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)

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

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

Learning of basic competencies in dentistry through collaborative multidisciplinary problem-based learning strategies

The former spanish ministry of Science and Education (MEC) established, by royal decree 1393/2007, five basic competencies whose acquisition must be guaranteed for all new graduates finishing higher education studies. Although it is mandatory that these five basic competencies are included in all new curricular plans of spanish universities, no official directives were provided for their development and implementation. We report on a new strategy to improve learning of these five basic competencies in the context of higher education in dentristr