A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.

Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.

Multiple Reaction Monitoring Profiling (MRM-Profiling) of Lipids To Distinguish Strain-Level Differences in Microbial Resistance in Escherichia coli.

The worldwide increase in antimicrobial resistance is due to antibiotic overuse in agriculture and overprescription in medicine. For appropriate and timely patient support, faster diagnosis of antimicrobial resistance is required. Current methods for bacterial identification rely on genomics and proteomics and use comparisons with databases of known strains, but the diagnostic value of metabolites and lipids has not been explored significantly. Standard mass spectrometry/chromatography methods involve multiple dilutions during sample preparation and separation. To increase the amount of chemical information acquired and the speed of analysis of lipids, multiple reaction monitoring profiling (MRM-Profiling) has been applied. The MRM-Profiling workflow includes a discovery stage and a screening stage. The discovery stage employs precursor (PREC) ion and neutral loss (NL) scans to screen representative pooled samples for functional groups associated with particular lipid classes. The information from the first stage is organized in precursor/product ion pairs, or MRMs, and the screening stage rapidly interrogates individual samples for these MRMs. In this study, we performed MRM-Profiling of lipid extracts from four different strains of Escherichia coli cultured with amoxicillin or amoxicillin/clavulanate, a ß-lactam and ß-lactamase inhibitor, respectively. t tests, analysis of variance and receiver operating characteristic (ROC) curves were used to determine the significance of each MRM. Principal component analysis was applied to distinguish different strains cultured under conditions that allowed or disallowed development of bacterial resistance. The results demonstrate that MRM-Profiling distinguishes the lipid profiles of resistant and nonresistant E. coli strains.

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans.

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans-cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow-derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin-layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)-12, transforming growth factor-beta (TGF-ß) and IL-10. Similarly, EV-treated DC produced IL-12p40, IL-10 and tumour necrosis factor-alpha. In addition, EV treatment induced the up-regulation of CD86 and major histocompatibility complex class-II (MHC-II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.

Identification of Salmonella Typhimurium Deubiquitinase SseL Substrates by Immunoaffinity Enrichment and Quantitative Proteomic Analysis.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here, we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification, and global quantitative proteomic analysis. As a model system to identify substrates, we used a virulence-related deubiquitinase, SseL, secreted by Salmonella enterica serovar Typhimurium into host cells. Using this approach, two SseL substrates were identified in the RAW 264.7 murine macrophage-like cell line, S100A6 and heterogeneous nuclear ribonuclear protein K, in addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. This method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.

Characterization of Lipids and Proteins Associated to the Cell Wall of the Acapsular Mutant Cryptococcus neoformans Cap 67.

Cryptococcus neoformans is an opportunistic human pathogen that causes life-threatening meningitis. In this fungus, the cell wall is exceptionally not the outermost structure due to the presence of a surrounding polysaccharide capsule, which has been highly studied. Considering that there is little information about C. neoformans cell wall composition, we aimed at describing proteins and lipids extractable from this organelle, using as model the acapsular mutant C. neoformans cap 67. Purified cell wall preparations were extracted with either chloroform/methanol or hot sodium dodecyl sulfate. Total lipids fractionated in silica gel 60 were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS), while trypsin digested proteins were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We detected 25 phospholipid species among phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. Two glycolipid species were identified as monohexosyl ceramides. We identified 192 noncovalently linked proteins belonging to different metabolic processes. Most proteins were classified as secretory, mainly via nonclassical mechanisms, suggesting a role for extracellular vesicles (EV) in transwall transportation. In concert with that, orthologs from 86% of these proteins have previously been reported both in fungal cell wall and/or in EV. The possible role of the presently described structures in fungal-host relationship is discussed.

Multi-omic data integration links deleted in breast cancer 1 (DBC1) degradation to chromatin remodeling in inflammatory response.

