Dba-free" Palladium Intermediates Of The Heck-matsuda Reaction."

The dba-free Heck-Matsuda reaction was investigated via direct ESI-MS(/MS) monitoring. Palladium species involved in the reduction of Pd(II) during a Wacker type reaction and several dba-free arylpalladium transient complexes were detected and characterized. Based on these findings, a more comprehensible catalytic cycle for this pivotal reaction is suggested.113277-8

Spatial Segmentation and Feature Selection for Desi Imaging Mass Spectrometry Data with Spatially-Aware Sparse Clustering.

Recent experimental advances in matrix-assisted laser desorption/ionization (MALDI) and desorption electrospray ionization (DESI) have demonstrated the usefulness of these technologies in the molecular imaging of biological samples. However, development of computational methods for the statistical interpretation and analysis of the chemical differences present in the distinct regions of these samples is still a major challenge. In this poster, we propose statistically-minded methods and computational tools for analyzing DESI imaging experiments. Specifically, we present techniques for signal processing and unsupervised multivariate image segmentation, which are also applicable to other imaging mass spectrometry (IMS) methods such as MALDI

Pancreatic Cancer Surgical Resection Margins: Molecular Assessment by Mass Spectrometry Imaging

International audienceBackgroundSurgical resection with microscopically negative margins remains the main curative option for pancreatic cancer; however, in practice intraoperative delineation of resection margins is challenging. Ambient mass spectrometry imaging has emerged as a powerful technique for chemical imaging and real-time diagnosis of tissue samples. We applied an approach combining desorption electrospray ionization mass spectrometry imaging (DESI-MSI) with the least absolute shrinkage and selection operator (Lasso) statistical method to diagnose pancreatic tissue sections and prospectively evaluate surgical resection margins from pancreatic cancer surgery.Methods and FindingsOur methodology was developed and tested using 63 banked pancreatic cancer samples and 65 samples (tumor and specimen margins) collected prospectively during 32 pancreatectomies from February 27, 2013, to January 16, 2015. In total, mass spectra for 254,235 individual pixels were evaluated. When cross-validation was employed in the training set of samples, 98.1% agreement with histopathology was obtained. Using an independent set of samples, 98.6% agreement was achieved. We used a statistical approach to evaluate 177,727 mass spectra from samples with complex, mixed histology, achieving an agreement of 81%. The developed method showed agreement with frozen section evaluation of specimen margins in 24 of 32 surgical cases prospectively evaluated. In the remaining eight patients, margins were found to be positive by DESI-MSI/Lasso, but negative by frozen section analysis. The median overall survival after resection was only 10 mo for these eight patients as opposed to 26 mo for patients with negative margins by both techniques. This observation suggests that our method (as opposed to the standard method to date) was able to detect tumor involvement at the margin in patients who developed early recurrence. Nonetheless, a larger cohort of samples is needed to validate the findings described in this study. Careful evaluation of the long-term benefits to patients of the use of DESI-MSI for surgical margin evaluation is also needed to determine its value in clinical practice.ConclusionsOur findings provide evidence that the molecular information obtained by DESI-MSI/Lasso from pancreatic tissue samples has the potential to transform the evaluation of surgical specimens. With further development, we believe the described methodology could be routinely used for intraoperative surgical margin assessment of pancreatic cancer

Desorption Electrospray Ionization Mass Spectrometry Reveals Lipid Metabolism of Individual Oocytes and Embryos.

Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i) lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS), using the bovine species as biological model, (ii) the metabolically most relevant lipid compounds by multivariate data analysis and (iii) lipid upstream metabolism by quantitative real-time PCR (qRT-PCR) analysis of several target genes (ACAT1, CPT 1b, FASN, SREBP1 and SCAP). Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerols (TAG). Principal component analysis (PCA) and linear discriminant analysis (LDA), performed for the first time on DESI-MS fused data, allowed unequivocal discrimination between oocytes and blastocysts based on specific lipid profiles. This analytical approach resulted in broad and detailed lipid annotation of single oocytes and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate that blastocysts produced in vitro and their in vivo counterparts differed significantly in the homeostasis of cholesterol and FFA metabolism. These results should assist in the production of viable and healthy embryos by elucidating in vivo embryonic lipid metabolism

Absence of the caspases 1/11 modulates liver global lipid profile and gut microbiota in high-fat-diet-induced obese mice

