Mass Spectrometry Analysis Of Proteins Research Articles

Hemolysin BL from novel Bacillus toyonensis BV-17 induces antitumor activity both in vitro and in vivo

ABSTRACT The gut microbiota plays an important role in cancer development and immunotherapy. Bacterial toxins have enormous antitumor potential due to their cytotoxicity and ability to activate the immune system. Using 16S rRNA gene sequencing, we compared the gut microbiota composition of fecal samples from healthy individuals and patients with colorectal cancer (CRC) and observed that the genus Bacillus was common in the healthy donors but was absent in the CRC patients. Further, we isolated a novel Bacillus toyonensis BV-17 from the fecal samples of the healthy individuals. Our results showed that the supernatant of the Bacillus toyonensis BV-17 cultures could quickly kill various tumor cell lines within minutes in vitro, by causing cell membrane disruption, blebbing, and leakage of cytoplasmic content. Fast protein liquid chromatography (FPLC) and mass spectrometry analysis identified hemolysin BL (HBL) as the effector molecule, which exhibits a different cytotoxicity mechanism compared to previous studies. Intra-tumor injection of low dose HBL inhibited the growth of both treated and untreated tumors in mice. The outcomes of this pioneer study suggest that HBL exhibits antitumor activity and is a potential chemotherapeutic agent that could be engineered to target only tumor cells in future.

Capn3 depletion causes Chk1 and Wee1 accumulation and disrupts synchronization of cell cycle reentry during liver regeneration after partial hepatectomy

Recovery of liver mass to a healthy liver donor by compensatory regeneration after partial hepatectomy (PH) is a prerequisite for liver transplantation. Synchronized cell cycle reentry of the existing hepatocytes after PH is seemingly a hallmark of liver compensatory regeneration. Although the molecular control of the PH-triggered cell cycle reentry has been extensively studied, little is known about how the synchronization is achieved after PH. The nucleolus-localized protein cleavage complex formed by the nucleolar protein Digestive-organ expansion factor (Def) and cysteine proteinase Calpain 3 (Capn3) has been implicated to control wounding healing during liver regeneration through selectively cleaving the tumor suppressor p53 in the nucleolus. However, whether the Def-Capn3 complex participates in regulating the synchronization of cell cycle reentry after PH is unknown. In this report, we generated a zebrafish capn3b null mutant (capn3b∆19∆14). The homozygous mutant was viable and fertile, but suffered from a delayed liver regeneration after PH. Delayed liver regeneration in capn3b∆19∆14 was due to disruption of synchronized cell proliferation after PH. Mass spectrometry (MS) analysis of nuclear proteins revealed that a number of negative regulators of cell cycle are accumulated in the capn3b∆19∆14 liver after PH. Moreover, we demonstrated that Check-point kinase 1 (Chk1) and Wee1, two key negative regulators of G2 to M transition, are substrates of Capn3. We also demonstrated that Chk1 and Wee1 were abnormally accumulated in the nucleoli of amputated capn3b∆19∆14 liver. In conclusion, our findings suggest that the nucleolar-localized Def-Capn3 complex acts as a novel regulatory pathway for the synchronization of cell cycle reentry, at least partially, through inactivating Chk1 and Wee1 during liver regeneration after PH.

Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling.

Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex. The small RNA-mediated depletion of kalirin diminished activities in cellular membranes for activating Rab11 and resulted in a shift in size of Rab11 positive structures from small to larger ones and tubulation of recycling endosomes. Our study suggests that kalirin and TRAPP form a dual GEF complex to choreograph actions of Rab11 and Rac1 at recycling endosomes.

Characterization of extracellular vesicles derived from two populations of human placenta derived mesenchymal stem/stromal cells

