Tri-Reagent Homogenate Is a Suitable Starting Material for UHPLC-MS Lipidomic Analysis

Background: Transcriptomic and lipidomic dual analyses usually initiate with independent extractive procedures. That entails a difficulty in aligning results from both omics platforms, especially in the case of highly heterogeneous tissues, such as the kidney. Methods: Bligh and Dyer lipid extraction was performed using rat kidney homogenates prepared in PBS or commercially available Tri-reagent used for RNA extraction. Samples were analyzed by ultrahigh performance liquid chromatography-mass spectrometry (UHPLC-MS) lipidomic analysis. Results: Comparison of the lipidome obtained from phosphate-buffered saline (PBS) and Tri-reagent homogenates showed qualitative and quantitative validity of the Tri-reagent homogenate with the exception of ether lipids; the acidic nature of the mix seems to promote the hydrolysis of the ether bond, especially in plasmalogens. We tested several conditions in the sample processing, which allowed to optimize the procedure. Conclusions: Aiming to implement a method that allows the extraction of RNA and lipids from the same tissue homogenate not using external tracers, we here report the use of Tri-reagent homogenates as a suitable starting material for UHPLC-MS lipidomic analysis.This research was funded by the Basque Government (grants IT971-16, IT1476-22 and KK-2020/00069) and the Spanish Ministry of Science and Innovation (PID2021-124425OB-100)

Behazun-gatzen garraioaren fisiologia

Heste mehera isurtzen diren behazun-gatzek lipidoen digestioan eta xurgapenean laguntzen dute. Behazun-gatzak hepatozitoek sintetizatzen dituzte kolesterola erabiliz; beren izaera anfipatikoa eta egitura molekularra direla-eta, detergente gisa jokatzen dute, eta beraz toxikoak izan daitezke kontzentrazio handietan. Hori horrela izanda, beren sintesia eta garraioa oso modu finean erregulatu behar dira, bai gibelean bai behazun-gatzen garraioan parte hartzen duten beste ehunetan. Lan honetan gainbegirada bat egin diegu behazun-gatzen metabolismoari eta garraio-prozesuei, azken hauen transkripzio mailako kontrolari buruz argitaratu diren azken lanak ere bilduz

Expression of Adenosine A2B Receptor and Adenosine Deaminase in Rabbit Gastric Mucosa ECL Cells

Adenosine is readily available to the glandular epithelium of the stomach. Formed continuously in intracellular and extracellular locations, it is notably produced from ATP released in enteric cotransmission. Adenosine analogs modulate chloride secretion in gastric glands and activate acid secretion in isolated parietal cells through A2B adenosine receptor (A2BR) binding. A functional link between surface A2BR and adenosine deaminase (ADA) was found in parietal cells, but whether this connection is a general feature of gastric mucosa cells is unknown. Here we examine whether A2BR is expressed at the membrane of histamine-producing enterochromaffin-like (ECL) cells, the major endocrine cell type in the oxyntic mucosa, and if so, whether it has a vicinity relationship with ADA. We used a highly homogeneous population of rabbit ECL cells (size 7.5-10 mu m) after purification by elutriation centrifugation. The surface expression of A2BR and ADA proteins was assessed by flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are partially coexpressed at the gastric ECL cell surface and that A2BR is functional, with regard to binding of adenosine analogs and adenylate cyclase activation. The physiological relevance of A2BR and ADA association in regulating histamine release is yet to be explained.We thank all the members of R. Franco and C. Lluis research group (University of Barcelona, Spain) for assistance with immunostaining experiments and the advice received. We thank E. Castro and M.T. Miras-Portugal (Complutense University of Madrid, Spain) for assistance with microfluorimetry experiments. This work was supported by grants from the Basque Government (IT-971-16) and the University of the Basque Country (UFI11/20)

Proteinen desnaturalizazio termalaren bidezko analisia konposatu bioeraginkor berriak aurkitzeko

Bioactive compounds are either natural or synthetic chemicals that can affect both the cellular and/or physiological activity in living organisms. Included among these are, for example, drugs that can have beneficial effects on health, through the modulation of biological processes. The development of new strategies to search new bioactive compounds and their new biological targets is a high impact challenge. In order to find target proteins and mechanisms of action, several different methodologies have been developed, including the method discussed in this paper. It is based on the analysis of the modifications caused by bioactive compounds on the protein thermal denaturation process. This work describes the basics of this methodology suitable in the search of bioactive compounds as well as the criteria for its implementation.; Konposatu bioeraginkorrak bizidunetan jarduera zelularraren edota fisiologikoaren gain eragin dezaketen substantzia kimiko naturalak zein sintetikoak dira. Horien artean daude, adibidez, farmakoak, prozesu biologikoen modulazioaren bitartez osasunaren gain efektu onuragarriak bideratu ditzaketenak. Konposatu bioeraginkor berrien eta horien itu biologikoak bilatzeko estrategia berrien garapena inpaktu handiko erronka da. Itu proteinak edota ekintza mekanismoak aurkitzeko metodologia ezberdinak garatu dira. Itu proteinak aurkitzeko metodoen artean, lan honetan jorratzen dena daukagu: konposatu bioeraginkorrek proteinen desnaturalizazio termalean eragindako aldaketen azterketan oinarritutako metodoa. Lan honetan, konposatu bioeraginkorrak bilatzeko metodologia horren oinarriak eta haren inplementaziorako irizpideak azaltzen dira

