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The embryonic environment can modify cancer cell metabolism, and it is reported to induce the loss of tumorigenic properties and even affect the differentiation of cancer cells into normal tissues. The cellular mechanisms related to this remarkable phenomenon, which is likely mediated by cell-to-cell communication, have been previously investigated with particular focus on the proteins and genes involved. In this study we report the optimization and results of a straightforward

Made available in DSpace on 2022-04-29T08:32:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) The aim of this study was to examine the effects of long-term dietary supplementation of young Nellore bulls with rumen-protected polyunsaturated fatty acids (PUFAs) and of the inclusion of catalase in the semen extender on semen quality, in vitro sperm fertilizing ability, and intracytoplasmic lipid content in the resulting embryos. Twelve Nellore bulls were supplemented with rumen-protected PUFAs or with a basal diet from 14 to 24 months of age. The semen was collected at the end of supplementation. For cryopreservation, the ejaculate was divided into two equal volumes and catalase was added to the extender in one of the fractions. Thus, the experimental design consisted of a 2 × 2 factorial scheme with two diets (control and PUFA) and two extenders (Cat+ and Cat-). Total motility and the percentage of rapid cells in fresh semen were negatively affected by dietary supplementation with PUFAs (P < 0.05), but these effects did not persist after freezing. The frozen/thawed semen of animals fed PUFAs exhibited an increase in the percentages of damaged plasma and acrosomal membranes, as well as an increase in the proportion of lipids ions at m/z 578 and m/z 757 detected by MALDI-MS. Nevertheless, there was no effect of the treatments on in vitro embryo development. However, embryos derived from bulls supplemented with PUFAs exhibited higher lipid accumulation compared to control (P < 0.05). In conclusion, PUFA supplementation promoted worsening of semen quality without affecting the in vitro sperm fertilizing ability; however, the paternal diet affected the intracytoplasmic lipid content in the resulting embryos. São Paulo State University (UNESP) Department of Animal Reproduction Graduate Program in Genetics and Animal Breeding School of Agrarian and Veterinary Sciences Campus Jaboticabal São Paulo State University (UNESP) Department of Animal Reproduction Graduate Program in Veterinary Medicine School of Agrarian and Veterinary Sciences Campus Jaboticabal Proteomics Center of Excellence Northwestern University, 2170 Campus Dr ThoMSon Mass Spectrometry Laboratory Institute of Chemistry University of Campinas Cidade Universitaária Zeferino Vaz, Campinas Department of Chemistry Purdue University Instituto de Zootecnia (IZ/APTA), Sertãozinho São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science, Campus Botucatu São Paulo State University (UNESP) School of Veterinary Medicine Laboratory of Reproductive Physiology, Campus Araçatuba São Paulo State University (UNESP) Department of Animal Reproduction Graduate Program in Genetics and Animal Breeding School of Agrarian and Veterinary Sciences Campus Jaboticabal São Paulo State University (UNESP) Department of Animal Reproduction Graduate Program in Veterinary Medicine School of Agrarian and Veterinary Sciences Campus Jaboticabal São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science, Campus Botucatu São Paulo State University (UNESP) School of Veterinary Medicine Laboratory of Reproductive Physiology, Campus Araçatuba FAPESP: #2013/13696–3 FAPESP: #2015/06733–5 FAPESP: #2019/11174–6 CNPq: #307416/2015–1 CNPq: #314136/2018–5 CAPES: 001

Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed in vitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing in vivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.

Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme highly active in cancer. Here we studied how the balance between SFAs and UFAs maintained by SCD impacts cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman spectroscopy (SRS). Further, the loss of equilibrium between UFAs and SFAs resulting from SCD knock down triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Stiff and disorganized ER membrane was visualized by electron microscopy and SRS imaging in cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and re-equilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed in vitro, translated into suppression of intraperitoneal tumor growth in xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA enriched diet, initiated ER stress in tumors growing in vivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to new treatment strategies targeting the lipid balance.Significance StatementWe show that the balance between saturated and unsaturated fatty acids tightly regulated by the desaturase SCD impacts the survival of cancer cells; increased levels of unsaturation being protective against ER stress induced apoptosis. Decreasing fatty acid unsaturation, either through SCD depletion or through SCD inhibition coupled with a dietary intervention blocks tumor progression in vivo. Our findings support the concept of targeting the lipid balance as a new target in cancer.

Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time-points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at −80.0°C without solvent for less than two weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.

This study describes the latest generation of a high-throughput screening system that is capable of screening thousands of organic reactions in a single day. This system combines a liquid handling ...

Thousands of chemical compounds produced by industry are dispersed in the human environment widely enough to reach the world population, and the introduction of new chemicals constantly occurs. As new synthetic molecules emerge, rapid analytical workflows for screening possible presence of exogenous compounds in biofluids can be useful as a first pass analysis to detect chemical exposure and guide the development and application of more elaborate LC-MS/MS methods for quantification. In this study, a suspect screening workflow using the multiple reaction monitoring (MRM) profiling method is proposed as a first pass exploratory technique to survey selected exogenous molecules in human urine samples. The workflow was applied to investigate 12 human urine samples using 310 MRMs related to the chemical functionalities of 87 exogenous compounds present in the METLIN database and reported in the literature. A total of 11 MRMs associated with five different compounds were detected in the samples. Product ion scans for the precursor ions of the selected MRMs were acquired as a further identification step for these chemicals. The suspect screening results suggested the presence of five exogenous compounds in the human urine samples analyzed, namely metformin, metoprolol, acetaminophen, paraxanthine and acrylamide. LC-MS/MS was applied as a last step to confirm these results, and the presence of four out of the five targets selected by MRM profiling were corroborated, indicating that this workflow can support the selection of suspect compounds to screen complex samples and guide more time-consuming and specific quantification analyses.

