Retinyl Ester Analysis by Orbitrap Mass Spectrometry.

Retinoids are light-sensitive molecules that are normally detected by UV absorption techniques. Here we describe the identification and quantification of retinyl ester species by high-resolution mass spectrometry. Retinyl esters are extracted by the method of Bligh and Dyer and subsequently separated by HPLC in runs of 40 min. The retinyl esters are identified and quantified by mass spectrometry analysis. This procedure enables the highly sensitive detection and characterization of retinyl esters in biological samples such as hepatic stellate cells.

Targeting coenzyme Q10 synthesis overcomes bortezomib resistance in multiple myeloma.

During the development of drug resistance, multiple myeloma (MM) cells undergo changes to their metabolism. However, how these metabolic changes can be exploited to improve treatment efficacy is not known. Here we demonstrate that targeting coenzyme Q10 (CoQ) biosynthesis through the mevalonate pathway works in synergy with the proteasome inhibitor bortezomib (BTZ) in MM. We show that gene expression signatures relating to the mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC) predispose to clinical BTZ resistance and poor prognosis in MM patients. Mechanistically, BTZ-resistant cells show increased activity of glutamine-driven TCA cycle and oxidative phosphorylation, together with an increased vulnerability towards ETC inhibition. Moreover, BTZ resistance is accompanied by high levels of the mitochondrial electron carrier CoQ, while the mevalonate pathway inhibitor simvastatin increases cell death and decreases CoQ levels, specifically in BTZ-resistant cells. Both in vitro and in vivo, simvastatin enhances the effect of bortezomib treatment. Our study links CoQ synthesis to drug resistance in MM and provides a novel avenue for improving BTZ responses through statin-induced inhibition of mitochondrial metabolism.

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa.

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Outer Membrane Vesicles Protect Gram-Negative Bacteria against Host Defense Peptides.

Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of defense against invading pathogens. They possess antimicrobial activity against a broad spectrum of pathogens. However, pathogens have been known to adapt to hostile environments. Therefore, the bacterial response to treatment with HDPs was investigated. Previous observations suggested that sublethal concentrations of HDPs increase the release of outer membrane vesicles (OMVs) in Escherichia coli. First, the effects of sublethal treatment with HDPs CATH-2, PMAP-36, and LL-37 on OMV release of several Gram-negative bacteria were analyzed. Treatment with PMAP-36 and CATH-2 induced release of OMVs, but treatment with LL-37 did not. The OMVs were further characterized with respect to morphological properties. The HDP-induced OMVs often had disc-like shapes. The beneficial effect of bacterial OMV release was studied by determining the susceptibility of E. coli toward HDPs in the presence of OMVs. The minimal bactericidal concentration was increased in the presence of OMVs. It is concluded that OMV release is a means of bacteria to dispose of HDP-affected membrane. Furthermore, OMVs act as a decoy for HDPs and thereby protect the bacterium. IMPORTANCE Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development. Some resistance mechanisms have developed in bacteria, and complete understanding of bacterial defense against HDPs will aid their use in the clinic. This study provides insight into outer membrane vesicles (OMVs) as potential defense mechanisms against HDPs, which will allow anticipation of unforeseen resistance to HDPs in clinical use and possibly prevention of bacterial resistance by the means of OMVs.

A Comprehensive Functional Characterization of Escherichia coli Lipid Genes.

Lipid membranes are the border between living cells and their environments. The membrane's lipid composition defines fluidity, thickness, and protein activity and is controlled by the intricate actions of lipid gene-encoded enzymes. However, a comprehensive analysis of each protein's contribution to the lipidome is lacking. Here, we present such a comprehensive and functional overview of lipid genes in Escherichia coli by individual overexpression or deletion of these genes. We developed a high-throughput lipidomic platform, combining growth analysis, one-step lipid extraction, rapid LC-MS, and bioinformatic analysis into one streamlined procedure. This allowed the processing of more than 300 samples per day and revealed interesting functions of known enzymes and distinct effects of individual proteins on the phospholipidome. Our data demonstrate the plasticity of the phospholipidome and unexpected relations between lipid classes and cell growth. Modeling of lipidomic responses to short-chain alcohols provides a rationale for targeted membrane engineering.

Distinct lipid compositions of two types of human prostasomes.

Prostasomes are vesicles secreted by prostate epithelial cells and found in abundance in seminal plasma. They regulate aspects of sperm cell function and are also thought to prevent immune-mediated destruction of sperm cells within the female reproductive tract. In a previous study, we isolated two distinct populations of prostasomes, differing both in size and protein composition, from the seminal fluid of vasectomized men. In the current study, we characterized the lipid content of these two prostasome populations. Both prostasome types had an unusual lipid composition, with high levels of sphingomyelin (SM), cholesterol, and glycosphingolipids at the expense of, in particular, phosphatidylcholine. The different classes of glycerophospholipids consisted mainly of mono-unsaturated species. The sphingosine-based lipids, SM and the hexosylceramides, were characterized by a near absence of unsaturated species. The two types of prostasome differed in lipid composition, particularly with regard to the relative contributions of SM and hexosylceramides. Potential implications of the lipid compositions of prostasomes for the mechanisms of their formation and function are discussed.

Comparative proteome analysis to explore p53 pathway disruption in head and neck carcinogenesis.

The 5-year-survival rate of head and neck squamous cell carcinoma (HNSCC) has been only moderately improved over the last few decades. HNSCC develops in precursor fields of genetically altered mucosal cells, typically characterized by p53 pathway disruption, that mostly do not give any clinical symptoms. Patients present therefore often with invasive carcinomas in an advanced stage. After tumor resection, part of these fields frequently stays behind unnoticed, causing secondary tumors. Identification of these precursor fields would allow screening and early detection of both primary and secondary tumors. Our aim was to identify differential proteins related to p53 dysfunction. These proteins may serve as valuable biomarkers that can predict the presence of a precursor field. We used a squamous cell model for p53 inactivation, which was analyzed by 2D-DIGE and LC-MS/MS. This approach enabled us to identify a set of 74 proteins that were differentially expressed in cells with normal versus disrupted p53 function. For six proteins the major changes in expression were verified with immunohistochemical staining. The most promising result was the identification of peroxiredoxin-1 which allowed immunohistochemical discrimination between normal epithelium and precursor field tissue with a TP53 mutation.