Corrected and republished from: "Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence".

Small molecules are components of fungal extracellular vesicles (EVs), but their biological roles are only superficially known. NOP16 is a eukaryotic gene that is required for the activity of benzimidazoles against Cryptococcus deuterogattii. In this study, during the phenotypic characterization of C. deuterogattii mutants expected to lack NOP16 expression, we observed a reduced EV production. Whole-genome sequencing, RNA-Seq, and cellular proteomics revealed that, contrary to our initial findings, these mutants expressed Nop16 but exhibited altered expression of 14 genes potentially involved in sugar transport. Based on this observation, we designated these mutant strains as Past1 and Past2, representing potentially altered sugar transport. Analysis of the small molecule composition of EVs produced by wild-type cells and the Past1 and Past2 mutant strains revealed not only a reduced number of EVs but also an altered small molecule composition. In a Galleria mellonella model of infection, the Past1 and Past2 mutant strains were hypovirulent. The hypovirulent phenotype was reverted when EVs produced by wild-type cells, but not mutant EVs, were co-injected with the mutant cells in G. mellonella. These results connect EV biogenesis, cargo, and cryptococcal virulence.

Proteomic Analysis of a Rat Streptozotocin Model Shows Dysregulated Biological Pathways Implicated in Alzheimer's Disease.

Alzheimer's Disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory loss and cognitive impairment, affecting 35 million individuals worldwide. Intracerebroventricular (ICV) injection of low to moderate doses of streptozotocin (STZ) in adult male Wistar rats can reproduce classical physiopathological hallmarks of AD. This biological model is known as ICV-STZ. Most studies are focused on the description of behavioral and morphological aspects of the ICV-STZ model. However, knowledge regarding the molecular aspects of the ICV-STZ model is still incipient. Therefore, this work is a first attempt to provide a wide proteome description of the ICV-STZ model based on mass spectrometry (MS). To achieve that, samples from the pre-frontal cortex (PFC) and hippocampus (HPC) of the ICV-STZ model and control (wild-type) were used. Differential protein abundance, pathway, and network analysis were performed based on the protein identification and quantification of the samples. Our analysis revealed dysregulated biological pathways implicated in the early stages of late-onset Alzheimer's disease (LOAD), based on differentially abundant proteins (DAPs). Some of these DAPs had their mRNA expression further investigated through qRT-PCR. Our results shed light on the AD onset and demonstrate the ICV-STZ as a valid model for LOAD proteome description.

Increasing confidence in proteomic spectral deconvolution through mass defect.

MOTIVATION: Confident deconvolution of proteomic spectra is critical for several applications such as de novo sequencing, cross-linking mass spectrometry and handling chimeric mass spectra. RESULTS: In general, all deconvolution algorithms may eventually report mass peaks that are not compatible with the chemical formula of any peptide. We show how to remove these artifacts by considering their mass defects. We introduce Y.A.D.A. 3.0, a fast deconvolution algorithm that can remove peaks with unacceptable mass defects. Our approach is effective for polypeptides with less than 10 kDa, and its essence can be easily incorporated into any deconvolution algorithm. AVAILABILITY AND IMPLEMENTATION: Y.A.D.A. 3.0 is freely available for academic use at http://patternlabforproteomics.org/yada3. SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.

Characterizing protein conformers by cross-linking mass spectrometry and pattern recognition.

MOTIVATION: Chemical cross-linking coupled to mass spectrometry (XLMS) emerged as a powerful technique for studying protein structures and large-scale protein-protein interactions. Nonetheless, XLMS lacks software tailored toward dealing with multiple conformers; this scenario can lead to high-quality identifications that are mutually exclusive. This limitation hampers the applicability of XLMS in structural experiments of dynamic protein systems, where less abundant conformers of the target protein are expected in the sample. RESULTS: We present QUIN-XL, a software that uses unsupervised clustering to group cross-link identifications by their quantitative profile across multiple samples. QUIN-XL highlights regions of the protein or system presenting changes in its conformation when comparing different biological conditions. We demonstrate our software's usefulness by revisiting the HSP90 protein, comparing three of its different conformers. QUIN-XL's clusters correlate directly to known protein 3D structures of the conformers and therefore validates our software. AVAILABILITYAND IMPLEMENTATION: QUIN-XL and a user tutorial are freely available at http://patternlabforproteomics.org/quinxl for academic users. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Advances in knowledge of Manihot (Euphorbiaceae) from Brazil.