The aim of this work was to compare the lipidome and metabolome profiling in the Longissimus thoracis muscle early and late postmortem from high and normal ultimate pH (pHu) beef. Lipid profiling discriminated between high and normal pHu beef based on fatty acid metabolism and mitochondrial beta-oxidation of long chain saturated fatty acids at 30 min postmortem, and phospholipid biosynthesis at 44 h postmortem. Metabolite profiling also discriminated between high and normal pHu beef, mainly through glutathione, purine, arginine and proline, and glycine, serine and threonine metabolisms at 30 min postmortem, and glycolysis, TCA cycle, glutathione, tyrosine, and pyruvate metabolisms at 44 h postmortem. Lipid and metabolite profiles showed reduced glycolysis and increased use of alternative energy metabolic processes that were central to differentiating high and normal pHu beef. Phospholipid biosynthesis modification suggested high pHu beef experienced greater oxidative stress.

Can exposure time to equilibration solutions during oocyte vitrification affect the lipid profile of oocytes and embryonic development? Could vitrification media supplemented with oleic, linoleic acids and L-carnitine effectively minimize damage induced by vitrification on embryo development and oocyte membrane lipid profile?Experimental study including 936 oocytes from C57BL/6J mice, randomly divided into fresh IVF (control) and equilibration solution groups. Oocytes were exposed to equilibration solution from Irvine Scientific, Tvitri-4 or Tvitri-4 supplemented with L-carnitine and fatty acids for 7 or 10 min, vitrified-warmed, and submitted to IVF. The lipid profile of oocytes immediately after equilibration solution exposure was also asessed using the same equilibration times and solution compositions.Longer equilibration time resulted in lower oocyte survival and blastocyst rates, and reduced relative abundance of structural lipids, i.e. phosphatidylcholines and sphingomyelins, varying according to equilibration solution composition. It also induced membrane disruptions resembling bubbles in the oocyte surface predominantly in equilibration solution from Irvine Scientific, rarely in Tvitri-4 and absent in Tvitri-4 supplemented with L-carnitine and fatty acids. To reveal the metabolic pathways associated with the equilibration phase of vitrification, lipid pathway analysis was conducted; both P-values and pathway impact values showed that the linoleic acid metabolism (P = 0.00223; impact =1) and alpha-Linolenic acid metabolism (P = 0.00084; impact = 0.33) were the most pathway perturbed, followed by glycerophospholipid metabolism (P = 0.0167; impact = 0.25) CONCLUSION: A longer equilibration phase pre-vitrification can influence embryo development and induce changes in oocyte lipid composition related to membrane integrity. The results suggest internalization of oleic and linoleic acids added to equilibration solution by the oocyte, which, to some extent, contributed to membrane phospholipids preservation, regardless of the equilibration times assessed.

Merging optical images of tissue sections with the spatial distributions of molecules seen by imaging mass spectrometry is a powerful approach to better understand the metabolic roles of the mapped molecules. Here, we use histologically friendly desorption electrospray ionization–mass spectrometry (DESI-MS) to map the lipid distribution in tissue sections of ovaries from cows (N = 8), sows (N = 3), and mice (N = 12). Morphologically friendly DESI-MS imaging allows the same sections to be examined for morphological information. Independent of the species, ovarian follicles, corpora lutea, and stroma could be differentiated by principal component analysis, showing that lipid profiles are well conserved among species. As examples of specific findings, arachidonic acid and the phosphatidylinositol PI(38:4), were both found concentrated in the follicles and corpora lutea, structures that promoted ovulation and implantation, respectively. Adrenic acid was spatially located in the corpora lutea, suggesting the importance of this fatty acid in the ovary luteal phase. In summary, lipid information captured by DESI-MS imaging could be related to ovarian structures and data were all conserved among cows, sows, and mice. Further application of DESI-MS imaging to either physiological or pathophysiological models of reproductive conditions will likely expand knowledge of the roles of specific lipids and pathways in ovarian activity and mammalian fertility.

This study describes an automated system used for high throughput screening of reaction conditions based on accelerated reactions occurring in small volumes of reagents. Reaction mixtures are prepared in array format using a fluid handling robot and spotted on a flat polytetrafluoroethylene plate at densities up to 6144 per plate. The reaction and analysis steps are performed simultaneously using desorption electrospray ionization (DESI) to release microdroplets containing the reaction mixture from the plate for reaction prior to arrival at a mass spectrometer. Analysis rates are up to 1 reaction mixture per second and data are recorded in real time using an ion trap mass spectrometer. Beacon compounds are used to triangulate position on the plate and this allows tandem mass spectrometry (MS/MS) to be performed on confirm products of interest. Custom software allows the user to control the system. It is also used to receive data from the DESI mass spectrometer to screen the spectra for compounds of interest, to perform MS/MS and to save data. This custom software also communicates with the software controlling the fluid handling robot (Biomek i7) as well as the Beckman software used to prepare reaction mixtures and also the software that controls the solvent used as the DESI spray. Data were recorded for N-alkylation, N-acylation and N-sulfonylation reactions in three 8 hour experiments on successive days to establish the ruggedness and repeatability of the system. Repeatability is high (94-97%) over this period with false negative 6% (depending on noise threshold chosen). Plates containing 384 reaction mixtures are analyzed in 7 min by moving the DESI sprayer in steps under the sprayer instead of continuously.