Obesity is a chronic disease with rising worldwide prevalence and largely associated with several other comorbidities, such as cancer, non-alcoholic fatty liver disease (NAFLD), and metabolic syndrome. Hepatic steatosis, a hallmark of NAFLD, is strongly correlated with obesity and has been correlated with changes in the gut microbiota, which can promote its development through the production of short-chain fatty acids (SCFAs) that regulate insulin resistance, bile acid, choline metabolism, and inflammation. Recent studies have suggested a controversial role for the inflammasome/caspase-1 in the development of obesity and non-alcoholic steatohepatitis (NASH). Here, we evaluated the role of inflammasome NLRP3 and caspases 1/11 in the establishment of obesity and hepatic steatosis in diet-induced obese mice, correlating them with the global lipid profile of the liver and gut microbiota diversity. After feeding wild-type, caspases 1/11, and NLRP3 knockout mice with a standard fat diet (SFD) or a high-fat diet (HFD), we found that the caspases 1/11 knockout mice, but not NLRP3 knockout mice, were more susceptible to HFD-induced obesity, and developed enhanced hepatic steatosis even under SFD conditions. Lipidomics analysis of the liver, assessed by MALDI-MS analysis, revealed that the HFD triggered a significant change in global lipid profile in the liver of WT mice compared to those fed an SFD, and this profile was modified by the lack of caspases 1/11 and NLRP3. The absence of caspases 1/11 was also correlated with an increased presence of triacylglycerol in the liver. Gut microbial diversity analysis, using 16S rRNA gene sequencing, showed that there was also an increase of Proteobacteria and a higher Firmicutes/Bacteroidetes ratio in the gut of caspases 1/11 knockout mice fed an HFD. Overall, mice without caspases 1/11 harbored gut bacterial phyla involved with weight gain, obesity, and hepatic steatosis. Taken together, our data suggest an important role for caspases 1/11 in the lipid composition of the liver and in the modulation of the gut microbial community composition. Our results further suggest that HFD-induced obesity and the absence of caspases 1/11 may regulate both lipid metabolism and gut microbial diversity, and therefore may be associated with NAFLD and obesity10CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP312359/2016-02016/22577-6This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq -#312359/2016-0). CM was funded by the Canada Research Chair Program, the Canadian Foundation for Innovation, McGill University, and the Canadian Institutes for Health Research (PJT-149098). ME was funded by the São Paulo Research Foundation (FAPESP) (2016/22577-6)

Cholesterol Sulfonation Enzyme, SULT2B1b, Modulates AR and Cell Growth Properties in Prostate Cancer

Cholesterol accumulates in prostate lesions and has been linked to prostate cancer (PCa) incidence and progression. However, how accumulated cholesterol contributes to PCa development and progression is not completely understood. Cholesterol sulfate (CS), the primary sulfonation product of cholesterol sulfotransferase (SULT2B1b), accumulates in human prostate adenocarcinoma and precancerous prostatic intraepithelial neoplasia (PIN) lesions compared to normal regions of the same tissue sample. Given the enhanced accumulation of CS in these lesions, it was hypothesized that SULT2B1b-mediated production of CS provides a growth advantage to these cells. To address this, PCa cells with RNAi-mediated knockdown (KD) of SULT2B1b were used to assess the impact on cell growth and survival. SULT2B1b is expressed and functional in a variety of prostate cells and the data demonstrate that SULT2B1b KD, in LNCaP and other androgen-responsive (VCaP and C4-2) cells, results in decreased cell growth/viability and induces cell death. SULT2B1b KD also decreases androgen receptor (AR) activity and expression at mRNA and protein levels. While AR overexpression has no impact on SULT2B1b KD-mediated cell death, addition of exogenous androgen is able to partially rescue the growth inhibition induced by SULT2B1b KD in LNCaP cells. These results suggest that SULT2B1b positively regulates the AR either through alterations in ligand availability or by interaction with critical co-regulators that influence AR activity

A PTHi pode prever as variações do cálcio após tiroidectomia total?

