function and novel CAF biomarkers. We used isobaric labeling, liquid chromatography and mass spectrometry to evaluate the proteome of 9 cancer and 3 CAF cell lines. Of the 9460 proteins evaluated, functional enrichment analysis revealed an upregulation of N-glycan biosynthesis and extracellular matrix proteins in CAFs. 85 proteins had 16-fold higher expression in CAFs compared to cancer cells, including previously known CAF markers like fibroblast activating protein (FAP). Novel overexpressed CAF biomarkers included heat shock protein β-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1). SiRNA knockdown of the genes encoding these proteins did not reduce contractility in lung CAFs, suggesting they were not crucial to this function. Immunohistochemical analysis of 30 tumor samples (10 lung, 10 colorectal and 10 pancreatic) showed restricted HSPB6 and PTGS1 expression in the stroma. Therefore, we describe an unbiased differential proteome analysis of CAFs compared to cancer cells, which revealed higher expression of HSPB6 and PTGS1 in CAFs. Data are available via ProteomeXchange (PXD040360). SIGNIFICANCE: Cancer-associated fibroblasts (CAFs) are highly abundant stromal cells present in tumors. CAFs are known to influence tumor progression and drug resistance. Characterizing the proteome of CAFs could give potential insights into new stromal drug targets and biomarkers. Mass spectrometry-based analysis comparing proteomic profiles of CAFs and cancers characterized 9460 proteins of which 85 proteins had 16-fold higher expression in CAFs compared to cancer cells. Further interrogation of this rich resource could provide insight into the function of CAFs and could reveal putative stromal targets. We describe for the first time that heat shock protein β-6 (HSPB6/HSP20) and cyclooxygenase 1 (PTGS1/COX1) are overexpressed in CAFs compared to cancer cells.

Danshen, belongs to the Lamiaceae family, and its scientific name is Salvia miltiorrhiza Bunge. It is a valuable medicinal plant to prevent and treat cardiovascular and cerebrovascular diseases. Lysine succinylation, a widespread modification found in various organisms, plays a critical role in regulating secondary metabolism in plants. The hairy roots of Salvia miltiorrhiza were subject to proteomic analysis to identify lysine succinylation sites using affinity purification and HPLC-MS/MS in this investigation. Our findings reveal 566 lysine succinylation sites in 348 protein sequences. We observed 110 succinylated proteins related to secondary metabolism, totaling 210 modification sites. Our analysis identified 53 types of enzymes among the succinylated proteins, including phenylalanine ammonia-lyase (PAL) and aldehyde dehydrogenase (ALDH). PAL, a crucial enzyme involved in the biosynthesis of rosmarinic acid and flavonoids, displayed succinylation at two sites. ALDH, which participates in the phenylpropane metabolic pathway, was succinylated at 8 eight sites. These observations suggest that lysine succinylation may play a vital role in regulating the production of secondary metabolites in Salvia miltiorrhiza. Our study may provide valuable insights for further investigation on plant succinylation, specifically as a reference point. SIGNIFICANCE: Salvia miltiorrhiza Bunge is a valuable medicinal plant that prevents and treats cardiovascular and cerebrovascular diseases. Lysine succinylation plays a critical role in regulating secondary metabolism in plants. The hairy roots of Salvia miltiorrhiza were subject to proteomic analysis to identify lysine succinylation sites using affinity purification and HPLC-MS/MS in this investigation. These observations suggest that lysine succinylation may act as a vital role in regulating the production of secondary metabolites in Salvia miltiorrhiza. Our study may provide valuable insights for further investigation on succinylation in plants, specifically as a reference point.