Background & Aim Growing evidence shows that extracellular vesicles (EVs) are responsible for the therapeutic effect of stem cell therapy. EVs are a promising, potential cell-free therapy in regenerative medicine. The human placenta is a rich source of mesenchymal stem/stromal cells (MSCs). The first aim of this study was to isolate and characterize EVs derived from two well-characterised placental hTERT-transformed MSC lines; chorionic MSC line (CMSC29) and decidual MSC line (DMSC23). The second aim was to analyse the protein, lipid and small RNA content of isolated EVs from both cell lines, and to assess their biological activities. Methods, Results & Conclusion EVs were isolated and manufactured using the proprietary LEAP technology of Exopharm Ltd. Isolated EVs were then characterized at the biophysical level by microfluidic resistive pulse sensing (MRPS, particle size and number), transmission electron microscopy (morphology), and Western blot (EVs markers). The protein and lipid contents were analysed by mass spectrometry and the nucleic acid content by RNA sequencing. In addition, the biological activities of purified EVs were evaluated in a BioMAP® Diversity PLUS® screen and in vitro cell proliferation and migration assays. Spectradyne analysis showed that LEAP-purified EVs from CMSC29 had higher concentration of particles compared with DMSC23-derived EVs. Protein mass spectrometry analysis identified a large number of proteins which were mainly associated with binding. Traditional EV marker proteins were identified, including CD9 (DMSC-derived EV) and CD81 (CMSC-derived EV), and CD44 a marker for MSC was also identified on the DMSC23-derived EVs. There were several growth factors identified in both DMSC23- and CMSC29-derived EVs including fibroblast growth factor 2 (FGF2), insulin, and connective tissue growth factor. Lipid analysis identified the major species to be phosphatidylcholine, triacylglycerides and diacylglycerides in both DMSC23- and CMSC29-derived EVs. The nucleic acid content of both groups has been sequenced and bioinformatic analysis is currently underway. The EVs from both cell sources demonstrated biological activity in the BioMAP® Diversity PLUS® screen as well as by stimulating the proliferation and migration of dermal fibroblasts in in vitro assays. In conclusion, these data revealed differences between LEAP-purified EVs from human placenta-derived MSC cell lines DMSC23 and CMSC29, which may be potentially useful for different disease models.

6-Arylmethylidene Penicillin-Based Sulfone Inhibitors for Repurposing Antibiotic Efficiency in Priority Pathogens.

The ability of 6-(aryl)methylidene penicillin-based sulfones 1-7 to repurpose β-lactam antibiotics activity with bacterial species that carry carbapenem-hydrolyzing class D β-lactamases (OXA-23, OXA-24/40 and OXA-48), as well as with class A (TEM-1, CTX-M-2) and class C (CMY-2, DHA-1) enzymes, is reported. The combinations imipenem/3 and imipenem/4 restored almost completely the antibiotic efficacy in OXA-23 and OXA-24/40 carbapenemase-producing A. baumannii strains (1 μg mL-1) and also provided good results for OXA-48 carbapenemase-producing K. pneumoniae strains (4 μg mL-1). Compounds 2-6 in combinations with ceftazidime and ampicillin were also efficient in restoring antibiotic efficacy in E. coli strains carrying class C (CMY-2 and DHA-1) and class A (TEM-1 and CTX-M-2) β-lactamase enzymes, respectively. Kinetic and inhibition studies with the OXA-24/40 enzyme, protein mass spectrometry analysis and docking studies allowed us to gain an insight into the inhibition mechanism and the experimentally observed differences between the ligands.

Elevated myocardial SORBS2 and the underlying implications in left ventricular noncompaction cardiomyopathy.

BackgroundLeft ventricular noncompaction cardiomyopathy (LVNC) is a hereditary heart disease characterized by an excessive trabecular meshwork of deep intertrabecular recesses within the ventricular myocardium. The guidelines for management of LVNC patients aim to improve quality of life by preventing cardiac heart failure. However, the mechanism underlying LVNC-associated heart failure remains poorly understood.MethodsUsing protein mass spectrometry analysis, we established that Sorbin And SH3 Domain Containing 2 (SORBS2) is up-regulated in LVNC hearts without changes to structure proteins. We conducted in vivo experiments wherein the heart tissues of wild-type mice were injected with an AAV9 vector to overexpress SORBS2, followed by analysis using echocardiography, T-tubule analysis and Ca2+ imaging to identify functional and morphological changes. In addition, we analyzed the function and structure of SORBS2 overexpressing human embryonic stem cell (hESC) derived cardiomyocytes (hESC-CM) via immunoblotting, immunohistochemistry, immunofluorescence, and confocal Ca2+ imaging.FindingsLVNC myocardial tissues feature strongly elevated expression of SORBS2, microtubule densification and redistribution of Junctophilin 2 (JP2). SORBS2 interacts with β-tubulin, promoting its polymerization in 293T cells and hESC-derived CMs. In vivo, cardiac dysfunction, β-tubulin densification, JP2 translocation, T-tubule disorganization and Ca2+ handling dysfunction were observed in mice overexpressing SORBS2.InterpretationWe identified a novel mechanism through which SORBS2 interacts with β-tubulin and promotes microtubule densification, eventually effecting JP2 distribution and T-tubule, potentially contributing to heart failure in LVNC disease.FundThis work was supported by a CAMS Initiative for Innovative Medicine grant (CAMS-I2M, 2016-I2M-1-015 to Y.J.Wei)

