Triphenylphosphonium-Conjugated Palmitic Acid for Mitochondrial Targeting of Pancreatic Cancer Cells: Proteomic and Molecular Evidence.

Pancreatic ductal adenocarcinoma (PDAC)'s resistance to therapies is mainly attributed to pancreatic cancer stem cells (PCSCs). Mitochondria-impairing agents can be used to hamper PCSC propagation and reduce PDAC progression. Therefore, to develop an efficient vector for delivering drugs to the mitochondria, we synthesized tris(3,5-dimethylphenyl)phosphonium-conjugated palmitic acid. Triphenylphosphonium (TPP) is a lipophilic cationic moiety that promotes the accumulation of conjugated agents in the mitochondrion. Palmitic acid (PA), the most common saturated fatty acid, has pro-apoptotic activity in different types of cancer cells. TPP-PA was prepared by the reaction of 16-bromopalmitic acid with TPP, and its structure was characterized by 1H and 13C NMR and HRMS. We compared the proteomes of TPP-PA-treated and untreated PDAC cells and PCSCs, identifying dysregulated proteins and pathways. Furthermore, assessments of mitochondrial membrane potential, intracellular ROS, cardiolipin content and lipid peroxidation, ER stress, and autophagy markers provided information on the mechanism of action of TPP-PA. The findings showed that TPP-PA reduces PDAC cell proliferation through mitochondrial disruption that leads to increased ROS, activation of ER stress, and autophagy. Hence, TPP-PA might offer a new approach for eliminating both the primary population of cancer cells and PCSCs, which highlights the promise of TPP-derived compounds as anticancer agents for PDAC.

Multi-Omics Approaches for Freshness Estimation and Detection of Illicit Conservation Treatments in Sea Bass (Dicentrarchus Labrax): Data Fusion Applications.

Fish freshness consists of complex endogenous and exogenous processes; therefore, the use of a few parameters to unravel illicit practices could be insufficient. Moreover, the development of strategies for the identification of such practices based on additives known to prevent and/or delay fish spoilage is still limited. The paper deals with the identification of the effect played by a Cafodos solution on the conservation state of sea bass at both short-term (3 h) and long-term (24 h). Controls and treated samples were characterized by a multi-omic approach involving proteomics, lipidomics, metabolomics, and metagenomics. Different parts of the fish samples were studied (muscle, skin, eye, and gills) and sampled through a non-invasive procedure based on EVA strips functionalized by ionic exchange resins. Data fusion methods were then applied to build models able to discriminate between controls and treated samples and identify the possible markers of the applied treatment. The approach was effective in the identification of the effect played by Cafodos that proved to be different in the short- and long-term and complex, involving proteins, lipids, and small molecules to a different extent.

Glucose/Ribitol Dehydrogenase and 16.9 kDa Class I Heat Shock Protein 1 as Novel Wheat Allergens in Baker's Respiratory Allergy.

Wheat allergens are responsible for symptoms in 60-70% of bakers with work-related allergy, and knowledge, at the molecular level, of this disorder is progressively accumulating. The aim of the present study is to investigate the panel of wheat IgE positivity in allergic Italian bakers, evaluating a possible contribution of novel wheat allergens included in the water/salt soluble fraction. The water/salt-soluble wheat flour proteins from the Italian wheat cultivar Bolero were separated by using 1-DE and 2-DE gel electrophoresis. IgE-binding proteins were detected using the pooled sera of 26 wheat allergic bakers by immunoblotting and directly recognized in Coomassie stained gel. After a preparative electrophoretic step, two enriched fractions were furtherly separated in 2-DE allowing for detection, by Coomassie, of three different proteins in the range of 21-27 kDa that were recognized by the pooled baker's IgE. Recovered spots were analyzed by nanoHPLC Chip tandem mass spectrometry (MS/MS). The immunodetected spots in 2D were subjected to mass spectrometry (MS) analysis identifying two new allergenic proteins: a glucose/ribitol dehydrogenase and a 16.9 kDa class I heat shock protein 1. Mass spectrometer testing of flour proteins of the wheat cultivars utilized by allergic bakers improves the identification of until now unknown occupational wheat allergens.