Made available in DSpace on 2020-12-12T02:26:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The effectiveness of the use of natriuretic peptide C (NPPC) in the blocking of meiosis has already been proven in several species. However, there are no reports on the use of NPPC in the activation of metabolic processes in embryos. Whereas modulations of cAMP concentrations alter the lipid metabolism of bovine oocytes, the present study aims to evaluate the effect of NPPC on the development, lipid content and transcript levels of genes related to lipid metabolism of IVP bovine embryos. For this purpose, ovaries were obtained from a slaughterhouse, and oocytes were fertilized in vitro (D0). From D5 of in vitro culture, embryos were treated with 100 nM NPPC (NPPC group) or with no NPPC (Control group) and evaluated in terms of Blastocyst (D7) and hatching rates (D10). For the assessment of the cytoplasmatic lipid amounts, blastocysts were stained with Sudan Black B dye. The embryonic lipid profile was investigated by electrospray ionization desorption-mass spectrometry (DESI-MS). The abundance of nine transcripts related to lipid metabolism were assessed using the Biomark HD system. For statistical analysis, blastocyst and hatching rates, lipid content by the Sudan Black B and variation of gene expression between groups were compared by Student t-test. For lipid profile analysis, principal component analysis (PCA) and fold-change were performed. The embryo lipid content was similar between NPPC (881 ± 3.7) and Control (883 ± 5.2) groups (p > 0.05). However, cholesteryl esters and TAGs were downregulated by NPPC at multiple levels according to the DESI-MS profiles. Of the analyzed genes, ELOVL6 and SREBF1 showed an up-regulation in the control group (p < 0.05), while CPT2 was observed to be up-regulated in the NPPC-treated embryos. There was no significant difference in the blastocyst production rate between NPPC (44.4%) and Control (42.4%), however the hatching rate at D10 was higher (p < 0.05) in the NPPC group (69.77%) when compared to the Control group (48.33%). These findings demonstrate that NPPC alters the mRNA expression of genes related to lipid metabolism and that it exerts a positive effect on the hatching rates of IVP Bos taurus indicus embryos. University of Londrina (UEL) Laboratory of Animal Reproduction São Paulo State University (Unesp) Institute of Biosciences Department of Pharmacology Center for Natural and Human Sciences University ABC Federal São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Science Laboratório de Virologia Animal Departamento de Medicina Veterinária Preventiva Universidade Estadual de Londrina Department of Chemistry and Center for Analytical Instrumentation Development Purdue University Instituto de Ciências Biomédicas Universidade de São Paulo Centro de Ciências Naturais e Humanas Universidade Federal Do ABC São Paulo State University (Unesp) Institute of Biosciences Department of Pharmacology São Paulo State University (UNESP) School of Sciences Humanities and Languages Department of Biological Science CNPq: 403862/2016-7

The worldwide increase in antimicrobial resistance is due to antibiotic overuse in agriculture and overprescription in medicine. For appropriate and timely patient support, faster diagnosis of antimicrobial resistance is required. Current methods for bacterial identification rely on genomics and proteomics and use comparisons with databases of known strains, but the diagnostic value of metabolites and lipids has not been explored significantly. Standard mass spectrometry/chromatography methods involve multiple dilutions during sample preparation and separation. To increase the amount of chemical information acquired and the speed of analysis of lipids, multiple reaction monitoring profiling (MRM-Profiling) has been applied. The MRM-Profiling workflow includes a discovery stage and a screening stage. The discovery stage employs precursor (PREC) ion and neutral loss (NL) scans to screen representative pooled samples for functional groups associated with particular lipid classes. The information from the first stage is organized in precursor/product ion pairs, or MRMs, and the screening stage rapidly interrogates individual samples for these MRMs. In this study, we performed MRM-Profiling of lipid extracts from four different strains of Escherichia coli cultured with amoxicillin or amoxicillin/clavulanate, a β-lactam and β-lactamase inhibitor, respectively. t tests, analysis of variance and receiver operating characteristic (ROC) curves were used to determine the significance of each MRM. Principal component analysis was applied to distinguish different strains cultured under conditions that allowed or disallowed development of bacterial resistance. The results demonstrate that MRM-Profiling distinguishes the lipid profiles of resistant and nonresistant E. coli strains.

Lomustine, an important agent for treatment of brain tumors and Hodgkin’s lymphoma, has been synthesized using continuous flow methodology. Desorption electrospray ionization mass spectrometry (DESI-MS) was used to quickly explore a large number of reaction conditions for one of the reaction steps and guide the efficient translation of optimized conditions to continuous lomustine production. Using only four inexpensive commercially available starting materials and a total residence time of 9 min, lomustine was prepared via a linear sequence of two chemical reactions performed separately in two telescoped flow reactors. Sequential offline extraction and filtration resulted in a 63% overall yield of pure lomustine at a production rate of 110 mg/h. The primary advantages of this approach are the rapid manufacture of lomustine with two telescoped steps to avoid isolation and purification of a labile intermediate and the mild conditions used in the nitrosylation step, thereby significantly increasing the purit...