Metabolites are the final products of metabolism and provide insights into the biochemical balance of tissue systems. A cascade of reactions involving proteins, carbohydrates, and lipids affects meat color, tenderness, and flavor, specifically, metabolites that are key biomolecules in biochemical reactions associated with attainment of acceptable meat quality. Bioinformatics platforms, such as Kyoto Encyclopedia of Genes and Genomes (KEGG) databases and MetaboAnalyst, are utilized to help understanding the role of differentially abundant metabolites and characterizing their roles in cellular function/metabolism. However, the inability to identify all metabolites using a single platform and limited metabolite libraries specifically for meat/food remains a challenge. Therefore, the advances in metabolite separation, easy-to-use data processing, increased resolution of mass-spectrometry, and data analysis will help to make inferences or develop biomarkers related to meat quality. This review discusses how metabolomics can be exploited to characterize meat quality, the challenges, and current trends. SIGNIFICANCE: Metabolites play an important role in attaining consumer-preferred meat quality traits and nutritive value of foods. Visual appearance of fresh foods, such as muscle foods, are utilized by consumers to assess the quality at the retail market before making purchases. Similarly, tenderness and flavor of meats influence eating satisfaction and re-purchase decisions. Inconsistencies in meat quality lead to enormous economic losses to the food industry. For instance, consumers often associate a bright-cherry red color with freshness, and the US beef industry loses $3.74 billion annually due to discoloration during storage. Both pre-and post-harvest factors influence the extent of meat quality changes. Metabolomics offer robust tools to get a snapshot of small molecules such as acids, amino acids, glycolytic- and tricarboxylic acids, fatty acids, and sugars present in post-mortem muscle tissue and their role in meat quality. Further, using bioinformatics platforms enables characterizing the role of differentially present metabolites in meat quality as well as identifying biomarkers for desirable quality traits such as tender meat or color-stable carcasses. Innovative applications of metabolomics can be exploited to elucidate the underpinnings of meat quality and to develop novel strategies to enhance marketability of retail fresh meats.

Tuberous sclerosis complex (TSC) is a rare, multisystem genetic disorder that leads to the development of benign tumors in multiple organs and neurological symptoms. TSC clinical manifestations show a great heterogenicity, with most patients presenting severe neuropsychiatric and neurological disorders. TSC is caused by loss-of-function mutations in either TSC1 or TSC2 genes, leading to overexpression of the mechanistic target of rapamycin (mTOR) and, consequently, abnormal cellular growth, proliferation and differentiation as well as to cell migration defects. Beside the growing interest, TSC remains a disorder poorly understood, with limited perspectives in the field of therapeutic strategies. Here we used murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene as a TSC model to unravel novel molecular aspects of the pathophysiology of this disease. 2D-DIGE-based proteomic analysis detected 55 differently represented spots in Tsc1-deficient cells, compared to wild-type counterparts, which were associated with 36 protein entries after corresponding trypsinolysis and nanoLC-ESI-Q-Orbitrap-MS/MS analysis. Proteomic results were validated using various experimental approaches. Bioinformatics associated differently represented proteins with oxidative stress and redox pathways, methylglyoxal biosynthesis, myelin sheath, protein S-nitrosylation and carbohydrate metabolism. Because most of these cellular pathways have already been linked to TSC features, these results were useful to clarify some molecular aspects of TSC etiopathogenesis and suggested novel promising therapeutic protein targets. SIGNIFICANCE: Tuberous Sclerosis Complex (TSC) is a multisystemic disorder caused by inactivating mutations of TSC1 or TSC2 genes, which induce overactivation of the mTOR component. The molecular mechanisms underlying the pathogenesis of TSC remain unclear, probably due to complexity of mTOR signaling network. To have a picture of protein abundance changes occurring in TSC disorder, murine postnatal subventricular zone (SVZ) neural stem progenitor cells (NSPCs) deficient of Tsc1 gene were used as a model of disease. Thus, Tsc1-deficient SVZ NSPCs and wild-type cells were comparatively evaluated by proteomics. This analysis evidenced changes in the abundance of proteins involved in oxidative/nitrosative stress, cytoskeleton remodelling, neurotransmission, neurogenesis and carbohydrate metabolism. These proteins might clarify novel molecular aspects of TSC etiopathogenesis and constitute putative molecular targets for novel therapeutic management of TSC-related disorders.