Proteomic approaches to decipher cancer cell secretome.

In this review, we give an overview of the actual proteomic approaches used in the study of cancer cells secretome. In particular, we describe the proteomic strategies to decipher cancer cell secretome initially focusing on the different aspects of sample preparation. We examine the issues related to the presence of low abundant proteins, the analysis of secreted proteins in the conditioned media with or without the removal of fetal bovine serum and strategies developed to reduce intracellular protein contamination. As regards the identification and quantification of secreted proteins, we described the different proteomic approaches used, i.e. gel-based, MS-based (label-based and label-free), and the antibody and array-based methods, together with some of the most recent applications in the field of cancer research. Moreover, we describe the bioinformatics tools developed for the in silico validation and characterization of cancer cells secretome. We also discuss the most important available tools for protein annotation and for prediction of classical and non-classical secreted proteins. In summary in this review advances, concerns and challenges in the field of cancer secretome analysis are discussed.

Proteomic and Ultrastructural Analysis of Cellulite-New Findings on an Old Topic.

: Background: Cellulite is a condition in which the skin has a dimpled lumpy appearance. The main causes of cellulite development, studied until now, comprehends modified sensitivity to estrogens, the damage of microvasculature present among dermis and hypodermis. The differences of adipose tissue architecture between male and female might make female more susceptible to cellulite. Adipose tissue is seen to be deeply modified during cellulite development. Our study tried to understand the overall features within and surrounding cellulite to apply the best therapeutic approach. METHODS: Samples of gluteal femoral area were collected from cadavers and women who had undergone surgical treatment to remove orange peel characteristics on the skin. Samples from cadavers were employed for an accurate study of cellulite using magnetic resonance imaging at 7 Tesla and for light microscopy. Specimens from patients were employed for the proteomic analysis, which was performed using high resolution mass spectroscopy (MS). Stromal vascular fraction (SVF) was obtained from the samples, which was studied using MS and flow cytometry. RESULTS: light and electron microscopy of the cellulite affected area showed a morphology completely different from the other usual adipose depots. In cellulite affected tissues, sweat glands associated with adipocytes were found. In particular, there were vesicles in the extracellular matrix, indicating a crosstalk between the two different components. Proteomic analysis showed that adipose tissue affected by cellulite is characterized by high degree of oxidative stress and by remodeling phenomena. CONCLUSIONS: The novel aspects of this study are the peculiar morphology of adipose tissue affected by cellulite, which could influence the surgical procedures finalized to the reduction of dimpling, based on the collagen fibers cutting. The second novel aspect is the role played by the mesenchymal stem cells isolated from stromal vascular fraction of adipose tissue affected by cellulite.

Mining cancer biology through bioinformatic analysis of proteomic data.

Introduction: Discovery proteomics for cancer research generates complex datasets of diagnostic, prognostic, and therapeutic significance in human cancer. With the advent of high-resolution mass spectrometers, able to identify thousands of proteins in complex biological samples, only the application of bioinformatics can lead to the interpretation of data which can be relevant for cancer research. Areas covered: Here, we give an overview of the current bioinformatic tools used in cancer proteomics. Moreover, we describe their applications in cancer proteomics studies of cell lines, serum, and tissues, highlighting recent results and critically evaluating their outcomes. Expert opinion: The use of bioinformatic tools is a fundamental step in order to manage the large amount of proteins (from hundreds to thousands) that can be identified and quantified in a cancer biological samples by proteomics. To handle this challenge and obtain useful data for translational medicine, it is important the combined use of different bioinformatic tools. Moreover, a particular attention to the global experimental design, and the integration of multidisciplinary skills are essential for best setting of tool parameters and best interpretation of bioinformatics output.