Desorption electrospray ionization mass spectrometry (DESI-MS) is a powerful tool for ultra-high throughput chemical reaction screening having enormous scientific and practical implications. DESI-MS enables considerably faster chemical reaction and/or product discovery than most current screening methods using very small amount of materials. It has the advantage of bringing the reagents in contact inside charged micro-droplets, that can accelerate reaction by compared to bulk or flow reaction approaches. Since both the desorption efficiency from the surface and the reaction kinetics can be significantly influenced by the DESI spray solvent flowrate and composition, these parameters are expected to play key roles in the overall performance of the ultrahigh-throughput screening. Syringe pumps are frequently used to deliver the DESI spray. They are robust, but inflexible and slow when it comes to manipulating solvent composition and varying flowrate setpoints. This study addresses the importance of designing a high performance, flexible solvent delivery system that enables more rapid solvent composition and flowrate setpoint changes than the routinely used syringe pumps. Our system utilizes a piezoelectric-based pressure controller and valve system employed with fine-tuned adaptive proportional-integrative-derivative (PID) controller. N-alkylation reactions were used to demonstrate the impact of spray-solvent composition and flowrate on the DESI-MS outcome. In particular, the results showed that ions resulting from solvent mixtures can induce reactions that would be otherwise unobserved when using the corresponding neat solvents. This further signifies the need for a flexible spray-solvent delivery system with fast dynamics in the context of ultra-high throughput DESI-MS analysis.

Newborn calves rely on lipids in colostrum for energy and immune function. The lipid concentration in colostrum, however, is highly variable, and little is known about its composition and maternal factors that influence its composition. The first objective was to measure plasma lipid composition of multiparous cows at 35 d before calving (BC; 35 ± 3 d; ± standard deviation) and 7 d BC (7 ± 2 d), their colostrum, and serum lipid composition of calves (24 h after birth) using multiple reaction monitoring profiling, which is an exploratory and highly sensitive lipidomic analysis method that screens lipids based on chemical functionality. Second, data were analyzed to determine if there were relationships between circulating lipids in the cow, colostrum lipids, and calf serum lipids. Third, relationships between markers of metabolic status of the cows and circulating and colostrum lipids were analyzed with correlation analysis. Blood was sampled and plasma prepared from multiparous cows (n = 16) at 35 and 7 d BC. Within 3 h of parturition, colostrum was collected from cows and fed to her calf. Calves received another feeding of colostrum within 12 h after birth and a serum sample was collected from each calf 24 h after the first feeding of colostrum. The metabolic status of cows was evaluated using insulin, glucose, and nonesterified fatty acid area under the curve in response to an intravenous glucose tolerance test performed at 3 wk BC. Lipids were extracted from plasma, colostrum, and calf serum and were analyzed using multiple reaction monitoring profiling. Concentration of lipids were calculated using spiked in standards and expressed as percent of lipids identified. Data were uploaded into MetaboAnalyst 5.0 for multivariate and univariate analysis. Principal component analysis indicated that circulating lipids in the cow and calf were distinct from lipids in colostrum. Phosphatidylglycerol (PG) concentration was greater in colostrum and calf serum than in cow plasma, with 23 of the 24 PG found in colostrum also found in calf serum. In response to intravenous glucose tolerance test in late gestation, nonesterified fatty acid area under the curve was positively related to total triacylglycerols lipids in 7 d BC plasma (r = 0.63) but negatively related to total membrane lipids in colostrum (r = -0.55). Thus, the metabolic status of the dam influences circulating lipids and colostrum lipid content. Moreover, the circulating lipidome of the cow and calf are similar to one another and distinct from the colostrum lipidome, except for PG, where it appears that colostrum serves as the source for PG in the calf's circulation.

In this study, we applied multiple reaction monitoring (MRM)-profiling to explore the relative ion intensity of lipid classes in plasma samples from sea turtles in order to profile lipids relevant to sea turtle physiology and investigate how dynamic ocean environments affect these profiles. We collected plasma samples from foraging green (Chelonia mydas, n = 28) and hawksbill (Eretmochelys imbricata, n = 16) turtles live captured in North Pacific Costa Rica in 2017. From these samples, we identified 623 MRMs belonging to 10 lipid classes (sphingomyelin, phosphatidylcholine, free fatty acid, cholesteryl ester, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, ceramide, and triacylglyceride) and one metabolite group (acyl-carnitine) present in sea turtle plasma. The relative ion intensities of most lipids (80%) were consistent between species, across seasons, and were not correlated to body size or estimated sex. Of the differences we observed, the most pronounced was the differences in relative ion intensity between species. We identified 123 lipids that had species-specific relative ion intensities. While some of this variability is likely due to green and hawksbill turtles consuming different food items, we found indications of a phylogenetic component as well. Of these, we identified 47 lipids that varied by season, most belonging to the structural phospholipid classes. Overall, more lipids (n = 39) had higher relative ion intensity in the upwelling (colder) season compared to the non-upwelling season (n = 8). Further, we found more variability in hawksbill turtles than green turtles. Here, we provide the framework in which to apply future lipid profiling in the assessment of health, physiology, and behavior in endangered sea turtles.

Purpose We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types. Methods Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation – extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability. Results 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45–195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3). Conclusions Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.

Background A marker indicative of fertility potential of replacement gilts early in development would decrease culling rates in the sow herd, improve sow herd reproductive efficiency, and reduce production costs. The objective of this study was to determine if vaginal lipid profiles at 21 d postnatal (PN) could predict sow reproductive performance. Methods Vaginal swabs of the anterior vagina were taken at 21 ± 4 d PN from gilts born on a commercial sow production facility for lipidomic analysis. Animals were followed prospectively for two years and assigned to reproductive performance categories based on observation of estrus or piglets weaned per sow per year (PSY) across two farrowings. Results Lipids were extracted from cellular material collected with swabs taken from high fertility (HF; n = 28; ≥26 PSY) and infertile (IF; n = 34; no estrus, no pregnancy) animals and multiple reaction monitoring (MRM) profiling was used for lipidome analysis. Relative abundance of arachidonic acid (ARA, C20:4) and docosahexaenoic acid (DHA, C22:6) were lower (P P P

Extracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 μl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 μg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.

