New insights into protein S-nitrosylation. Mitochondria as a model system

The biological effects of nitric oxide (NO) are in significant part mediated through S-nitrosylation of cysteine thiol. Work on model thiol substrates has raised the idea that molecular oxygen (O(2)) is required for S-nitrosylation by NO; however, the relevance of this mechanism at the low physiological pO(2) of tissues is unclear. Here we have used a proteomic approach to study S-nitrosylation reactions in situ. We identify endogenously S-nitrosylated proteins in subcellular organelles, including dihydrolipoamide dehydrogenase and catalase, and show that these, as well as hydroxymethylglutaryl-CoA synthase and sarcosine dehydrogenase (SarDH), are S-nitrosylated by NO under strictly anaerobic conditions. S-Nitrosylation of SarDH by NO is best rationalized by a novel mechanism involving the covalently bound flavin of the enzyme. We also identify a set of mitochondrial proteins that can be S-nitrosylated through multiple reaction channels, including anaerobic/oxidative, NO/O(2), and GSNO-mediated transnitrosation. Finally, we demonstrate that steady state levels of S-nitrosylation are higher in mitochondrial extracts than the intact organelles, suggesting the importance of denitrosylation reactions. Collectively, our results provide new insight into the determinants of S-nitrosothiol levels in subcellular compartments.     

New insights into developing antibiofouling surfaces for industrial photobioreactors

The biofouling formation of the marine microalga Nannochloropsis gaditana on nontoxic surfaces was quantified on rigid materials, both coated (with fouling release coatings and nanoparticle coatings) and noncoated, to cover a wide range of surface properties from strongly hydrophobic to markedly hydrophilic under conditions similar to those prevailing in outdoor massive cultures of marine microalgae. The effect of seawater on surfaces that presented the best antibiofouling properties was also evaluated. The adhesion intensity on the different surfaces was compared with the predictions of the biocompatibility theories developed by Baier and Vogler using water adhesion tension (τ⁰) as the quantitative parameter of surface wettability. For the most hydrophobic surfaces, τ⁰ ≤ 0, the microalgae adhesion density increased linearly with τ⁰, following the Baier's theory trend. However, for the rest of the surfaces, τ⁰ ≥ 0, a tendency toward minimum adhesion was observed for amphiphilic surfaces with a τ⁰ = 36 mJ/m², a value close to that which minimizes cell adhesion according to Vogler's theory. The understanding and combination of the two biocompatibility theories could help to design universal antibiofouling surfaces that minimize the van der Waals forces and prevent foulant adsorption by using a thin layer of hydration.     

Metavinculin: New insights into functional properties of a muscle adhesion protein

Metavinculin is a muscle-specific splice variant of the ubiquitously expressed cytoskeletal adaptor protein vinculin. Both proteins are thought to be co-expressed in all muscle types where they co-localize to microfilament-associated adhesion sites. It has been shown that a metavinculin-specific insertion of 68 amino acids alters the biochemical properties of the five-helix bundle in the tail domain. Here, we demonstrate that the metavinculin-specific helix H1′ plays an important role for protein stability of the tail domain, since a point mutation in this helix, R975W, which is associated with the occurrence of dilated cardiomyopathy in man, further decreases thermal stability of the metavinculin tail domain. In striated muscle progenitor cells (myoblasts), both, metavinculin and the R975W mutant show significantly reduced, albeit distinctive residency and exchange rates in adhesion sites as compared to vinculin. In contrast to previous studies, we show that metavinculin is localized in a muscle fiber type-dependent fashion to the costameres of striated muscle, reflecting the individual metabolic and physiological status of a given muscle fiber. Metavinculin expression is highest in fast, glycolytic muscle fibers and virtually absent in M. diaphragmaticus, a skeletal muscle entirely lacking fast, glycolytic fibers. In summary, our data suggest that metavinculin enrichment in attachment sites of muscle cells leads to higher mechanical stability of adhesion complexes allowing for greater shear force resistance.     

The citrus fruit proteome: insights into citrus fruit metabolism

Fruit development and ripening are key processes in the production of the phytonutrients that are essential for a balanced diet and for disease prevention. The pathways involved in these processes are unique to plants and vary between species. Climacteric fruit ripening, especially in tomato, has been extensively studied; yet, ripening of non-climacteric fruit is poorly understood. Although the different species share common pathways; developmental programs, physiological, anatomical, biochemical composition and structural differences must contribute to the operation of unique pathways, genes and proteins. Citrus has a non-climacteric fruit ripening behavior and has a unique anatomical fruit structure. For the last few years a citrus genome-wide ESTs project has been initiated and consists of 222,911 clones corresponding to 19,854 contigs and 37,138 singletons. Taking advantage of the citrus database we analyzed the citrus proteome. Using LC-MS/MS we analyzed soluble and enriched membrane fractions of mature citrus fruit to identify the proteome of fruit juice cells. We have identified ca. 1,400 proteins from these fractions by searching NCBI-nr (green plants) and citrus ESTs databases, classified these proteins according to their putative function and assigned function according to known biosynthetic pathways.     