Trichostatin A alters cytoskeleton and energy metabolism of pancreatic adenocarcinoma cells: An in depth proteomic study.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of all human cancers with a high mortality rate. Resistance to conventional treatments and chemotherapeutics is a typical feature of PDAC. To investigate the causes of drug resistance it is essential to deeply investigate the mechanism of action of chemotherapeutics. In this study, we performed an in depth shotgun proteomic approach using the label-free proteomic SWATH-MS analysis to investigate novel insights of the mechanism of action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in PDAC cells. This proteomic analysis in PaCa44 cells and data elaboration of TSA-regulated proteins by bioinformatics showed an overall up-regulation of cytokeratins and other proteins related to the cytoskeleton organization, keratinization, and apoptotic cell death. On the contrary, a large amount of the down-regulated proteins by TSA treatment belongs to the cellular energetic metabolism and to the machinery of protein synthesis, such as ribosomal proteins, determining synergistic cell growth inhibition by the combined treatment of TSA and the glycolytic inhibitor 2-deoxy-d-glucose in a panel of PDAC cell lines. Data are available via ProteomeXchange with identifier PXD007801.

IEF peptide fractionation method combined to shotgun proteomics enhances the exploration of rice milk proteome.

We conducted a proteomics study in order to detect the proteomic method which provides the most complete characterization of the proteins of rice milk. In particular, we compared the results obtained from LC-MS/MS after protein precipitation with acetone or TCA, as well as the results obtained from LC-MS/MS after protein prefractionation based on SDS-PAGE (GeLC-MS/MS) or ProteoMiner™ technology (ProteoMiner-LC-MS/MS), and after peptide prefractionation based on IEF (pIEF-LC-MS/MS). A total of 158 protein species have been detect in rice milk. The physical-chemical analysis and classification of the identified proteins were also reported. In particular, we showed that pIEF-LC-MS/MS method led to a significant increase in the proteome coverage, allowing the identification of a total of 96 proteins of milk rice. This study demonstrates the utility of a prefractionation step based on pIEF before the shotgun proteomic analysis and offers an in-depth insight into the rice milk proteome.

Tissue proteomics of splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL) is a rare chronic B lymphoproliferative disease, whose molecular pathogenesis has still not been well established. For the first time, a proteomic approach was undertaken to analyse the protein profiles of SMZL tissue. 1D and 2D Western blot, immunohistochemical analysis, and functional data mining were also performed in order to validate results, investigate protein species specific regulation, classify proteins, and explore their potential relationships. We demonstrated that SMZL is characterized by modulation of protein species related to energetic metabolism and apoptosis pathways. We also reported specific protein species (such as biliverdin reductase A, manganese superoxide dismutase, beta-2 microglobulin, growth factor receptor-bound protein 2, acidic leucine-rich nuclear phosphoprotein 32 family member A, and Set nuclear oncogene) directly involved in NF-kB and BCR pathways, as well as in chromatin remodelling and cytoskeleton. Our findings shed new light on SMZL pathogenesis and provide a basis for the future development of novel biomarkers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD001124.

Comparative proteomic and phosphoproteomic profiling of pancreatic adenocarcinoma cells treated with CB1 or CB2 agonists.

The pancreatic adenocarcinoma cell line Panc1 was treated with cannabinoid receptor ligands (arachidonylcyclopropylamide or GW405833) in order to elucidate the molecular mechanism of their anticancer effect. A proteomic approach was used to analyze the protein and phosphoprotein profiles. Western blot and functional data mining were also employed in order to validate results, classify proteins, and explore their potential relationships. We demonstrated that the two cannabinoids act through a widely common mechanism involving up- and down-regulation of proteins related to energetic metabolism and cell growth regulation. Overall, the results reported might contribute to the development of a therapy based on cannabinoids for pancreatic adenocarcinoma.

Advances in enrichment methods for mass spectrometry-based proteomics analysis of post-translational modifications.