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder that leads to lower natural reproductive potential and presents a challenge for assisted reproductive medicine because patients may exhibit immature oocyte retrieval and a higher risk of ovarian hyper stimulation syndrome during in vitro fertilization (IVF) treatment. This study aimed to identify potential lipid biomarkers for women with PCOS and a hyper response to controlled ovarian stimulation. Follicular fluid samples were collected from patients who underwent IVF, including normal responder women who became pregnant (control group, n = 11), women with PCOS and a hyper response to gonadotropins (PCOS group, n = 7) and women with only hyper response to gonadotropins (HR group, n = 7). A lipidomic analysis was performed by electrospray ionization mass spectrometry, and candidate biomarkers were analyzed by tandem mass spectrometry experiment. The lipid profiles indicated particularities related to differences in phosphatidylcholine (PCOS and HR), phosphatidylserine, phosphatydilinositol and phosphatidylglycerol (control), sphingolipids (PCOS) and phosphatidylethanolamine (control and HR). These findings contribute to the understanding of the molecular mechanisms associated with lipid metabolism in the PCOS-related hyper response, and strongly suggest that these lipids may be useful as biomarkers, leading to the development of more individualized treatment for pregnancy outcome.Polycystic ovary syndrome (PCOS) is an endocrine-metabolic disorder that leads to lower natural reproductive potential and presents a challenge for assisted reproductive medicine because patients may exhibit immature oocyte retrieval and a higher risk of324554CNPQ - CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2012/06389-4]sem informação2012/06389-4This work was supported by CNPq (National Council for Scientific and Technological Development), Brazil and by grant 2012/06389-4, São Paulo Research Foundation (FAPESP

Developments in ambient mass spectrometry imaging and its applications in biomedical research and cancer diagnosis

My dissertation research has focused on developing and applying innovative mass spectrometry methods to the biomedical field, specifically in the area of cancer detection and intraoperative surgical-guidance. We used desorption electrospray ionization (DESI-MS) imaging to chemically characterize different types of cancers. The characterization was performed using particular chemical compounds found to be markers of disease through chemical imaging of human biopsy tissue. In particular, we have focused on investigating human genitourinary cancers, such as prostate, bladder and kidney, as well as human brain cancers. Our goal now is to translate this technology as validated in the laboratory to clinical and surgical settings, offering chemical information on disease state that could increase patient survival and improve disease management. We believe that this methodology can assist clinicians by offering analytical tools and molecular information that can provide improved intraoperative diagnosis of tumors and their margins, augmenting but not interfering with established surgical workflow. We have successfully shown that DESI-MS enables fast, accurate diagnosis and grading of human cancers based on lipid profiles, and have developed statistical classifiers based on molecular data. DESI-MS also provides valuable information on tumor cell concentration in tissue and on tumor margin position, especially in the case of human brain cancers. In addition to diagnostic capabilities, we have been able to contribute new knowledge on lipid expression in human cancers. Many of the studies included in this dissertation involved identification and structural characterization of various lipids by tandem MS analysis. For example, we detected cholesterol sulfate (CS) as a potential biomarker for prostate cancer. These studies have evolved and in collaboration with scientists in the Purdue Cancer Center we have now identified the enzyme cholesterol sulfotransferase 2B1b as a novel regulator of a malignant phenotype in prostate cancer. We are now also investigating the possibility of detecting CS and other lipid markers in urine samples as a non-invasive method for prostate cancer diagnosis. In human brain cancers, we have detected distinctive variations in lipid profiles related to malignancy, such as higher abundance of lipid phosphatidylserine (40:4) in high grade oligodendroglioma subtype, information which is potentially important for better understanding the biochemical processes related to cancer development. It is remarkable how the changes in lipid profiles observed in DESI-MS data provide reliable and accurate information on tumor subtype and grade. We are currently revising and expanding the classifier to include other types of brain tumors, such as meningiomas and metastatic brain tumors, and normal brain tissue with the goal of further improving diagnostic capabilities. We have also recently identified the oncometabolite 2-hydroxygluterate (2HG) directly from tissue by negative ion mode DESI-MS imaging in human gliomas; overproduction and accumulation of 2HG has been recently associated with a genetic mutation of the isocitrate dehydrogenase 1 (IDH1) enzyme, an indicator of increased survival rates for glioma patients. The exceptional ability to rapidly detect 2HG from tissue by DESI will add to the diagnostic capabilities of the technology by providing valuable prognostic information to surgeon and patient, as well as additional information on tumor margins. The development of novel methodologies and capabilities for tissue analysis by DESI-MS has also been pursued. The capability for full 3D molecular image construction using DESI-MS imaging was developed, allowing direct correlation and easy visualization of endogenous compounds in substructures of an entire organ, as demonstrated for a mouse brain. More recently, new solvent systems were developed which allow for a new capability for DESI-MS imaging - non-destructive tissue analysis. Sequential analysis, using for example immunohistochemistry or MALDI, can now be performed on the same tissue section previously imaged by DESI-MS. This allows lipid and metabolite information obtained by DESI-MS to be unambiguously correlated to protein and morphological information. This progress has greatly expanded the applications of DESI technology, especially in the biological field, and provides means for better understanding the molecular information derived from tissue. Moreover, this development also allows DESI-MS imaging to be now fully integrated into the pathological procedures, in clinical and surgical practice. (Abstract shortened by UMI.