Optimization of a MALDI-Imaging protocol for studying adipose tissue-associated disorders.

Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is increasingly recognized for its potential in the discovery of novel biomarkers directly from tissue sections. However, there are no MALDI IMS studies as yet on the adipose tissue, a lipid-enriched tissue that plays a pivotal role in the development of obesity-associated disorders. Herein, we aimed at developing an optimized method for analyzing adipose tissue lipid composition under both physiological and pathological conditions by MALDI IMS. Our studies showed an exacerbated lipid delocalization from adipose tissue sections when conventional strategies were applied. However, our optimized method using conductive-tape sampling and 2,5-dihydroxybenzoic acid (DHB) as a matrix, preserved the anatomical organization and minimized lipid diffusion from sample sections. This method enabled the identification of a total of 625 down-regulated and 328 up-regulated m/z values in the adipose tissue from a rat model of extreme obesity as compared to lean animals. Combination of MALDI IMS and liquid chromatography (LC)-MS/MS data identified 44 differentially expressed lipid species between lean and obese animals, including phospholipids and sphingomyelins. Among the lipids identified, SM(d18:0_18:2), PE(P-16:0_20:0), and PC(O-16:0_16:1) showed a differential spatial distribution in the adipose tissue of lean vs. obese animals. In sum, our method provides a valuable new tool for research on adipose tissue that may pave the way for the identification of novel biomarkers of obesity and metabolic disease.

Influence of sample preparation on lipidomics analysis of polar lipids in adipose tissue.

The main limitations of lipidomics analysis are the chemical complexity of the lipids, the range of concentrations at which they exist, and the variety of samples usually analyzed. These limitations particularly affect the characterization of polar lipids owing to the interference of neutral lipids, essentially acylglycerides, which are at high concentration and suppress ionization of low concentrated lipids in mass spectrometry detection. The influence of sample preparation on lipidomics analysis of polar lipids in adipose tissue by LC-MS/MS was the aim of this research. Two common extractants used for lipids isolation, methanol:chloroform (MeOH:CHCl3) and methyl tert-butyl ether (MTBE), were qualitatively and quantitatively compared for the extraction of the main families of lipids. The obtained results showed that each family of lipids is influenced differently by the extractant used. However, as a general trend, the use of MTBE as extractant led to higher extraction efficiency for unsaturated fatty acids, glycerophospholipids and ceramides, while MeOH:CHCl3 favored the isolation of saturated fatty acids and plasmalogens. The implementation of a solid-phase extraction (SPE) step for selective isolation of glycerophospholipids prior to LC-MS/MS analysis was assayed to evaluate its influence on lipids detection coverage as compared to direct analysis. This step was critical to enhance the detection coverage of glycerophospholipids by removal of ionization suppression effects caused by acylglycerides.

[Survey on the use and behaviour of metal-metal hip replacements in Spain]. / Encuesta sobre el uso y comportamiento de las prótesis de cadera metal-metal en España.

BACKGROUND: Following medical device alerts published in different countries of problems with metal-on-metal total hip replacements, the Spanish Agency of Medicines and Medical Devices (AEMPS) in collaboration with the Spanish Hip Society Surgery designed a national survey to gather information on the use and behaviour of these hip implants. METHODS: The survey consisted of a questionnaire sent by e-mail to 283 clinical centre recipients of metal-on-metal hips to be filled in by surgeons with expertise in the field. RESULTS: A total of 257 questionnaires were completed. The response rate of the clinical centres was 36.7%. A total of 97.7% of the responses reported that clinical and radiological follow-ups are carried out, and 79.6% undertook metal ion analyses (chromium and cobalt). A large majority (83.6%) of the responders who had who used surface implants, and 70% of those with large-head implants reported peri-operative complications. The most common complication was pain (25% with surface implants and 30.8% with large-head implants). Currently 80.8% of those responding were considering abandoning implanting of these hip replacements. CONCLUSIONS: Despite the many limitations to this study, the survey has allowed us to obtain in a quick first view of the implant scenario of Metal on Metal hip implants in Spain, and to determine the type of patient implanted, the time of implantation, and the experience/expertise of the surgeons, and the type of follow-up carried out.

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis.

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit ß4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na(+)/Ca(2+) exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na(+)/K(+) pump subunit ß was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.

Surgical repair of paralytic lagophthalmos by medial tarsal suspension of the lower lid.

BACKGROUND: The seventh nerve palsy causes loss of function in the affected orbicularis oculi muscle. There is exposure and dessication of the corneal surface and obvious aesthetic changes in facial symmetry. METHOD: A surgical procedure has been used in 11 patients to substantially improve palpebral closure. The technique consists of a small upper lid tarsoconjunctival flap sutured into a nasal pretarsal pocket of the lower lid, achieving a medial tarsal suspension of the lower lid. RESULTS: This technique improves cosmesis, is reversible, and combinable with other procedures. The signs and symptoms of exposure keratitis are satisfactorily resolved. There have been no complications in an average follow up of 5 1/2 years after this surgical procedure. CONCLUSION: This technique could be considered an appropriate treatment for patients suffering temporary or permanent facial palsy.