Post-translational modifications (PTMs) occur during or after protein biosynthesis and increase the functional diversity of proteome. They comprise phosphorylation, acetylation, methylation, glycosylation, ubiquitination, sumoylation (among many other modifications), and influence all aspects of cell biology. Mass-spectrometry (MS)-based proteomics is the most powerful approach for PTM analysis. Despite this, it is challenging due to low abundance and labile nature of many PTMs. Hence, enrichment of modified peptides is required for MS analysis. This review provides an overview of most common PTMs and a discussion of current enrichment methods for MS-based proteomics analysis. The traditional affinity strategies, including immunoenrichment, chromatography and protein pull-down, are outlined together with their strengths and shortcomings. Moreover, a special attention is paid to chemical enrichment strategies, such as capture by chemoselective probes, metabolic and chemoenzymatic labelling, which are discussed with an emphasis on their recent progress. Finally, the challenges and future trends in the field are discussed.

Plant Signals Anticipate the Induction of the Type III Secretion System in Pseudomonas syringae pv. actinidiae, Facilitating Efficient Temperature-Dependent Effector Translocation.

Disease resistance in plants depends on a molecular dialogue with microbes that involves many known chemical effectors, but the time course of the interaction and the influence of the environment are largely unknown. The outcome of host-pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Here, we strengthen the paradigm of the plant-pathogen molecular dialogue by addressing overlooked details concerning the timing of interactions, specifically the role of plant signals and temperature on the regulation of bacterial virulence during the first few hours of the interaction. Whole-genome expression profiling after 1 h revealed that the perception of plant signals from kiwifruit or tomato extracts anticipated T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions, facilitating more efficient effector transport in planta, as revealed by the induction of a temperature-dependent hypersensitive response in the nonhost plant Arabidopsis thaliana Columbia-0 (Col-0). Our results show that in the arms race between plants and bacteria, the temperature-dependent timing of bacterial virulence versus the induction of plant defenses is probably one of the fundamental parameters governing the outcome of the interaction. IMPORTANCE Plant diseases-their occurrence and severity-result from the impact of three factors: the host, the pathogen, and the environmental conditions, interconnected in the disease triangle. Time was further included as a fourth factor accounting for plant disease, leading to a more realistic three-dimensional disease pyramid to represent the evolution of disease over time. However, this representation still considers time only as a parameter determining when and to what extent a disease will occur, at a scale from days to months. Here, we show that time is a factor regulating the arms race between plants and pathogens, at a scale from minutes to hours, and strictly depends on environmental factors. Thus, besides the arms possessed by pathogens and plants per se, the opportunity and the timing of arms mobilization make the difference in determining the outcome of an interaction and thus the occurrence of plant disease.

Tumor Suppressor Role of Wild-Type P53-Dependent Secretome and Its Proteomic Identification in PDAC.

The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC.

Protein Secretion Prediction Tools and Extracellular Vesicles Databases.

Secreted proteins play important roles in several biological processes such as growth, proliferation differentiation, cell-cell communication, migration, and apoptosis; moreover, these extracellular molecules mediate homeostasis by influencing the cross-talking within the surrounding tissues. Currently, the research area of cell secretome has become of great interest since the profiling of secreted proteins could be essential for the biomarker discovery and for the identification of new therapeutic strategies. Several bioinformatic platforms have been implemented for the in silico characterization of secreted proteins: this chapter describes a typical workflow for the analysis of proteins secreted by cultured cells through bioinformatic approaches. Central issue is related to discrimination between proteins secreted by classical and non-classical pathways. Therefore, specific prediction tools for the classification of candidate secreted proteins are here presented.

Kohonen Artificial Neural Network and Multivariate Analysis in the Identification of Proteome Changes during Early and Long Aging of Bovine Longissimus dorsi Muscle Using SWATH Mass Spectrometry.