This study investigated the dynamics of ubiquitinated proteins after the inflammatory stimulation of RAW 264.7 macrophage-like cells with bacterial lipopolysaccharide. Ubiquitination is a common protein post-translational modification that regulates many key cellular functions. We demonstrated that levels of global ubiquitination and K48 and K63 polyubiquitin chains change after lipopolysaccharide stimulation. Quantitative proteomic analysis identified 1199 ubiquitinated proteins, 78 of which exhibited significant changes in ubiquitination levels following stimulation. Integrating the ubiquitinome data with global proteomic and transcriptomic results allowed us to identify a subset of 88 proteins that were targeted for degradation after lipopolysaccharide stimulation. Using cellular assays and Western blot analyses, we biochemically validated DBC1 (a histone deacetylase inhibitor) as a degradation substrate that is targeted via an orchestrated mechanism utilizing caspases and the proteasome. The degradation of DBC1 releases histone deacetylase activity, linking lipopolysaccharide activation to chromatin remodeling in caspase- and proteasome-mediated signaling.

The gp82 surface molecule of Trypanosoma cruzi metacyclic forms.

Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the parasite forms from the insect vector that initiate infection in the mammalian host. Studies with metacyclic forms generated in vitro, as counterparts of insect-borne parasites, have shown that gp82 plays an essential role in host cell invasion and in the establishment of infection by the oral route. Among the gp82 properties relevant for infection are the gastric mucin-binding capacity and the ability to induce the target cell signaling cascades that result in actin cytoskeleton disruption and lysosome exocytosis, events that facilitate parasite internalization. The gp82 sequences from genetically divergent T. cruzi strains are highly conserved, displaying >90 % identity. Both the host cell-binding sites, as well as the gastric mucin-binding sequence of gp82, are localized in the C-terminal domain of the molecule. In the gp82 structure model, the main cell-binding site consists of an α-helix, which connects the N-terminal ß-propeller domain to the C-terminal ß-sandwich domain, where the second cell binding site is nested. The two cell binding sites are fully exposed on gp82 surface. Downstream and close to the α-helix is the gp82 gastric mucin-binding site, which is partially exposed. All available data support the notion that gp82 is structurally suited for metacyclic trypomastigote invasion of host cells and for initiating infection by the oral route.

KVFinder: steered identification of protein cavities as a PyMOL plugin.

BACKGROUND: The characterization of protein binding sites is a major challenge in computational biology. Proteins interact with a wide variety of molecules and understanding of such complex interactions is essential to gain deeper knowledge of protein function. Shape complementarity is known to be important in determining protein-ligand interactions. Furthermore, these protein structural features have been shown to be useful in assisting medicinal chemists during lead discovery and optimization. RESULTS: We developed KVFinder, a highly versatile and easy-to-use tool for cavity prospection and spatial characterization. KVFinder is a geometry-based method that has an innovative customization of the search space. This feature provides the possibility of cavity segmentation, which alongside with the large set of customizable parameters, allows detailed cavity analyses. Although the main focus of KVFinder is the steered prospection of cavities, we tested it against a benchmark dataset of 198 known drug targets in order to validate our software and compare it with some of the largely accepted methods. Using the one click mode, we performed better than most of the other methods, staying behind only of hybrid prospection methods. When using just one of KVFinder's customizable features, we were able to outperform all other compared methods. KVFinder is also user friendly, as it is available as a PyMOL plugin, or command-line version. CONCLUSION: KVFinder presents novel usability features, granting full customizable and highly detailed cavity prospection on proteins, alongside with a friendly graphical interface. KVFinder is freely available on http://lnbio.cnpem.br/bioinformatics/main/software/.

Structural shifts of aldehyde dehydrogenase enzymes were instrumental for the early evolution of retinoid-dependent axial patterning in metazoans.