Extracellular vesicles (EVs) continue to attract interest for their potential role in targeted therapeutics and as biomarkers of disease and drug response. In order to achieve clinical utility, it is important to determine the pharmacokinetic parameters of candidate EVs during preclinical development using in vivo animal models. To date, no methods exist for studying EV kinetics without modification to surface ligands that may affect normal behavior. Here we introduce an accessible method for labeling and quantifying EVs administered to conscious animals, without disrupting endogenous ligands. Our method relies upon established laboratory techniques and can be tailored to a variety of biological questions. Digital PCR is leveraged to detect a non-homologous oligonucleotide tracer introduced into the vesicles, allowing for quantification over a wide dynamic range. Using an application of this method, we found differences in the in vivo kinetics of EVs from three different cell types using non-linear mixed effects modeling. We propose that this method will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.

Small molecules, including metabolites and lipids, provide information on metabolic pathways and active biological processes in living organisms. They are often diagnostic of disease. Current exploratory methods for metabolomics and lipidomics mostly rely on separation using liquid or gas chromatography (LC or GC) coupled with mass spectrometers capable of acquiring high resolution data to generate an enormous data, but at the cost of lengthy processing and data acquisition. Even though many molecules can be identified and quantified by these methods, the laborious protocols for purification, identification, and validation limit the accessible sample chemical information. To improve the speed and efficiency of exploratory metabolomics and lipidomics, multiple reaction monitoring profiling (MRM profiling) has been developed. This strategy involves a three-stage workflow which starts by considering the metabolome as a collection of functional groups. The Discovery Stage interrogates a representative sample mixture for functional groups using the functional group specific precursor ion (Prec) scans and neutral loss (NL) scans. This experiment usually uses a triple quadrupole mass spectrometer without chromatography, i.e. by direct sample infusion. In the second Screening Stage, the main features seen in the Prec and NL scans are organized into lists of precursor ion/product ion transitions (MRMs) which are then used for the fast, specific, and sensitive interrogation of each individual sample. Data analysis by univariate and multivariate statistical methods is used to identify the most informative MRMs and so classify the individual samples. The compounds (biomarkers) which are responsible for the most informative MRMs in particular sample classes can be investigated in an optional third Identification Stage i.e. in a structural identification study. MRM profiling benefits from the much smaller number of functional groups compared to the number of individual metabolites existing in biological samples (where most metabolites are still unknown), resulting in acquisition of a much smaller data set and a shorter analysis time. The application of MRM Profiling to several biological and clinical problems is used to illustrate its features.

Nanotechnology has the capacity to revolutionize numerous fields and processes, however, exposure-induced health effects are of concern. The majority of nanoparticle (NP) safety evaluations have been performed utilizing healthy models and have demonstrated the potential for pulmonary toxicity. A growing proportion of individuals suffer diseases that may enhance their susceptibility to exposures. Specifically, metabolic syndrome (MetS) is increasingly prevalent and is a risk factor for the development of chronic diseases including type-2 diabetes, cardiovascular disease, and cancer. MetS is a combination of conditions which includes dyslipidemia, obesity, hypertension, and insulin resistance. Due to the role of lipids in inflammatory signaling, we hypothesize that MetS-associated dyslipidemia may modulate NP-induced immune responses. To examine this hypothesis, mice were fed either a control diet or a high-fat western diet (HFWD) for 14-weeks. A subset of mice were treated with atorvastatin for the final 7-weeks to modulate lipids. Mice were exposed to silver NPs (AgNPs) via oropharyngeal aspiration and acute toxicity endpoints were evaluated 24-h post-exposure. Mice on the HFWD demonstrated MetS-associated alterations such as increased body weight and cholesterol compared to control-diet mice. Cytometry analysis of bronchoalveolar lavage fluid (BALF) demonstrated exacerbation of AgNP-induced neutrophilic influx in MetS mice compared to healthy. Additionally, enhanced proinflammatory mRNA expression and protein levels of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and interleukin-6 were observed in MetS mice compared to healthy following exposure. AgNP exposure reduced mRNA expression of enzymes involved in lipid metabolism, such as arachidonate 5-lipoxygenase and arachidonate 15-lipoxygenase in both mouse models. Exposure to AgNPs decreased inducible nitric oxide synthase gene expression in MetS mice. An exploratory lipidomic profiling approach was utilized to screen lipid mediators involved in pulmonary inflammation. This assessment indicates the potential for reduced levels of lipids mediators of inflammatory resolution (LMIR) in the MetS model compared to healthy mice following AgNP exposure. Statin treatment inhibited enhanced inflammatory responses as well as alterations in LMIR observed in the MetS model due to AgNP exposure. Taken together our data suggests that MetS exacerbates the acute toxicity induced by AgNPs exposure possibly via a disruption of LMIR leading to enhanced pulmonary inflammation.