To study proteomic changes involved in tenderization of Longissimus dorsi, Charolais heifers and bulls muscles were sampled after early and long aging (12 or 26 days). Sensory evaluation and instrumental tenderness measurement were performed. Proteins were analyzed by gel-free proteomics. By pattern recognition (principal component analysis and Kohonen's self-organizing maps) and classification (partial least squares-discriminant analysis) tools, 58 and 86 dysregulated proteins were detected after 12 and 26 days of aging, respectively. Tenderness was positively correlated mainly with metabolic enzymes (PYGM, PGAM2, TPI1, PGK1, and PFKM) and negatively with keratins. Downregulation in hemoglobin subunits and carbonic anhydrase 3 levels was relevant after 12 days of aging, while mimecan and collagen chains levels were reduced after 26 days of aging. Bioinformatics indicated that aging involves a prevalence of metabolic pathways after late and long periods. These findings provide a deeper understanding of changes involved in aging of beef and indicate a powerful method for future proteomics studies.

Integrated lipidomics and proteomics reveal cardiolipin alterations, upregulation of HADHA and long chain fatty acids in pancreatic cancer stem cells.

Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC.

The Mutant p53-Driven Secretome Has Oncogenic Functions in Pancreatic Ductal Adenocarcinoma Cells.

The cancer secretome is a rich repository of useful information for both cancer biology and clinical oncology. A better understanding of cancer secretome is particularly relevant for pancreatic ductal adenocarcinoma (PDAC), whose extremely high mortality rate is mainly due to early metastasis, resistance to conventional treatments, lack of recognizable symptoms, and assays for early detection. TP53 gene is a master transcriptional regulator controlling several key cellular pathways and it is mutated in ~75% of PDACs. We report the functional effect of the hot-spot p53 mutant isoforms R175H and R273H on cancer cell secretome, showing their influence on proliferation, chemoresistance, apoptosis, and autophagy, as well as cell migration and epithelial-mesenchymal transition. We compared the secretome of p53-null AsPC-1 PDAC cells after ectopic over-expression of R175H-mutp53 or R273H-mutp53 to identify the differentially secreted proteins by mutant p53. By using high-resolution SWATH-MS technology, we found a great number of differentially secreted proteins by the two p53 mutants, 15 of which are common to both mutants. Most of these secreted proteins are reported to promote cancer progression and epithelial-mesenchymal transition and might constitute a biomarker secreted signature that is driven by the hot-spot p53 mutants in PDAC.

Exploring the wound healing, anti-inflammatory, anti-pathogenic and proteomic effects of lactic acid bacteria on keratinocytes.

The topical application of lactic acid bacteria (LAB) is recognized as a useful approach to improve skin health. This work aims to characterize by a multidisciplinary approach, the wound healing, anti-inflammatory, anti-pathogens and proteomic effects of six LAB lysates, belonging to the genus Lactobacillus. Our results demonstrated that the lysates of tested LAB stimulated the proliferation of keratinocytes, and that L. plantarum SGL 07 and L. salivarius SGL 19 accelerated the re-epithelization by inducing keratinocyte migration. The bacterial lysates also reduced the secretion of specific pro-inflammatory mediators from keratinocytes. Furthermore, viable L. salivarius SGL 19 and L. fermentum SGL 10 had anti-pathogenic effects against S. aureus and S. pyogenes, while L. brevis SGL 12 and L. paracasei SGL 04 inhibited S. aureus and S. pyogenes, respectively. The tested lactobacilli lysates also induced specific proteome modulation of the exposed keratinocytes, involving dysregulation of proteins (such as interleukin enhancer-binding factor 2 and ATP-dependent RNA helicase) and pathways (such as cytokine, NF-kB, Hedgehog, and RUNX signaling) associated with their specific wound healing and anti-inflammatory effects. This study indicates the different potential of selected lactobacilli, suggesting that they may be successfully used in the future together with conventional therapies to bring relief from skin disorders.

Progressively De-Differentiated Pancreatic Cancer Cells Shift from Glycolysis to Oxidative Metabolism and Gain a Quiescent Stem State.

Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients.

Publisher Correction: Runx2 stimulates neoangiogenesis through the Runt domain in melanoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.