Aldehyde dehydrogenases (ALDHs) catabolize toxic aldehydes and process the vitamin A-derived retinaldehyde into retinoic acid (RA), a small diffusible molecule and a pivotal chordate morphogen. In this study, we combine phylogenetic, structural, genomic, and developmental gene expression analyses to examine the evolutionary origins of ALDH substrate preference. Structural modeling reveals that processing of small aldehydes, such as acetaldehyde, by ALDH2, versus large aldehydes, including retinaldehyde, by ALDH1A is associated with small versus large substrate entry channels (SECs), respectively. Moreover, we show that metazoan ALDH1s and ALDH2s are members of a single ALDH1/2 clade and that during evolution, eukaryote ALDH1/2s often switched between large and small SECs after gene duplication, transforming constricted channels into wide opened ones and vice versa. Ancestral sequence reconstructions suggest that during the evolutionary emergence of RA signaling, the ancestral, narrow-channeled metazoan ALDH1/2 gave rise to large ALDH1 channels capable of accommodating bulky aldehydes, such as retinaldehyde, supporting the view that retinoid-dependent signaling arose from ancestral cellular detoxification mechanisms. Our analyses also indicate that, on a more restricted evolutionary scale, ALDH1 duplicates from invertebrate chordates (amphioxus and ascidian tunicates) underwent switches to smaller and narrower SECs. When combined with alterations in gene expression, these switches led to neofunctionalization from ALDH1-like roles in embryonic patterning to systemic, ALDH2-like roles, suggesting functional shifts from signaling to detoxification.

SUMOylation pathway in Trypanosoma cruzi: functional characterization and proteomic analysis of target proteins.

SUMOylation is a relevant protein post-translational modification in eukaryotes. The C terminus of proteolytically activated small ubiquitin-like modifier (SUMO) is covalently linked to a lysine residue of the target protein by an isopeptide bond, through a mechanism that includes an E1-activating enzyme, an E2-conjugating enzyme, and transfer to the target, sometimes with the assistance of a ligase. The modification is reversed by a protease, also responsible for SUMO maturation. A number of proteins have been identified as SUMO targets, participating in the regulation of cell cycle progression, transcription, translation, ubiquitination, and DNA repair. In this study, we report that orthologous genes corresponding to the SUMOylation pathway are present in the etiological agent of Chagas disease, Trypanosoma cruzi. Furthermore, the SUMOylation system is functionally active in this protozoan parasite, having the requirements for SUMO maturation and conjugation. Immunofluorescence analysis showed that T. cruzi SUMO (TcSUMO) is predominantly found in the nucleus. To identify SUMOylation targets and get an insight into their physiological roles we generated transfectant T. cruzi epimastigote lines expressing a double-tagged T. cruzi SUMO, and SUMOylated proteins were enriched by tandem affinity chromatography. By two-dimensional liquid chromatography-mass spectrometry a total of 236 proteins with diverse biological functions were identified as potential T. cruzi SUMO targets. Of these, metacaspase-3 was biochemically validated as a bona fide SUMOylation substrate. Proteomic studies in other organisms have reported that orthologs of putative T. cruzi SUMOylated proteins are similarly modified, indicating conserved functions for protein SUMOylation in this early divergent eukaryote.

Step-by-Step Approach to Build Multiple Reaction Monitoring (MRM) Profiling Instrument Acquisition Methods for Class-based Lipid Exploratory Analysis by Mass Spectrometry.

Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.

Improved proteomic approach for the discovery of potential vaccine targets in Trypanosoma cruzi.

Chagas disease, caused by Trypanosoma cruzi, is a devastating parasitic infection affecting millions of people. Although many efforts have been made for the development of immunotherapies, there is no available vaccine against this deadly infection. One major hurdle for the rational approach to develop a T. cruzi vaccine is the limited information about the proteins produced by different phylogenetic lineages, strains, and stages of the parasite. Here, we have adapted a 1D nanoHPLC system to perform online 2D LC-MS/MS, using the autosampler to inject the eluting salt solutions in the first dimension separation. The application of this methodology for the proteomic analysis of the infective trypomastigote stage of T. cruzi led to the identification of 1448 nonredundant proteins. Furthermore, about 14% of the identified sequences comprise surface proteins, most of them glycosylphosphatidylinositol (GPI)-anchored and related to parasite pathogenesis. Immunoinformatic analysis revealed thousands of potential peptides with predicted high-binding affinity for major histocompatibility complex (MHC) class I and II molecules. The high diversity of proteins expressed on the trypomastigote surface may have many implications for host-cell invasion and immunoevasion mechanisms triggered by the parasite. Finally, we performed a rational approach to filter potential T-cell epitopes that could be further tested and validated for development of a Chagas disease vaccine.

Vesicle and vesicle-free extracellular proteome of Paracoccidioides brasiliensis: comparative analysis with other pathogenic fungi.