Proteomics in the fruit tree science arena: New insights into fruit defense,development, and ripening

Fruit tree crops are agricultural commodities of high economic importance, while fruits also represent one of the most vital components of the human diet. Therefore, a great effort has been made to understand the molecular mechanisms covering fundamental biological processes in fruit tree physiology and fruit biology. Thanks to the development of cutting‐edge “omics” technologies such as proteomic analysis, scientists now have powerful tools to support traditional fruit tree research. Such proteomic analyses are establishing high‐density 2DE reference maps and peptide mass fingerprint databases that can lead fruit science into a new postgenomic research era. Here, an overview of the application of proteomics in key aspects of fruit tree physiology as well as in fruit biology, including defense responses to abiotic and biotic stress factors, is presented. Α panoramic view of ripening‐related proteins is also discussed, as an example of proteomic application in fruit science.     

New insights into water-phospholipid model membrane interactions

Modulating the relative humidity (RH) of the ambient gas phase of a phospholipid/water sample for modifying the activity of phospholipid-sorbed water [humidity-controlled osmotic stress methods, J. Chem. Phys. 92 (1990) 4519 and J. Phys. Chem. 96 (1992) 446] has opened a new field of research of paramount importance. New types of phase transitions, occurring at specific values of this activity, have been then disclosed. Hence, it is become recognized that this activity, like the temperature T, is an intensive parameter of the thermodynamical state of these samples. This state can be therefore changed (phase transition) either, by modulating T at a given water activity (a given hydration level), or, by modulating the water activity, at a given T. The underlying mechanisms of these two types of transition differ, especially when they appear as disorderings of fatty chains. In lyotropic transitions, this disordering follows from two thermodynamical laws. First, acting on the activity (the chemical potential) of water external to a phospholipid/water sample, a transbilayer gradient of water chemical potential is created, leading to a transbilayer flux of water (Fick's law). Second, water molecules present within the hydrocarbon region of this phospholipid bilayer interact with phospholipid molecules through their chemical potential (Gibbs-Duhem relation): the conformational state of fatty chains (the thermodynamical state of the phospholipid molecules) changes. This process is slow, as revealed by osmotic stress time-resolved experiments. In thermal chain-melting transitions, the first rapid step is the disordering of fatty chains of a fraction of phospholipid molecules. It occurs a few degrees before the main transition temperature, T(m), during the pretransition and the sub-main transition. The second step, less rapid, is the redistribution of water molecules between the different parts of the sample, as revealed by T-jump time-resolved experiments. Finally, in lyotropic and thermal transitions, hydration and conformation are linked but the order of anteriority of their change, in each case, is probably not the same. In this review, first, the interactions of phospholipid submolecular fragments and water molecules, in the interfacial and hydrocarbon regions of phospholipid/water multibilayer stacks, will be described. Second, the coupling of the conformational states of phospholipid and water molecules, during thermal and lyotropic transitions, will be demonstrated through examples.     

The hamster as a model for embryo implantation: insights into a multifaceted process

Defects in preimplantation embryonic development, uterine receptivity, and implantation are the leading cause of infertility, pregnancy problems and birth defects. Significant progress has been made in our basic understanding of these processes using the mouse model, where implantation is ovarian estrogen-dependent in the presence of progesterone. However, an animal model where implantation is progesterone-dependent must also be studied to gain a full understanding of the embryo and uterine events that are required for implantation. In this regard, the hamster is a useful model and this review summarizes the information currently available regarding mechanisms involved in synchronous preimplantation embryo and uterine development for implantation in this species.     