A Fast and Accurate Method to Identify and Quantify Enzymes in Brush-Border Membranes: In Situ Hydrolysis Followed by Nano LC-MS/MS

A simple method for the identification of brush-border membrane α-glucosidases is described. The proteins were first solubilized and separated in a gel under native, non-denaturing, conditions. The gel was then incubated in substrate solutions (maltose or sucrose), and the product (glucose) exposed in situ by the oxidation of o-dianisidine, which yields a brown-orange color. Nano-liquid chromatography coupled to mass spectrometry analyses of proteins (nano LC-MS/MS) present in the colored bands excised from the gels, was used to confirm the presence of the enzymes. The stain is inexpensive and the procedure permits testing several substrates in the same gel. Once enzymes are identified, their abundance, relative to that of other proteins in the brush border, can be semi-quantified using nano LC-MS/MS.

In situ mass spectrometry analysis of intact proteins and protein complexes from biological substrates.

Advances in sample preparation, ion sources and mass spectrometer technology have enabled the detection and characterisation of intact proteins. The challenges associated include an appropriately soft ionisation event, efficient transmission and detection of the often delicate macromolecules. Ambient ion sources, in particular, offer a wealth of strategies for analysis of proteins from solution environments, and directly from biological substrates. The last two decades have seen rapid development in this area. Innovations include liquid extraction surface analysis, desorption electrospray ionisation and nanospray desorption electrospray ionisation. Similarly, developments in native mass spectrometry allow protein–protein and protein–ligand complexes to be ionised and analysed. Identification and characterisation of these large ions involves a suite of hyphenated mass spectrometry techniques, often including the coupling of ion mobility spectrometry and fragmentation techniques. The latter include collision, electron and photon-induced methods, each with their own characteristics and benefits for intact protein identification. In this review, recent developments for in situ protein analysis are explored, with a focus on ion sources and tandem mass spectrometry techniques used for identification.

Allotype-specific processing of the CD16a N45-glycan from primary human natural killer cells and monocytes.

Fc γ receptor IIIa/CD16a is an activating cell surface receptor with a well-defined role in natural killer (NK) cell and monocyte effector function. The extracellular domain is decorated with five asparagine (N)-linked glycans; N-glycans at N162 and N45 directly contribute to high-affinity antibody binding and protein stability. N-glycan structures at N162 showed significant donor-dependent variation in a recent study of CD16a isolated from primary human NK cells, but structures at N45 were relatively homogeneous. In this study, we identified variations in N45 glycan structures associated with a polymorphism coding for histidine instead of leucine at position 48 of CD16a from two heterozygous donors. It is known that H48 homozygous individuals suffer from immunodeficiency and recurrent viral infections. A mass spectrometry analysis of protein isolated from the primary natural killer cells of individuals expressing both CD16a L48 and H48 variants demonstrated clear processing differences at N45. CD16a H48 displayed a greater proportion of complex-type N45 glycans compared to the more common L48 allotype with predominantly hybrid N45-glycoforms. Structures at the four other N-glycosylation sites showed minimal differences from data collected on donors expressing only the predominant L48 variant. CD16a H48 purified from a pool of monocytes similarly displayed increased processing at N45. Here, we provide evidence that CD16a processing is affected by the H48 residue in primary NK cells and monocytes from healthy human donors.

Facile Improvement of Negative Ion Mode Electrospray Ionization Using Capillary Vibrating Sharp-Edge Spray Ionization.