Microorganisms release effector molecules that modulate the host machinery enabling survival, replication, and dissemination of a pathogen. Here we characterized the extracellular proteome of Paracoccidioides brasiliensis at its pathogenic yeast phase. Cell-free culture supernatants from the Pb18 isolate, cultivated in defined medium, were separated into vesicle and vesicle-free fractions, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. In vesicle and vesicle-free preparations we identified, respectively, 205 and 260 proteins with two or more peptides, including 120 overlapping identifications. Almost 70% of the sequences were predicted as secretory, mostly using nonconventional secretory pathways, and many have previously been localized to fungal cell walls. A total of 72 proteins were considered as commonly transported by extracellular vesicles, considering that orthologues have been reported in at least two other fungal species. These sequences were mostly related to translation, carbohydrate and protein metabolism, oxidation/reduction, transport, response to stress, and signaling. This unique proteomic analysis of extracellular vesicles and vesicle-free released proteins in a pathogenic fungus provides full comparison with other fungal extracellular vesicle proteomes and broadens the current view on fungal secretomes.

Global analysis of protein palmitoylation in African trypanosomes.

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.

Suspect screening of exogenous compounds using multiple reaction screening (MRM) profiling in human urine samples.

Thousands of chemical compounds produced by industry are dispersed in the human environment widely enough to reach the world population, and the introduction of new chemicals constantly occurs. As new synthetic molecules emerge, rapid analytical workflows for screening possible presence of exogenous compounds in biofluids can be useful as a first pass analysis to detect chemical exposure and guide the development and application of more elaborate LC-MS/MS methods for quantification. In this study, a suspect screening workflow using the multiple reaction monitoring (MRM) profiling method is proposed as a first pass exploratory technique to survey selected exogenous molecules in human urine samples. The workflow was applied to investigate 12 human urine samples using 310 MRMs related to the chemical functionalities of 87 exogenous compounds present in the METLIN database and reported in the literature. A total of 11 MRMs associated with five different compounds were detected in the samples. Product ion scans for the precursor ions of the selected MRMs were acquired as a further identification step for these chemicals. The suspect screening results suggested the presence of five exogenous compounds in the human urine samples analyzed, namely metformin, metoprolol, acetaminophen, paraxanthine and acrylamide. LC-MS/MS was applied as a last step to confirm these results, and the presence of four out of the five targets selected by MRM profiling were corroborated, indicating that this workflow can support the selection of suspect compounds to screen complex samples and guide more time-consuming and specific quantification analyses.

Mutations in the human phospholamban gene in patients with heart failure.

BACKGROUND: Phospholamban (PLN) is a crucial Ca(2+) cycling protein and a primary mediator of the ß-adrenergic effects resulting in enhanced cardiac output. Mutations in the gene encoding PLN have been associated with idiopathic dilated cardiomyopathy; however, no systematic search for PLN mutations in heart failure has been conducted. METHODS: We screened a cohort of 1,014 Brazilian patients with heart failure for mutations in the PLN gene. Molecular modeling studies of the mutations found were developed. Different disease etiologies were present in our sample: idiopathic, ischemic, Chagas, valvular, hypertensive, and others. RESULTS: We identified 4 unrelated patients with PLN mutations (prevalence of 0.4%), 3 of them in the same amino acid residue (R9). Two patients presented a G-T missense mutation at the G26 nucleotide, which encodes an Arg-Leu substitution at codon 9 (R9L). One patient presented a G-A missense mutation at the same nucleotide, which encodes an Arg-His substitution at codon 9 (R9H). The fourth affected patient presented a T-G nonsense mutation at the nucleotide 116, substituting a termination codon for Leu-39 (L39stop). Molecular modeling studies suggested that R9L and R9H mutations might affect the region involved in protein kinase A docking and probably affect the mechanism modulating the release of phosphorylated PLN from the substrate binding site of protein kinase A. CONCLUSIONS: Mutations in the PLN gene are a rare cause of heart failure, present almost exclusively in patients with dilated cardiomyopathy etiology. The Arg9 and Leu39 residues are the leading location of mutations described at this locus to date. Despite the few mutated residues described to date, the clinical spectrum of presentation appears to vary considerably.