New insights into cancer-related proteins provided by the yeast model

Cancer is a devastating disease with a profound impact on society. In recent years, yeast has provided a valuable contribution with respect to uncovering the molecular mechanisms underlying this disease, allowing the identification of new targets and novel therapeutic opportunities. Indeed, several attributes make yeast an ideal model system for the study of human diseases. It combines a high level of conservation between its cellular processes and those of mammalian cells, with advantages such as a short generation time, ease of genetic manipulation and a wealth of experimental tools for genome- and proteome-wide analyses. Additionally, the heterologous expression of disease-causing proteins in yeast has been successfully used to gain an understanding of the functions of these proteins and also to provide clues about the mechanisms of disease progression. Yeast research performed in recent years has demonstrated the tremendous potential of this model system, especially with the validation of findings obtained with yeast in more physiologically relevant models. The present review covers the major aspects of the most recent developments in the yeast research area with respect to cancer. It summarizes our current knowledge on yeast as a cellular model for investigating the molecular mechanisms of action of the major cancer-related proteins that, even without yeast orthologues, still recapitulate in yeast some of the key aspects of this cellular pathology. Moreover, the most recent contributions of yeast genetics and high-throughput screening technologies that aim to identify some of the potential causes underpinning this disorder, as well as discover new therapeutic agents, are discussed.     

Analysis of citrate accumulation during peach fruit development via a model approach

Based on the citrate model of Lobit and colleagues and measured data, a new model, which is able to reproduce the variation over time of citrate concentration in two peach cultivars, has been proposed. As in the original one, the new model calculates the rate of citrate synthesis or degradation as the product of a 'synthesis potential' and an 'efficiency level'. While in the old model the 'efficiency level' was a simple linear function of temperature and respiration, in the new one its relationship with respiration is accounted for by a coefficient that decreases throughout fruit development. The differences in model parameters between the two cultivars were investigated: late-maturing cv. Suncrest had significantly lower citrate synthesis potential than mid-maturing cv. Fidelia. The responses of citrate concentration to model parameters, temperature, fruit respiration, and growth curves were studied. The most important parameter in the new model, k(4,2), represented the date when the relationship between respiration and 'efficiency level' changed from positive to negative. Raising mean temperature increased the citrate concentration at the beginning and decreased it near maturity for cv. Suncrest, while citrate concentration increased throughout fruit development and more strongly for cv. Fidelia. An increase in the mesocarp dry weight increased both fruit respiration and citrate concentration at the beginning of fruit development, while near maturity it increased fruit respiration but decreased citrate concentration. The model was also able to reproduce the effect of assimilate supply (leaf:fruit ratio). Further potential uses of the model were discussed.     

New insights into the allergenicity of tropomyosin: a bioinformatics approach

The invertebrate panallergen tropomyosin is a protein with an extremely simple folding. This makes it a perfect target for investigating structural differences between invertebrate and vertebrate tropomyosins, which are not considered allergenic. Phylogenetic and sequence analyses were conducted in order to explore the differences in primary structure between several tropomyosins and to promote an experimental development in the field of food allergy, based on the study of tropomyosin. The phylogenetic analyses showed that tropomyosin is a useful evolutionary marker. The phylogenetic trees obtained with tropomyosin were not always phylogenetically correct, but they might be useful for allergen avoidance by tropomyosin allergic individuals. Sequence analyses revealed that the probability of alpha helix folding in invertebrate tropomyosins was lower than in all the studied vertebrate ones, except for the Atlantic bluefin tuna Thunnus thynnus tropomyosin. This suggested that the lack of alpha helix folding may be involved in the immunogenicity of tropomyosins. More specifically, the regions adjacent to the positions 133–135 and 201 of the invertebrate tropomyosins, presented lower probability of alpha helix folding than those of vertebrates and are candidates to be responsible for their allergenicity.     

New insights into metabolic properties of marine bacteria encoding proteorhodopsins

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.     

New insights into 5q- syndrome as a ribosomopathy

Myelodysplastic Syndromes (MDS) are a heterogeneous group of acquired clonal bone marrow disorders, characterised by ineffective haematopoiesis. The mechanisms underlying many of these blood disorders have remained elusive due to the difficulty in pinpointing specific gene mutations or haploinsufficencies, which can occur within large deleted regions. However, there is an increasing interest in the classification of some of these diseases as ribosomopathies. Indeed, studies have implicated Ribosomal Protein (RP) S14 as a strong candidate for haploinsufficiency in 5q- syndrome, a particular form of MDS. Recently, two novel mouse models have provided evidence for the involvement of both RPS14 and the p53 pathway, and specific miRNAs in 5q- syndrome. In this review we will discuss: 5q- syndrome mouse models, the possible mechanisms underlying this blood disorder with respect to the candidate genes, and comparisons with other ribosomopathies, and the involvement of the p53 pathway in these diseases.     

Lipids and atrial fibrillation: New insights into a paradox

In this Perspective, Dimitrios Sagris, Stephanie Harrison, and Gregory Lip discuss new evidence concerning the paradoxical relationship between circulating lipids and atrial fibrillation.