Electrospray ionization (ESI) is often affected by corona discharge when spraying 100% aqueous solutions as the voltage that induces discharge can be well below the onset voltage of ESI. As a result, it is especially challenging to perform native mass spectrometry in negative ion mode where 100% aqueous solution is preferred. Here we report a simple instrumentation method to improve the performance of ESI in negative ion mode based on capillary vibrating sharp-edge spray ionization. By attaching a fused silica capillary emitter to a vibrating glass slide, improved signal quality is achieved for various analytes in aqueous solutions over applying ESI alone. Compared to commercial ESI sources using nebulization gas to reduce discharge, 10-100-fold enhancement in signal intensity and 3-10-fold improvement in S/N are achieved for various kinds of molecules including DNA, peptides, proteins, and oligosaccharides. Finally, the new method demonstrates utility for native mass spectrometry analysis of proteins and G-quadruplex DNA. The present method is expected to have great potential to be adopted by the scientific community because of its improved analytical performance, simplicity, and low cost.

Liquid Chromatography Tandem Mass Spectrometry Analysis of Proteins Associated with Age-Related Disorders in Human Pituitary Tissue.

The pituitary gland is involved in multiple physiological functions, including growth, development, reproduction, stress adaptation, osmotic balance, body composition, skin pigmentation, and aging. Advancing age is characterized by dysfunctions in multiple physiological signaling mechanisms, concomitant with perturbed patterns of pituitary hormone release as well as disrupted rhythmic secretion of virtually all pituitary hormones. This chapter presents a liquid chromatography tandem mass spectrometry (LC-MS/MS) protocol for analysis of the protein content in post-mortem pituitary glands. It was of special interest to determine if proteins previously linked with aging and age-related disorders could be identified to support further studies in this field using proteomic profiling approaches. This included hormones, hormone-processing enzymes, histones, oxidation-reduction enzymes, and others.

Antiproliferative and Immunoregulatory Effects of Azelaic Acid Against Acute Myeloid Leukemia via the Activation of Notch Signaling Pathway.

Acute myeloid leukemia (AML) is a common type of hematological malignancy that can progress rapidly. AML has a poor prognosis and a high incidence of relapse due to therapeutic resistance. Azelaic acid (AZA), a small molecular compound is known to exhibit antitumor effect on various tumor cells. This study aimed to evaluate the antiproliferative and immunoregulatory effects of AZA against AMLviathe activation of the notch signaling pathway. We found that AZA can inhibit the proliferation of AML cells. In addition, laser confocal microscopy showed AZA-treated AML cells began to swelling and undergo cytoplasmic vacuolization. Importantly, AZA promoted the proliferation of NK and T cells and increased the secretion of TNF-αand IFN-γ. AZA also increased the expression levels of CD107a and TRAIL in NK cells, and CD25 and CD69 in T cells to influence their activation and cytotoxic ability. AZA-treated NK cells can kill AML cells more efficiently at the single-cell level as observed under the microfluidic chips. Further mechanistic analysis using protein mass spectrometry analysis and Notch signaling reporter assay demonstrated that Notch1and Notch2 were up-regulated and the Notch signaling pathway was activated. Moreover, combining AZA with the Notch inhibitor, RO4929097, decreased the expression of Notch1and Notch2, and downstream HES1 and HEY1, which rendered AML cells insensitive to AZA-induced apoptosis and alleviated AZA-mediated cytotoxicity in AML. In vivo, AZA relieved the leukemic spleen infiltration and extended the survival. The percentage of CD3-CD56+NK cells and CD4+CD8+T cells as well as the secretion of cytotoxic cytokines was increased after the treatment of AZA. The overall findings reveal that AZA is a potential Notch agonist against AML in activating the Notch signaling pathway.

Preparation of Mitochondria from Ovarian Cancer Tissues and Control Ovarian Tissues for Quantitative Proteomics Analysis.