A UHPLC-Mass Spectrometry View of Human Melanocytic Cells Uncovers Potential Lipid Biomarkers of Melanoma

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the “malignancy lipid signature” features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma.This research was funded in part by grants from the Ministry of Economy; Industry and Competitiveness (RTC-2015-3693-1); Ministry of Science and Innovation (RTI-2018-095134-B-I00); Basque Government (IT971-16; IT1162-19; KK2016-036; KK2017-041 and KK2018-00090) and UPV/EHU (GIU17/066)

Atorvastatin Provides a New Lipidome Improving Early Regeneration After Partial Hepatectomy in Osteopontin Deficient Mice

Osteopontin (OPN), a multifunctional cytokine that controls liver glycerolipid metabolism, is involved in activation and proliferation of several liver cell types during regeneration, a condition of high metabolic demands. Here we investigated the role of OPN in modulating the liver lipidome during regeneration after partial-hepatectomy (PH) and the impact that atorvastatin treatment has over regeneration in OPN knockout (KO) mice. The results showed that OPN deficiency leads to remodeling of phosphatidylcholine and triacylglycerol (TG) species primarily during the first 24 h after PH, with minimal effects on regeneration. Changes in the quiescent liver lipidome in OPN-KO mice included TG enrichment with linoleic acid and were associated with higher lysosome TG-hydrolase activity that maintained 24 h after PH but increased in WT mice. OPN-KO mice showed increased beta-oxidation 24 h after PH with less body weight loss. In OPN-KO mice, atorvastatin treatment induced changes in the lipidome 24 h after PH and improved liver regeneration while no effect was observed 48 h post-PH. These results suggest that increased dietary-lipid uptake in OPN-KO mice provides the metabolic precursors required for regeneration 24 h and 48 h after PH. However, atorvastatin treatment offers a new metabolic program that improves early regeneration when OPN is deficient.This work was supported by IT-336-10 (Gobierno Vasco) (to PA) and SAF2015-64352-R (to PA), SAF2017-87301-R (to MLM-C) and EITB Maratoia BIO15/CA/014 (to MLM-C). MNG and DM were recipients of a predoctoral fellowship from the University of Basque Country UPV/EHU and BG-S and DS were recipients for predoctoral fellowships from the Basque Goverment. We thank technical support from Jose Antonio Lopez Gomez

Using the Synergy between HPLC-MS and MALDI-MS Imaging to Explore the Lipidomics of Clear Cell Renal Cell Carcinoma

Lipid imaging mass spectrometry (LIMS) has been tested in several pathological contexts, demonstrating its ability to segregate and isolate lipid signatures in complex tissues, thanks to the technique’s spatial resolution. However, it cannot yet compete with the superior identification power of high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS), and therefore, very often, the latter is used to refine the assignment of the species detected by LIMS. Also, it is not clear if the differences in sensitivity and spatial resolution between the two techniques lead to a similar panel of biomarkers for a given disease. Here, we explore the capabilities of LIMS and HPLC-MS to produce a panel of lipid biomarkers to screen nephrectomy samples from 40 clear cell renal cell carcinoma patients. The same set of samples was explored by both techniques, and despite the important differences between them in terms of the number of detected and identified species (148 by LIMS and 344 by HPLC-MS in negative-ion mode) and the presence/absence of image capabilities, similar conclusions were reached: using the lipid fingerprint, it is possible to set up classifiers that correctly identify the samples as either healthy or tumor samples. The spatial resolution of LIMS enables extraction of additional information, such as the existence of necrotic areas or the existence of different tumor cell populations, but such information does not seem determinant for the correct classification of the samples, or it may be somehow compensated by the higher analytical power of HPLC-MS. Similar conclusions were reached with two very different techniques, validating their use for the discovery of lipid biomarkers.The work was funded by the Basque Government (IT971-16, IT1162-19, and ELKARTEK KK2018-00090) and has been developed as a Ph.D. project of LMS, who is the recipient of a Predoctoral Fellowship from the Spanish Government (BES- 2016-078721) . The authors are grateful to SGiker Lipidomic Service (UPV/EHU, MICINN, GV/EG, ESF) for the expert advice and technical and human support in MALDI and HPLC- MS analysis