Sow milk fat content is crucial to neonatal survival, as it is utilized for thermogenesis and nutrition. However, fat is the most variable component of milk in concentration and lipid species. Characterizing lipid changes across the course of a sow’s lactation may help identify molecules or systems to target to help enhance milk fat quality and quantity for neonatal survival and growth. Percent fat variation is greatest in colostrum, the first milk. Little is known regarding colostrum synthesis, other than it accumulates in the gland beginning in mid-late pregnancy, which is prior to the initiation of fatty acid synthesis in lactocytes. The objective of this study was to characterize changes in lipid composition of milk across the course of lactation and determine if there was a relationship between fat percent and lipid species in colostrum and mature milk. Milk was collected from 9 multiparous sows on days 0, 3, 7, and 14 relative to birth. Percent fat was determined by creamatocrit, and found to be different (p< 0.05) between day 0 (12.36 ± 5.90%) and day 3 (16.22 ± 3.65%) but not between day 7 (13.13 ± 2.19%) and 14 (12.13 ± 2.45%). Fat was extracted from milk using the Bligh-Dyer method and profiled using multiple reaction monitoring. Amounts of lipid species were calculated relative to standards and data analysis was performed using Metaboanalyst 4.0. Principle component analysis revealed lactation day had a significant effect on distribution of fats. Triacylglycerides (TAG), phosphatidylglycerol (PG), and plasma membrane lipids were modified from colostrum to mature milk, with a significant increase in PGs and TAGs across the course of lactation. Correlation analysis of percent fat with lipid concentration indicated strong relationships (P < 0.05; |r| >0.80) with eight lipids. No differences are found in the abundance of plasma membrane phospholipids, sphingomyelin, or cholesterol esters across lactation days.

Pre-existing conditions modulate sensitivity to numerous xenobiotic exposures such as air pollution. Specifically, individuals suffering from metabolic syndrome (MetS) demonstrate enhanced acute inflammatory responses following particulate matter inhalation. The mechanisms associated with these exacerbated inflammatory responses are unknown, impairing interventional strategies and our understanding of susceptible populations. We hypothesize MetS-associated lipid dysregulation influences mediators of inflammatory resolution signaling contributing to increased acute pulmonary toxicity. To evaluate this hypothesis, healthy and MetS mouse models were treated with either 18-hydroxy eicosapentaenoic acid (18-HEPE), 14-hydroxy docosahexaenoic acid (14-HDHA), 17-hydroxy docosahexaenoic acid (17-HDHA), or saline (control) via intraperitoneal injection prior to oropharyngeal aspiration of silver nanoparticles (AgNP). In mice receiving saline treatment, AgNP exposure resulted in an acute pulmonary inflammatory response that was exacerbated in MetS mice. A targeted lipid assessment demonstrated 18-HEPE, 14-HDHA, and 17-HDHA treatments altered lung levels of specialized pro-resolving lipid mediators (SPMs). 14-HDHA and 17-HDHA treatments more efficiently reduced the exacerbated acute inflammatory response in AgNP exposed MetS mice as compared to 18-HEPE. This included decreased neutrophilic influx, diminished induction of inflammatory cytokines/chemokines, and reduced alterations in SPMs. Examination of SPM receptors determined baseline reductions in MetS mice compared to healthy as well as decreases due to AgNP exposure. Overall, these results demonstrate AgNP exposure disrupts inflammatory resolution, specifically 14-HDHA and 17-HDHA derived SPMs, in MetS contributing to exacerbated acute inflammatory responses. Our findings identify a potential mechanism responsible for enhanced susceptibility in MetS that can be targeted for interventional therapeutic approaches.

Metabolite profiling by mass spectrometry (MS) is an area of interest for disease diagnostics, biomarker discovery, and therapeutic evaluation. A recently developed approach, multiple reaction monitoring (MRM)-profiling, searches for metabolites with precursor (Prec) and neutral loss (NL) scans in a representative sample and creates a list of ion transitions. These are then used in an MRM method for fast screening of individual samples and discrimination between healthy and diseased. A large variety of functional groups are considered and all signals discovered are recorded in the individual samples, making this a largely unsupervised method. MRM-profiling is described here and then demonstrated with data for over 900 human plasma coronary artery disease (CAD) samples. Representative pooled samples for each condition were interrogated using a library of over a hundred Prec and NL scans on a triple quadrupole MS. The data from the Prec and NL experiments were converted into ion transitions, initially some 1266 transitions. Each ion transition was examined in the individual samples on a time scale of milliseconds per transition, which allows for rapid screening of large sample sets (

We report the high throughput analysis of reaction mixture arrays using methods and data handling routines that were originally developed for biological tissue imaging. Desorption electrospray ionization (DESI) mass spectrometry (MS) is applied in a continuous on-line process at rates that approach 104 reactions per h at area densities of up to 1 spot per mm2 (6144 spots per standard microtiter plate) with the sprayer moving at ca. 104 microns per s. Data are analyzed automatically by MS using in-house software to create ion images of selected reagents and products as intensity plots in standard array format. Amine alkylation reactions were used to optimize the system performance on PTFE membrane substrates using methanol as the DESI spray/analysis solvent. Reaction times can be

Sow longevity affects economic returns to pork producers. The cost of gilt replacements is substantial and sows with greater than three litters have lower costs per pig produced. An early marker of reproductive potential would facilitate early identification of superior females, and likely increase sow longevity. Gilts raised in small litters have greater reproductive competence, but mechanisms associated with increased reproductive responses are not fully understood. Here, early postnatal development of the gilt's reproductive tract is described, and a brief review of literature is presented to support that factors in colostrum regulate the developmental trajectory of the gilt's uterine tissues. We propose that, similar to the uterus, nutritional environment likely affects the postnatal developmental programme of the vagina. A metabolomics approach, multiple reaction monitoring -profiling, for biomarker discovery is described, along with evidence that lipids present in vaginal samples are differentially expressed in gilts exposed to colostrum versus milk replacer fed. These exploratory studies indicate that the vaginal cell lipidome may reflect the postnatal nutritional environment, which defines to a large extent the gilt's reproductive potential. Together findings support further investigations to identify biomarkers predictive of fertility outcomes in the metabolome of gilt reproductive tracts.