Ovarian cancer is a common gynecologic cancer with high mortality but unclear molecular mechanism. Most ovarian cancers are diagnosed in the advanced stage, which seriously hampers therapy. Mitochondrial changes are a hallmark of human ovarian cancers, and mitochondria are the centers of energy metabolism, cell signaling, and oxidative stress. In-depth insights into the changes of the mitochondrial proteome in ovarian cancers compared to control ovarian tissue will benefit in-depth understanding of the molecular mechanisms of ovarian cancer, and the discovery of effective and reliable biomarkers and therapeutic targets. An effective mitochondrial preparation method coupled with an isobaric tag for relative and absolute quantification (iTRAQ) quantitative proteomics are presented here to analyze human ovarian cancer and control mitochondrial proteomes, including differential-speed centrifugation, density gradient centrifugation, quality assessment of mitochondrial samples, protein digestion with trypsin, iTRAQ labeling, strong cation exchange fractionation (SCX), liquid chromatography (LC), tandem mass spectrometry (MS/MS), database analysis, and quantitative analysis of mitochondrial proteins. Many proteins have been successfully identified to maximize the coverage of the human ovarian cancer mitochondrial proteome and to achieve the differentially expressed mitochondrial protein profile in human ovarian cancers.

TH2BS11ph histone mark is enriched in the unsynapsed axes of the XY body and predominantly associates with H3K4me3-containing genomic regions in mammalian spermatocytes

BackgroundTH2B is a major histone variant that replaces about 80–85% of somatic H2B in mammalian spermatocytes and spermatids. The post-translational modifications (PTMs) on TH2B have been well characterised in spermatocytes and spermatids. However, the biological function(s) of these PTMs on TH2B have not been deciphered in great detail. In our attempt to decipher the unique function(s) of histone variant TH2B, we detected the modification in the N-terminal tail, Serine 11 phosphorylation on TH2B (TH2BS11ph) in spermatocytes.ResultsThe current study is aimed at understanding the function of the TH2BS11ph modification in the context of processes that occur during meiotic prophase I. Immunofluorescence studies with the highly specific antibodies revealed that TH2BS11ph histone mark is enriched in the unsynapsed axes of the sex body and is associated with XY body-associated proteins like Scp3, γH2AX, pATM, ATR, etc. Genome-wide occupancy studies as determined by ChIP sequencing experiments in P20 C57BL6 mouse testicular cells revealed that TH2BS11ph is enriched in X and Y chromosomes confirming the immunofluorescence staining pattern in the pachytene spermatocytes. Apart from the localisation of this modification in the XY body, TH2BS11ph is majorly associated with H3K4me3-containing genomic regions like gene promoters, etc. These data were also found to corroborate with the ChIP sequencing data of TH2BS11ph histone mark carried out in P12 C57BL6 mouse testicular cells, wherein we found the predominant localisation of this modification at H3K4me3-containing genomic regions. Mass spectrometry analysis of proteins that associate with TH2BS11ph-containing mononucleosomes revealed key proteins linked with the functions of XY body, pericentric heterochromatin and transcription.ConclusionsTH2BS11ph modification is densely localised in the unsynapsed axes of the XY body of the pachytene spermatocyte. By ChIP sequencing studies in mouse P12 and P20 testicular cells, we demonstrate that TH2BS11ph is predominantly associated with H3K4me3 positive genomic regions like gene promoters, etc. We propose that TH2BS11ph modification could act alone or in concert with other histone modifications to recruit the appropriate transcription or XY body recombination protein machinery at specific genomic loci.

German Government Official Methods Board Points the Way Forward: Launch of a New Working Group for Mass Spectrometry for Protein Analysis to Detect Food Fraud and Food Allergens

Abstract The detection of food fraud and undeclared food allergens is one of the major challenges for competent authorities. Because adulterations are continuously adapted to the methods used to uncover them, the accomplishment of this task has become increasingly difficult over time. In recent years, various new promising methods for the detection of multiple food adulterants and multiple food allergens have been developed. Some of them utilize LC–MS to identify specific marker peptides. However, these methods have yet to be validated and standardized. For this reason, the German officials have established a working group with the objective of validating methods through multilaboratory validation studies. The experts of the working group also aim for the first time to standardize validated methods and to develop general validation criteria. This manuscript will highlight the current work of the group. For this purpose, an overview is given on the principles and applications of the new mass spectrometric methods. Moreover, requirements and the present work of other institutions regarding method validation are described.

Gestational Tissue-Derived Human Mesenchymal Stem Cells Use Distinct Combinations of Bioactive Molecules to Suppress the Proliferation of Human Hepatoblastoma and Colorectal Cancer Cells.