Although the prevalence of cardiovascular comorbidities and associated risk factors such as diabetes mellitus, chronic kidney disease, and obesity increase with age, lipid levels may go through several changes over the course of a lifetime, associated with sex, ethnicity, and metabolic profile [1]. The association between lipid levels and atherosclerotic disease is well established [2], but the association between lipid levels and incidence of atrial fibrillation (AF) has not been fully elucidated.Despite the association between high lipoprotein levels and the increased risk of atherosclerosis and coronary artery disease (CAD), which, in turn, may lead to an increased risk of AF [3], several studies have suggested that high levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) are associated with a lower risk of AF [4]. The clinical significance and pathophysiological mechanisms of this paradoxical inverse association between lipid levels and AF risk remain unclear [4].In an accompanying study in PLOS Medicine, Mozhu Ding and colleagues conducted a large population-based study of >65,000 adults aged 45 to 60 years without any history of cardiovascular disease, using data from the Swedish National Patient Register and Cause of Death Register [5]. Using International Classification of Diseases (ICD) codes from discharge diagnoses of hospital visits and causes of death captured in these registries, participants were followed up for up to 35 years for incident AF. Higher levels of TC and LDL-C were associated with lower risk of AF within the first 5 years (hazard ratios [HRs]: 0.61, 95% confidence intervals [CIs]: 0.41 to –0.99; HR: 0.64, 95% CI: 0.45 to 0.92), but the effect was attenuated after 5 years of follow-up. Conversely, lower levels of HDL-C, high triglyceride (TG) levels and high TG/HDL-C ratio were consistently associated with a higher risk of AF over 3 decades of follow-up (HRs ranging from 1.13 [95% CI: 1.07 to 1.19, p < 0.001] to 1.53 [95% CI: 1.12 to 2.00]).Previous longitudinal studies have demonstrated that levels of the majority of lipoproteins increase significantly between the ages of 20 to 50 years before plateauing in older age; this pattern is observed mainly in men, while in women, increasing lipoproteins are associated with menopause [6]. These early findings were recently confirmed in a cross-sectional study in a Chinese population, in which TC and LDL-C were found to plateau between the ages of 40 and 60 in men and 60 years of age in women, before declining markedly [7]. Considering the inverse association of TC and LDL-C with aging, and the association between aging and higher prevalence of AF, this may partially explain the inverse association of TC and LDL-C with AF.Another interesting finding reported by Mozhu Ding and colleagues is the inverse association of HDL-C levels with AF incidence and the association of high TG levels and high TG/HDL-C ratio with increased risk of AF [8]. This association remained consistent for more than 10 years, suggesting a potentially strong association of HDL-C and TG with incident AF. Since HDL-C and high TG levels are important components of metabolic syndrome, this finding may demonstrate a role of metabolic syndrome and its components in the risk of AF. A recent meta-analysis of 6 cohort studies, including 30,810,460 patients, showed that metabolic syndrome and low HDL-C were associated with a significantly higher risk of AF (HR: 1.57; 95% CI: 1.40 to 1.77, and HR: 1.18; 95% CI: 1.06 to 1.32, respectively) [9]. Based on this evidence, we can speculate that a combination of low HDL-C and high non-HDL-C (i.e., TC excluding HDL-C) may have a potential association with AF risk. Nonetheless, a nationwide cross-sectional survey suggested that non-HDL-C may also be associated with a lower risk of AF [10].The associations observed in the study by Mozhu Ding and colleagues may have been related to lipid treatment therapies, but as the authors highlight, lipid-lowering medicines were uncommonly used in the first few years of the study period and it is unlikely that these could have influenced the associations observed at the beginning of the follow-up period.The association of lipid levels with incident AF remained consistent both in patients with and without heart failure (HF) or CAD. However, in the sensitivity analysis including only patients for whom data on use of medications were available, among those with HF or CAD being treated with lipid-lowering medication, the risk of AF was lower compared to those who were not treated [8]. It seems that although lipid levels are correlated with incident AF irrespective of the presence of HF or CAD, in this high-risk population, the use of lipid-lowering medication reduces the risk of AF, as was previously suggested [11].Mozhu Ding and colleagues have been able to conduct a large-scale study with a long follow-up period, and the findings agree with previous observational evidence. As with all observational studies, residual confounding may be present. In this study, baseline lipid levels were assessed, but variability over time was not examined. A previous nationwide study in Korea has suggested high variability in lipid levels is associated with a higher risk of AF development [12]. Additionally, smoking or physical activity, which represent important cardiovascular parameters, were not accounted for, which may have partially influenced the observed associations.Although the natural progression of vascular aging, chronic inflammation, and dynamic changes in cardiovascular risk factors, including dyslipidemia, may play an essential role in cardiovascular diseases and the risk of AF [13,14], the exact mechanisms of the potential inverse correlation of hyperlipidemia to AF remain elusive. The accompanying study of Mozhu Ding and colleagues supports the existing evidence on the paradoxical inverse correlation of TC, LDL-C, and HDL-C levels with the risk of future AF, providing further insights in the role of TG levels and their correlation to HDL-C levels. New insights may improve understanding of the pathophysiology behind this paradoxical observation [15]. Until then, hyperlipidemia should be assessed as part of the overall cardiovascular risk [16], and the AF paradox should not outweigh this risk.     