Lactic acid bacteria (LAB) and their metabolites such as bacteriocins have gained considerable interest in terms of their bio-preservative properties to improve food safety and quality. In this study, a quantitative proteomic investigation employing stable isotope labeling by peptide demethylation was carried out to investigate changes in intracellular proteins of bacteriocin-like substance (BLS) producing Lactococcus spp. 7.17 grown in vegetable or fruit juice culture media at 10 °C for 0, 3 or 7 days. In total, 1053 proteins in vegetable medium and 1113 in fruit medium were identified and quantified. Proteins that changed more than two- fold were identified as increased or decreased ones and grouped into four clusters. Those increased proteins were involved in the events of low temperature and ROS stress responses, DNA processing, transcription and translation, central carbon metabolism, fatty acid and phospholipid metabolism, amino acid and cell wall biosynthesis. Key proteins in relation to BLS producing property were also identified suggesting that at least one bacteriocin IIa production system exists in Lactococcus spp. 7.17. These findings provide insights into protein changes of L. lactis at low temperature and lay foundations for further investigations on BLS producing LAB using targeted quantitative proteomic approaches. SIGNIFICANCE OF THE RESEARCH: The inhibitory effects of Lactococcus spp. 7.17 on Listeria innocua in fruit and vegetable juice culture media were confirmed. Using a quantitative proteomic approach employing stable isotope labeling by peptide demethylation, 99 or 113 significantly changed proteins of Lactococcus spp. 7.17 grown in vegetable or fruit juice medium were determined, respectively. The significant change in protein abundance suggested an adaptive mechanism of Lactococcus spp. to culture condition at low temperatures. This research provides insights on protein changes of Lactococcus spp. which has potential application in fresh and fresh-cut fruit and vegetables at low temperature.

Eimeria necatrix is an obligate intracellular parasite that has a complex life cycle and causes significant economic losses to the poultry industry. To better understand the cellular invasion mechanism of E. necatrix and develop new measures against its infection, we conducted isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis to investigate protein abundance across different life cycle stages, including unsporulated oocysts (UO), sporozoites (SZ) and second-generation merozoites (MZ-2). Our analysis identified a total of 3606 proteins, among which 1725, 1724, 2143 and 2386 were annotated by the Gene Ontology (GO), EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) and InterPro (IPR) databases, respectively. We also identified 388, 300 and 592 differentially abundant proteins in SZ vs UO, SZ vs MZ-2 and MZ-2 vs UO, respectively. Further analysis revealed that 118 differentially abundant proteins were involved in cellular invasion and could be categorized into eight groups. These findings provide valuable insights into protein abundance across the different life cycle stages of E. necatrix and offer candidate proteins for future studies on cellular invasion and other biological processes. SIGNIFICANCE: Eimeria necatrix is an obligate intracellular parasite results in huge economic losses to the poultry industry. Understanding proteomic variations across the life cycle stages of E. necatrix may offer proteins related to cellular invasion of E. necatrix, and provide resources for the development of new treatment and prevention interventions against E. necatrix infection. The current data provide an overall summary of the protein abundance across the three life cycle stages of E. necatrix. We identified differentially abundant proteins potential related to cellular invasion. The candidate proteins we identified will form the basis of future studies for cellular invasion. This work also will help in the development of novel strategies for coccidiosis control.

The epidemiologically important food-borne trematode Opisthorchis felineus infests the liver biliary tract of fish-eating mammals and causes disorders, including bile duct neoplasia. Many parasitic species release extracellular vesicles (EVs) that mediate host-parasite interaction. Currently, there is no information on O. felineus EVs. Using gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry, we aimed to characterize the proteome of EVs released by the adult O. felineus liver fluke. Differential abundance of proteins between whole adult worms and EVs was assessed by semiquantitative iBAQ (intensity-based absolute quantification). Imaging, flow cytometry, inhibitor assays, and colocalization assays were performed to monitor the uptake of the EVs by H69 human cholangiocytes. The proteomic analysis reliably identified 168 proteins (at least two peptides matched a protein). Among major proteins of EVs were ferritin, tetraspanin CD63, helminth defense molecule 1, globin 3, saposin B type domain-containing protein, 60S ribosomal protein, glutathione S-transferase GST28, tubulin, and thioredoxin peroxidase. Moreover, as compared to the whole adult worm, EVs proved to be enriched with tetraspanin CD63, saposin B, helminth defense molecule 1, and Golgi-associated plant pathogenesis-related protein 1 (GAPR1). We showed that EVs are internalized by human H69 cholangiocytes via clathrin-dependent endocytosis, whereas phagocytosis and caveolin-dependent endocytosis do not play a substantial role in this process. Our study describes for the first time proteomes and differential abundance of proteins in whole adult O. felineus worms and EVs released by this food-borne trematode. Studies elucidating the regulatory role of individual components of EVs of liver flukes should be continued to determine which components of EV cargo play the most important part in the pathogenesis of fluke infection and in a closely linked pathology: bile duct neoplasia. SIGNIFICANCE: The food-borne trematode Opisthorchis felineus is a pathogen that causes hepatobiliary disorders in humans and animals. Our study describes for the first time the release of EVs by the liver fluke O. felineus, their microscopic and proteomic characterization, and internalization pathways by human cholangiocytes. Differential abundance of proteins between whole adult worms and EVs was assessed. EVs are enriched with canonical EV markers as well as parasite specific proteins, i.e. tetraspanin CD63, saposin B, helminth defense molecule 1, and others. Our findings will form the basis of the search for potential immunomodulatory candidates with therapeutic potential in the context of inflammatory diseases, as well as novel vaccine candidates.