Thin film formats are used to study the Claisen-Schmidt base-catalyzed condensation of 6-hydroxy-1-indanone with substituted benzaldehydes and to compare reaction acceleration relative to bulk. Acceleration factors relative exceeded 103 initially and were on the order of 102 at steady state, in spite of the fact that the confined volume reaction was not electrostatically driven. Substituent effects were muted compared to those in the corresponding bulk and microdroplet reactions and it is concluded that the rate-limiting step at steady state is reagent transport to the interface. Conditions were found that allowed product deposition from the thin film to occur continuously as reaction mixture was added and as solvent evaporated. Yields of 74% and production rates of 98 mg/hour were reached in a very simple experimental system which could be multiplexed to greater scales.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.Graphical AbstractSummary SentenceMaternal prepregnancy obesity induced by high-fat diet significantly impacts circulating lipids and similarly modifies the lipid composition of milk and circulating lipid of suckling neonates.

Secretions of the endometrium are vital for peri-implantation growth and development of the sheep conceptus. Extracellular vesicles (EVs) are present in the uterine lumen, emanate from both the endometrial epithelia of the uterus and trophectoderm of the conceptus, and hypothesized to mediate communication between those cell types during pregnancy establishment in sheep. Size-exclusion chromatography and nanoparticle tracking analysis determined that total EV number in the uterine lumen increased from days 10 to 14 of the cycle but was lower on days 12 and 14 of pregnancy in sheep. Intrauterine infusions of interferon tau (IFNT) did not affect total EV number in the uterine lumen. Quantitative mass spectrometric analyses defined proteins and lipids in EVs isolated from the uterine lumen of day 14 cyclic and pregnant sheep. In vitro analyses found that EVs decreased ovine trophectoderm cell proliferation and increased IFNT production without effects on gene expression as determined by RNA-seq. Collective results support the idea EVs impact conceptus growth during pregnancy establishment via effects on trophectoderm cell growth.

We demonstrate the use of accelerated reactions with desorption electrospray ionization mass spectrometry (DESI-MS) as a tool for predicting the outcome of microfluidic reactions. DESI-MS was employed as a high throughput experimentation tool to provide qualitative predictions of reaction outcomes, so that vast regions of chemical reactivity space may be more rapidly explored and areas of optimal efficiency identified. This work is part of a larger effort to accelerate reaction optimization to enable the rapid development of continuous-flow syntheses of small molecules in high yield. In order to build confidence in this approach, however, it is necessary to establish a robust predictive connection between reactions performed under analogous DESI-MS, batch, and microfluidic reaction conditions. In the present work, we explore the potential of high throughput DESI-MS experiments to identify trends in reactivity based on chemical structure, solvent, temperature, and stoichiometry that are consistent across these platforms. N-alkylation reactions were used as the test case due to their ease of reactant and product detection by electrospray ionization mass spectrometry (ESI-MS) and their great importance in API synthesis. While DESI-MS narrowed the scope of possibilities for reaction selection among some parameters such as solvent, others like stoichiometry and temperature still required further optimization under continuous synthesis conditions. DESI-MS high throughput experimentation (HTE) reaction evaluation significantly reduced the search space for flow chemistry optimization, thus representing a significant savings in time and materials to achieve a desired transformation with high efficiency.

Desorption electrospray ionization (DESI) is a spray-based ambient ionization method for mass spectrometry (MS) that generates ions in native atmospheric conditions (e.g., pressure and temperature). Ambient ionization allows in situ analysis of unmodified samples by eliminating analyte extraction and separation steps before MS. Lipid analysis of individual mammalian oocytes and preimplantation embryos is challenging because of their complex chemical composition and minute dimensions (100-300 μm in diameter). Nonetheless, DESI-MS can provide comprehensive lipidomic profiles of individual oocytes or embryos using a fast and simple workflow. DESI-MS lipid profiles include many classes of lipids such as phosphatidylcholines (PC), triacylglycerols (TAG), free fatty acids (FFA), phosphatidylethanolamines (PE), phosphatidylinositols (PI), phosphatidylserines (PS), diacylglycerols (DAG), ubiquinone, cholesterol and cholesterol derivatives (e.g., cholesterol sulfate and cholesterol esters). Depending on the mass spectrometer used, there is the additional possibility of obtaining structural information of lipids via MS

Desorption electrospray ionization (DESI) is a spray-based ambient ionization method for mass spectrometry (MS) that generates ions in native atmospheric conditions (e.g., pressure and temperature). Ambient ionization allows in situ analysis of unmodified samples by eliminating analyte extraction and separation steps before MS. Lipid analysis of individual mammalian oocytes and preimplantation embryos is challenging because of their complex chemical composition and minute dimensions (100-300 μm in diameter). Nonetheless, DESI-MS can provide comprehensive lipidomic profiles of individual oocytes or embryos using a fast and simple workflow. DESI-MS lipid profiles include many classes of lipids such as phosphatidylcholines (PC), triacylglycerols (TAG), free fatty acids (FFA), phosphatidylethanolamines (PE), phosphatidylinositols (PI), phosphatidylserines (PS), diacylglycerols (DAG), ubiquinone, cholesterol and cholesterol derivatives (e.g., cholesterol sulfate and cholesterol esters). Depending on the mass spectrometer used, there is the additional possibility of obtaining structural information of lipids via MSn, and molecular formulae via high resolution MS. Here, we describe the procedures for sample handling, the DESI-MS experimental arrangement, and the data collection and data analysis using low and high-resolution mass spectrometers (such as a linear ion trap and Orbitrap mass analyzer, respectively), as well as strategies for structural characterization of lipids. Lastly, we detail our workflow for relating the chemical information obtained by DESI-MS into the biological context of oocyte and embryo metabolism.