Background Cancer has been considered a serious global health problem and a leading cause of morbidity and mortality worldwide. Despite recent advances in cancer therapy, treatments of advance stage cancers are mostly ineffective resulting in poor survival of patients. Recent evidences suggest that multipotent human mesenchymal stem cells (hMSCs) play important roles in growth and metastasis of several cancers by enhancing their engraftment and inducing tumor neovascularization. However, the effect of hMSCs on cancer cells is still controversial because there are also evidences demonstrating that hMSCs inhibited growth and metastasis of some cancers. Methods In this study, we investigated the effects of bioactive molecules released from bone marrow and gestational tissue-derived hMSCs on the proliferation of various human cancer cells, including C3A, HT29, A549, Saos-2, and U251. We also characterized the hMSC-derived factors that inhibit cancer cell proliferation by protein fractionation and mass spectrometry analysis. Results We herein make a direct comparison and show that the effects of hMSCs on cancer cell proliferation and migration depend on both hMSC sources and cancer cell types and cancer-derived bioactive molecules did not affect the cancer suppressive capacity of hMSCs. Moreover, hMSCs use distinct combination of bioactive molecules to suppress the proliferation of human hepatoblastoma and colorectal cancer cells. Using protein fractionation and mass spectrometry analysis, we have identified several novel hMSC-derived factors that might be able to suppress cancer cell proliferation. Conclusion We believe that the procedure developed in this study could be used to discover other therapeutically useful molecules released by various hMSC sources for a future in vivo study.

Abstract LB-232: A novel approach for liquid biopsy by using nExo

Abstract Exosomes are small extracellular vesicles secreted from all living cells; its contents are highly varied, including proteins, small RNAs and recently discovered, genomic DNA (gDNA). The presence of nuclear content in exosomes suggests that different cell compartments can contribute to exosome cargo; however, little is known about this sub population of nuclear derived exosomes (nExo). Here, we analyzed nExo isolated from ovarian cancer cells and characterized them to explore potential clinical relevance such as cancer detection and therapeutic response. Exosomes were isolated from ovarian cancer cell lines, normal human fallopian epithelial tube cells and bio-fluids from mouse and human samples. To determine the purity of exosomes, nanoparticle tracking system, immunoblotting assay and cryo-transmission electron microscopy were used. To analyze the content of exosomes, protein mass spectrometry and imaging flow cytometry analysis was performed. First, we determined that mass spectrometry analyses for ovarian cancer cell exosomes revealed that 12.5% of proteins are derived from the cell nucleus sub-population. Additionally, using a novel imaging flow cytometry technique which enables us to identify single exosome and to quantify them, we determined that 10% of exosomes carried gDNA. Conversely, normal cells secreted only 0.2% of nExo. Then we hypothesized that the prevalence of gDNA is affected by genotoxic drugs. Treating cells with either the PARP inhibitor olaparib or topoisomerase inhibitor topotecan increased the proportion of nExo secreted from ovarian cancer cells. In addition, PARP proteins exist in ovarian cancer cell exosomes and 80.4 % of the exosome co-localized with gDNA. Using in vivo preclinical models, serum from ovarian cancer bearing mice contained a higher number of nExo than non-tumor controls, and this population was increased in response to treatment of genotoxic drugs. Furthermore, plasma and ascites from ovarian cancer patients contain around 0.3 % of nExo, which include nuclear proteins and gDNA. We demonstrate that nExo are predominantly secreted from cancer cells and could serve as an important biomarker. Although the subpopulation is relatively rare, their presence in bio-fluids isolated from cancer patients holds the potential of serving as novel biomarkers for early detection and therapeutic response to genotoxic agents. Citation Format: Akira Yokoi, Alejandro Villar-Prados, Paul Allen Oliphint, Theresa J. O'Halloran, Jason Roszik, Anil K. Sood. A novel approach for liquid biopsy by using nExo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-232.