Herbarium-based studies and extensive field work revealed the existence of a new species in the genus Manihot which is restricted to the municipality of Itirapina, in the state of São Paulo, Brazil. Characteristics such as the size and morphology of the leaves and bracts were important to recognize M. irregularis as a new taxon. Description, illustration, as well as comments on its morphology, distribution, conservation status, and a key for the identification of Manihot species for the state of São Paulo are presented.

Synthesis, LC-MS/MS analysis, and biological evaluation of two vaccine candidates against ticks based on the antigenic P0 peptide from R. sanguineus linked to the p64K carrier protein from Neisseria meningitidis.

A peptide from the P0 acidic ribosomal protein (pP0) of ticks conjugated to keyhole limpet hemocyanin from Megathura crenulata has shown to be effective against different tick species when used in host vaccination. Turning this peptide into a commercial anti-tick vaccine will depend on finding the appropriate, technically and economically feasible way to present it to the host immune system. Two conjugates (p64K-Cys1pP0 and p64K-ßAla1pP0) were synthesized using the p64K carrier protein from Neisseria meningitidis produced in Escherichia coli, the same cross-linking reagent, and two analogues of pP0. The SDS-PAGE analysis of p64K-Cys1pP0 showed a heterogeneous conjugate compared to p64K-ßAla1pP0 that was detected as a protein band at 91kDa. The pP0/p64K ratio determined by MALDI-MS for p64K-Cys1pP0 ranged from 1 to 8, being 3-5 the predominant ratio, while in the case of p64K-ßAla1pP0 this ratio was 5-7. Cys1pP0 was partially linked to 35 out of 39 Lys residues and the N-terminal end, while ßAla1pP0 was mostly linked to the six free cysteine residues, to the N-terminal end, and, in a lesser extent, to Lys residues. The assignment of the conjugation sites and side reactions were based on the identification of type 2 peptides. Rabbit immunizations showed the best anti-pP0 titers and the highest efficacy against Rhipicephalus sanguineus ticks when the p64K-Cys1pP0 was used as vaccine antigen. The presence of high molecular mass aggregates observed in the SDS-PAGE analysis of p64K-Cys1pP0 could be responsible for a better immune response against pP0 and consequently for its better efficacy as an anti-tick vaccine. Graphical abstract.

TopoLink: evaluation of structural models using chemical crosslinking distance constraints.

SUMMARY: A software was developed to evaluate structural models using chemical crosslinking experiments. The user provides the types of linkers used and their reactivity, and the observed crosslinks and dead-ends. The software computes the minimum length of a physically inspired linker that connects the reactive atoms of interest, and reports the consistency of each distance with the experimental observation. Statistics on model consistency with the links are provided. Tools to evaluate the correlation of crosslinks in ensembles of models were developed. TopoLink was used to evaluate the potential crosslinks of all structures of the CATH database. The number of crosslinks expected as a function of protein size and linker length can be used as guide for experimental design. AVAILABILITY AND IMPLEMENTATION: TopoLink is available as free software at http://m3g.iqm.unicamp.br/topolink, and distributed as source code with a user-friendly graphical interface for Windows. A web server is also provided. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Top-Down Garbage Collector: a tool for selecting high-quality top-down proteomics mass spectra.

MOTIVATION: We present the first tool for unbiased quality control of top-down proteomics datasets. Our tool can select high-quality top-down proteomics spectra, serve as a gateway for building top-down spectral libraries and, ultimately, improve identification rates. RESULTS: We demonstrate that a twofold rate increase for two E. coli top-down proteomics datasets may be achievable. AVAILABILITY AND IMPLEMENTATION: http://patternlabforproteomics.org/tdgc, freely available for academic use. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Unexpected Thiocyanate Adsorption onto Ferrihydrite Under Prebiotic Chemistry Conditions.