GntR10 is a transcriptional regulator in Brucella. Nuclear factor-kappa B (NF-κB) is involved in many cellular activities, playing major roles in orchestrating the expression of inflammatory genes and regulating protein function that is essential for pathogenic bacteria during infection. GntR10 deletion was previously found to affect the growth and the virulence of Brucella and expression levels of target genes of GntR10 in mice. However, the mechanisms of affection of NF-κB regulated by Brucella GntR10 are still unclear. Here, GntR10 deletion could regulate the expression of LuxR-type transcriptional activators (VjbR and BlxR) of the quorum sensing system (QSS) and type IV secretion system (T4SS) effectors (BspE and BspF) of Brucella. It could further inhibit the activation of the regulator NF-κB and affect the virulence of Brucella. This research provides new insights into the designing of Brucella vaccines and the screening of drug targets. SIGNIFICANCE: Transcriptional regulators are predominant bacterial signal transduction factors. The pathogenicity of Brucella is due to its ability to regulate the expression of virulence related genes including quorum sensing system (QSS) and type IV secretion system (T4SS). Transcriptional regulators are designed to regulate gene expression and enact an appropriate adaptive physiological response. Here, we show that Brucella transcriptional regulator GntR10 regulated the expression of QSS and T4SS effectors, which affected the activation of NF-κB.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense is a worldwide devastating fungal disease in the banana industry. The disease caused by Fusarium oxysporum f. sp. cubense is becoming more and more serious. The pathogen of Fusarium oxysporum f. sp. cubense tropical race 4 (Foc4) is the most harmful one. 'Guijiao 9' is a banana cultivar with good resistance to Foc4, which is identified by resistance screening of natural variant lines. It is of great significance to explore the resistance genes and key proteins of 'Guijiao 9' for banana cultivar improvement and disease resistance breeding. In this study, iTRAQ (isobaric Tags for Relative and Absolute quantitation) was used to analyze the xylem proteomic data of banana roots from the resistant variety 'Guijiao 9' and susceptible variety 'Williams', and the differences in protein accumulation profiles between these two varieties at 24, 48, and 72 h after infection with Foc4 were compared. The identified proteins were analyzed by the protein WGCNA (Weighted Gene Correlation Network Analysis), and the differentially expressed proteins (DEPs) were verified by qRT-PCR experiments. Proteomic analysis showed that there were differences in the protein accumulation profiles of the resistant cultivar 'Guijiao 9' and the susceptible cultivar 'Williams' after infection with Foc4, and there were differences in resistance-related proteins, biosynthesis of secondary metabolites, peroxidase, and pathogenesis-related proteins. The stress response of bananas to pathogens was affected by multiple factors. Protein co-expression analysis showed that there was a high correlation between the MEcyan module and resistance, and 'Guijiao 9' had a different resistance mechanism compared with 'Williams'. SIGNIFICANCE: 'Guijiao 9' is a banana variety with good resistance to Foc4, which is identified by screening the resistance of natural variant lines in the farmland where banana plants are seriously infected by Foc4. It is of great significance to excavate the resistance genes and key proteins of 'Guijiao 9' for banana variety improvement and disease resistance breeding. The aim of this paper is to identify the proteins and related functional modules controlling the pathogenicity differences of Foc4 by comparative proteomic analysis of 'Guijiao 9', so as to understand the resistance mechanism of banana to Fusarium wilt, and offer basis for the final identification, isolation and utilization of Foc4 resistance-related genes in banana variety improvement. The research results will also provide a basis for further understanding the host-pathogen interaction and revealing the resistance mechanism of bananas.