318-OR: Targeting PPARγ Phosphorylation Prevents Obesity by Inducing a Brown-Like Healthy Remodeling of Adipose Tissue

The prevalence of obesity—and thus the incidence of metabolic diseases, including type 2 diabetes and cardiovascular disease—is dramatically increasing worldwide. Despite the critical role of adipose tissues in nutrient metabolism, there are surprisingly few clinically-approved medications targeting adipose tissue. Thiazolidinediones (TZDs), PPARγ agonists are exceptions that act in adipose tissue, but they are associated with adverse effects such as weight gain, and heart failure. An exciting series of studies demonstrate that posttranslational modifications can selectively enhance the insulin sensitizing activity of PPARγ. Our laboratory previously showed that roscovitine (RS), a CDK inhibitor, prevents diet-induced obesity by blocking PPARγ S273 phosphorylation, which promotes formation of UCP1+ (brite) adipocytes in WAT. RNA profiling reveals that brite adipocytes are distinct from beige adipocytes, which arise through catecholamine signaling suggesting that brown-like adipocytes in WAT may arise from multiple origins. Using adipocytes and progenitor’s lineage tracing mice, we demonstrate that brite adipocytes originate from mature white adipocytes through trans-differentiation. Inhibition of S273 phosphorylation of PPARγ in obese mice prevents weight gain, restores insulin and glucose sensitivity and a normal pancreatic β-cells response to glucose as well as prevents liver steatosis and adipocytes formation in bone. Moreover, mass spectrometry analysis of extracellular matrix proteins showed a reduced adipose tissues fibrosis following RS treatment. Mechanistically, inhibition of PPARγ phosphorylation shifts its interaction with cofactors favoring a brown-like healthy remodeling of adipose tissue, which includes suppression of genes associated with inflammation and fibrosis. Overall, we demonstrate targeting PPARγ phosphorylation using RS can constitute a new therapy for obesity and its metabolic complications. Disclosure N. Rabhi: None. S.R. Farmer: None. Funding American Heart Association (17POST33660875); Francophone Society of Diabetes (R01DK102199-01)

German Government Official Methods Board Points the Way Forward: Launch of a New Working Group for Mass Spectrometry for Protein Analysis to Detect Food Fraud and Food Allergens.

The detection of food fraud and undeclared food allergens is one of the major challenges for competent authorities. Because adulterations are continuously adapted to the methods used to uncover them, the accomplishment of this task has become increasingly difficult over time. In recent years, various new promising methods for the detection of multiple food adulterants and multiple food allergens have been developed. Some of them utilize LC-MS to identify specific marker peptides. However, these methods have yet to be validated and standardized. For this reason, the German officials have established a working group with the objective of validating methods through multilaboratory validation studies. The experts of the working group also aim for the first time to standardize validated methods and to develop general validation criteria. This manuscript will highlight the current work of the group. For this purpose, an overview is given on the principles and applications of the new mass spectrometric methods. Moreover, requirements and the present work of other institutions regarding method validation are described.

Aryl hydrocarbon receptor pathway participates in myocardial ischemia reperfusion injury by regulating mitochondrial apoptosis

Reducing ischemia reperfusion (I/R) injury has been a major challenge in the treatment of cardiovascular disease. It is widely accepted that mitochondrial apoptosis is an important link in myocardial infarction induced by I/R damage. Studies suggested that aryl hydrocarbon receptor (AhR) pathway plays an important role in the development and function of the cardiovascular system and regulating the mitochondrial homeostasis. AhR played a critical role in regulating mitochondrial homeostasis in response to TCDD-induced stress through translocation of AhR from the cytoplasm into the cell nucleus. And a portion of AhR was found in the mitochondrial inter membrane space. Moreover, there were abundant AhR expression in myocardial cells induced by I/R, and ischaemic post-conditioning reversed apoptotic reperfusion injury involving the AhR signaling pathway. Additionally, AhR is involved in the mechanism of cardiac toxicity of many chemotherapeutic agents. Given the discovery mentioned above, we hypothesize that AhR pathway participates in myocardial ischemia reperfusion injury by regulating mitochondrial apoptosis. Meanwhile, how the AhR pathway is involved in I/R damage and regulating mitochondrial apoptosis is needed to further verified. To evaluate our hypothesis, we will measure the expression of AhR in mitochondria and cytoplasm after myocardial ischemia and reperfusion in vitro and in vivo. And then study the AhR pathway in regulating mitochondrial apoptosis to participate in myocardial I/R injury using mitochondrial protein mass spectrometry analysis and RNA interference technique. If our hypothesis is correct, AhR will be a key target in myocardial I/R injury and myocardial infarction, which could provide important agents.