New insights into the species problem

Historical opinions of the “species problem” are briefly reviewed, and four salient stages are recognized according to origin of species concepts. We propose that species is the unit preserving superior gene assembly and is maintained by specific mechanisms. Based on characteristics of plant evolution, we assume that understanding plant species may include three stages, i.e. morphological recognition stage, multidisciplinary verification stage, and illuminating mechanisms preserving superior gene assembly.     

Exosomes: New insights into cancer mechanisms

Exosomes are mobile extracellular vesicles with a diameter 40 to 150 nm. They play a critical role in several processes such as the development of cancers, intercellular signaling, drug resistance mechanisms, and cell-to-cell communication by fusion onto the cell membrane of recipient cells. These vesicles contain endogenous proteins and both noncoding and coding RNAs (microRNA and messenger RNAs) that can be delivered to various types of cells. Furthermore, exosomes exist in body fluids such as plasma, cerebrospinal fluid, and urine. Therefore, they could be used as a novel carrier to deliver therapeutic nucleic-acid drugs for cancer therapy. It was recently documented that, hypoxia promotes exosomes secretion in different tumor types leading to the activation of vascular cells and angiogenesis. Cancer cell-derived exosomes (CCEs) have been used as prognostic and diagnostic markers in many types of cancers because exosomes are stable at 4°C and −70°C. CCEs have many functional roles in tumorigenesis, metastasis, and invasion. Consequently, this review presents the data about the therapeutic application of exosomes and the role of CCEs in cancer invasion, drug resistance, and metastasis.     

New insights into the pathogenesis of glucocorticoid-induced avascular necrosis: microarray analysis of gene expression in a rat model

Introduction  

Avascular necrosis of the femoral head (ANFH) occurs variably after exposure to corticosteroids. Microvascular thrombosis is a common pathological finding. Since systemic thrombophilia is only weakly linked with ANFH, we propose that microvascular vessel pathology may be more related to local endothelial dysfunction and femoral head apoptosis. Corticosteroid effects on the endothelium and resultant apoptosis have been reported. We hypothesize that corticosteroids contribute to a differential gene expression in the femoral head in rats with early ANFH.     

New insights into the influence of monofluorination on dimyristoylphosphatidylcholine membrane properties: A solid-state NMR study

Solid-state ¹⁹F NMR spectroscopy is a method of choice to study the interactions between lipid membranes and other molecules such as peptides, proteins or drugs. Numerous fluorine-labeled NMR probes have been developed over the last few years, especially fluorine-labeled peptides. In order to develop a new kind of NMR reporter molecule and a complementary approach to fluorine-labeling of peptides, we synthesized six monofluorinated derivatives of the lipid dimyristoylphosphatidylcholine (F-DMPC), with the fluorine atom located along the acyl chain linked to the central glycerol position. To better understand the behavior of these fluorine-labeled lipids, we report here the investigation of F-DMPC membrane properties using solid-state ²H, ¹⁵N, ¹⁹F‐ and ³¹P‐NMR spectroscopy. This study was carried out on pure F-DMPC bilayers as well as F-DMPC/DMPC mixtures at various ratios. Slight perturbations were observed for pure F-DMPC multilamellar vesicles (MLVs), most noticeable for lipids with the fluorine atom located at the extremities of the acyl chain. On the other hand, no significant perturbations were observed for F-DMPC/DMPC MLVs containing up to 25% F-DMPC, nor for any fluorine-labeled bilayers that were prepared as macroscopically oriented samples. To test the interaction with some representative peptides, ¹⁵N-labeled α-helical antimicrobial peptides (AMPs) were incorporated into F-DMPC/DMPC (1/3) bilayers. ¹⁵N SS-NMR analyses confirmed that the known orientation of each AMP in pure DMPC was preserved in the presence of 25% monofluorinated DMPC, irrespective of the position of the ¹⁹F-label. In summary, F-DMPC/DMPC (1/3) model membranes can be used as NMR reporter to study membrane interactions with other molecules.