Elucidating mechanisms involved in flavor generation of dry-aged beef loins using metabolomics approach.

The present study was conducted to identify flavor-related chemical compounds and to elucidate beef flavor development in response to dry-aging. Paired grass-fed beef loins (n = 18) were obtained at 7 d postmortem, cut into two sections and assigned to 3 aging methods: conventional dry-aging (DA), vacuum packaged wet-aging (WA) and dry-aging in a bag (DW) for 28 days. Following aging, samples were analyzed for UPLC-MS metabolomics, volatile, fatty acid profiling, and consumer sensory comment analysis. Greater number of proteins and nucleotides derived metabolites were liberated in dry-aged samples compared to WA (P < 0.05). In particular, the liberation of gammaglutmayl peptides and glutamine metabolites through the glutathione metabolism were identified. While fatty acid profile was not affected by treatments (P > 0.05), higher concentrations of volatile compounds were found in the dry-aged (P < 0.05). Dry-aging process decreased the presence of terpenoid and steroid lipid group, which could possibly result in reducing undesirable flavor of grass-fed beef.

Shedding Light on the Dynamic Role of the "Target of Rapamycin" Kinase in the Fast-Growing C4 Species Setaria viridis, a Suitable Model for Biomass Crops.

The Target of Rapamycin (TOR) kinase pathway integrates energy and nutrient availability into metabolism promoting growth in eukaryotes. The overall higher efficiency on nutrient use translated into faster growth rates in C4 grass plants led to the investigation of differential transcriptional and metabolic responses to short-term chemical TOR complex (TORC) suppression in the model Setaria viridis. In addition to previously described responses to TORC inhibition (i.e., general growth arrest, translational repression, and primary metabolism reprogramming) in Arabidopsis thaliana (C3), the magnitude of changes was smaller in S. viridis, particularly regarding nutrient use efficiency and C allocation and partitioning that promote biosynthetic growth. Besides photosynthetic differences, S. viridis and A. thaliana present several specificities that classify them into distinct lineages, which also contribute to the observed alterations mediated by TOR. Indeed, cell wall metabolism seems to be distinctly regulated according to each cell wall type, as synthesis of non-pectic polysaccharides were affected in S. viridis, whilst assembly and structure in A. thaliana. Our results indicate that the metabolic network needed to achieve faster growth seems to be less stringently controlled by TORC in S. viridis.

Multiple reaction monitoring profiling as an analytical strategy to investigate lipids in extracellular vesicles.

Extracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 µl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 µg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.

HFE gene mutations in patients with primary iron overload: is there a significant improvement in molecular diagnosis yield with HFE sequencing?

Rare HFE variants have been shown to be associated with hereditary hemochromatosis (HH), an iron overload disease. The low frequency of the HFE p.C282Y mutation in HH-affected Brazilian patients may suggest that other HFE-related mutations may also be implicated in the pathogenesis of HH in this population. The main aim was to screen for new HFE mutations in Brazilian individuals with primary iron overload and to investigate their relationship with HH. Fifty Brazilian patients with primary iron overload (transferrin saturation>50% in females and 60% in males) were selected. Subsequent bidirectional sequencing for each HFE exon was performed. The effect of HFE mutations on protein structure were analyzed by molecular dynamics simulation and free binding energy calculations. p.C282Y in homozygosis or in heterozygosis with p.H63D were the most frequent genotypic combinations associated with HH in our sample population (present in 17 individuals, 34%). Thirty-six (72.0%) out of the 50 individuals presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n=11, 22.0%). One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior indicated that the p.V256I mutation does not reduce the binding affinity between HFE and ß2-microglobulin (ß2M) in the same way the p.C282Y mutation does compared with the native HFE protein. In conclusion, screening of HFE through direct sequencing, as compared to p.C282Y/p.H63D genotyping, was not able to increase the molecular diagnosis yield of HH. The novel p.V256I mutation could not be implicated in the molecular basis of the HH phenotype, although its role cannot be completely excluded in HH-phenotype development. Our molecular modeling analysis can help in the analysis of novel, previously undescribed, HFE mutations.