We hypothesized that postnatal development of the vagina is impacted by early nutritional environment. Our objective was to determine if lipid profiles of vaginal swabs were different between gilts suckled by sow or fed milk replacer the first 48 h postpartum, with and without a lard-based fat supplement. Gilts (>1.3 kg) were selected at birth across 8 litters and assigned to treatments: colostrum suckled (S, n=8); S plus fat supplement (SF, n=5); bottle-fed milk replacer (B, n=8); or B plus fat supplement (BF, n=7). At 48 h postnatal, vaginal swabs were taken with a cytology brush, immersed in ultrapure water to burst cells, and lipids extracted for analysis using multiple reaction monitoring (MRM)-profiling. Lipids extracted from serum collected at 48 h from gilts and milk collected from sows at 24 h were also analyzed with MRM-profiling. Receiver operating characteristic curve analysis found 18 lipids highly distinguished [area-under-the-curve (AUC) > 0.9] between S and B gilts, including phosphatidylethanolamine with 34 carbon and four unsaturations in the fatty acyl residues [PE(34:4)]. Twelve lipids from vaginal swabs highly correlated (r> 0.6;p< 0.01) with nutrition source. Lipids more abundant in milk replacer drove association. For example, mean intensity of PE (34:4) was 149-fold higher in milk replacer than colostrum, with 1.6- and 2.12-fold higher levels in serum and vaginal swab samples (p< 0.001), respectively, of B versus S gilts. Findings support that vaginal swabs can be used to noninvasively study effects of perinatal nutrition on tissue composition.Summary sentenceVaginal swab lipidome profiles at 48 h reflect the fat composition of neonatal diet during first two days postnatal.

The worldwide increase in antimicrobial resistance is due to antibiotic overuse in agriculture and overprescription in medicine. For appropriate and timely patient support, faster diagnosis of antimicrobial resistance is required. Current methods for bacterial identification rely on genomics and proteomics and use comparisons with databases of known strains, but the diagnostic value of metabolites and lipids has not been explored significantly. Standard mass spectrometry/chromatography methods involve multiple dilutions during sample preparation and separation. To increase the amount of chemical information acquired and the speed of analysis of lipids, multiple reaction monitoring profiling (MRM-Profiling) has been applied. The MRM-Profiling workflow includes a discovery stage and a screening stage. The discovery stage employs precursor (PREC) ion and neutral loss (NL) scans to screen representative pooled samples for functional groups associated with particular lipid classes. The information from the first stage is organized in precursor/product ion pairs, or MRMs, and the screening stage rapidly interrogates individual samples for these MRMs. In this study, we performed MRM-Profiling of lipid extracts from four different strains of

With the purpose of identifying factors involved in early stages of embryo development in the domestic cat, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) was used for the first time to describe the spatial localization of proteins in the oviducts of queens. Oviducts were obtained from two 2 and 4 years old cross-bred queens, divided into three segments, snap-frozen in liquid nitrogen and then stored at -80°C until use. Next, they were sectioned in a cryostat, fixed on ITO (indium tin oxide) conductive glass slides for MALDI-IMS and serial sections were collected on microscope slides for histology. As confirmed by histology, MALDI-IMS was able to show contrasting protein distributions in the oviductal infundibulum, ampulla and isthmus. Mass spectra were characterized by abundant ions of m/z 1,259, 4,939, 4,960 and 10,626, which have been tentatively attributed to keratin, thymosin β10, thymosin β4 and S100, respectively. Keratin and thymosins are involved in the biological response to tissue damage. S100 proteins are calcium-modulated proteins implicated in a variety of cellular activities, including cell differentiation and regulation of cell motility. These results suggest that protein composition differs between segments of the cat oviduct, which corresponds to morphological changes within these sections. Further functional studies could elucidate the effects of these proteins on feline reproductive physiology.

The differential expression of many productive and reproductive traits has already been associated with the genotype effect observed between Bos taurus indicus (Bos indicus) and Bos taurus taurus (Bos taurus) breeds. Lipids have a crucial role during oocyte development. The aim of this work was to characterize the lipid profiles of oocytes recovered from dairy breeds: Gir (Bos indicus), Holstein (Bos taurus) and their crossbred (Gir x Holstein). Cumulus-oocyte complexes were collected by ovum pick-up from Gir (n=21), Holstein (n=36), and crossbreed (n=33) cows and submitted to MALDI-MS lipid fingerprint analysis. Multi- and univariate analyses were used to characterize lipid profiles of oocytes derived from each breed. Third lipid profiles (Gir n=7; Holstein n=12; and crossbred n=11) were evaluated and allowed characterization of oocyte's lipids among breeds. The lipid profiles from Gir and Holstein were observed to be the most distinct, whereas Gir and crossbred oocytes presented greater similarities. Overall, a wide-range oocyte lipid profile characterization from distinctive bovine dairy breeds recovered by OPU were provided. We have identified two distinguishable lipid profile patterns among groups and revealed that phospholipids and triacylglycerol were the predominantly lipid assigned in Gir (Bos indicus) and Holstein (Bos taurus) oocytes, respectively.