The most crucial role played by minerals was in the preconcentration of biomolecules or precursors of biomolecules in prebiotic seas. If this step had not occurred, molecular evolution would not have occurred. Thiocyanate is an important molecule in the formation of biomolecules as well as a catalyst for prebiotic reactions. The adsorption of thiocyanate onto ferrihydrite was carried out under pH and ion composition conditions in seawater that resembled those of prebiotic Earth. The seawater used in this work had high Mg2+, Ca2+ and SO42- concentrations. The most important result of this work was that ferrihydrite adsorbed thiocyanateata pH value (7.2 ± 0.2) that usually does not adsorb thiocyanate. The high adsorptivity of Mg2+, Ca2+ and SO42-onto ferrihydrite showed that seawater ions can act as carriers of thiocyanate to the ferrihydrite surface, creating a huge outer-sphere complex. Kinetic adsorption and isotherm experiments showed the best fit for the pseudo-second-order model and an activation energy of 23.8 kJ mol-1forthe Langmuir-Freundlich model, respectively. Thermodynamic data showed positive ΔG values, which apparently contradict the adsorption isotherm data and kinetic data that was obtained. The adsorption of thiocyanate onto ferrihydrite could be explained by coupling with the exergonic SO42- adsorption onto ferrihydrite. The FTIR spectra showed no difference between the C≡N stretching peaks of adsorbed thiocyanate and free thiocyanate, corroborating the formation of an outer-sphere complex. All the results demonstrated the importance of the artificial seawater composition for the adsorption of thiocyanate and for understanding prebiotic chemistry.

DiagnoProt: a tool for discovery of new molecules by mass spectrometry.

MOTIVATION: Around 75% of all mass spectra remain unidentified by widely adopted proteomic strategies. We present DiagnoProt, an integrated computational environment that can efficiently cluster millions of spectra and use machine learning to shortlist high-quality unidentified mass spectra that are discriminative of different biological conditions. RESULTS: We exemplify the use of DiagnoProt by shortlisting 4366 high-quality unidentified tandem mass spectra that are discriminative of different types of the Aspergillus fungus. AVAILABILITY AND IMPLEMENTATION: DiagnoProt, a demonstration video and a user tutorial are available at http://patternlabforproteomics.org/diagnoprot . CONTACT: andrerfsilva@gmail.com or paulo@pcarvalho.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Proteomic Analysis of Peripheral Blood Mononuclear Cells after a High-Fat, High-Carbohydrate Meal with Orange Juice.

Oxidative stress and inflammation play a role in the physiopathology of insulin resistance, diabetes and cardiovascular disease. A single high-fat, high-carbohydrate (HFHC) meal induces an increase in inflammatory and oxidative stress markers in peripheral blood mononuclear cells (PBMC). Previous studies have shown that orange juice is able to prevent this response by inhibiting toll like receptors (TLR) expression and endotoxemia. Our goal was to study the proteome response in PBMC after the consumption of a HFHC meal consumed with water, orange juice or an isocaloric beverage (water with glucose). Twelve healthy individuals completed the protocol in a crossover design, and blood samples were obtained before and 1, 3, and 5 h after consumption. Proteomic profile, glucose, insulin, lipid and cytokines levels were investigated. The glycemic and insulinemic response was higher when the meal was consumed with glucose, while there was no difference in the response between water and orange juice. Proteome analysis in PBMC was carried out using TMT ten-plex. A total of 3813 proteins, originating from 15 662 peptides were identified. Three proteins showed significantly altered expression in the three treatments: apolipoprotein A-II, ceruloplasmin and hemopexin. When the HFHC meal was consumed with water there was an increase in some inflammatory pathways such as the Fc-gamma receptor dependent phagocytosis and the complement cascade, but the immune system as a whole was not significantly altered. However, when the meal was consumed with glucose, the immune system was up regulated. Among the pathways induced after 3 h were those of the adaptive immune system and cytokine signaling. Five hours after the meal, pathways of the complement cascade and classical antibody mediated complement activation were up regulated. When the meal was consumed with orange juice there was an up regulation of proteins involved in signal transduction, DNA replication and cell cycle. The promyelocytic leukemia protein (PML) showed a 28.2-fold increase. This protein was down regulated when the meal was consumed with water. Regarding the immune system, several of the pathways induced by glucose were down regulated when the meal was consumed with orange juice: proteins involved with the adaptive immune system and cytokine signaling. Therefore, we have shown that orange juice can not only suppress diet induced inflammation, but also regulate the expression of proteins such as PML, which may play a key role in the regulation of metabolism.

An Evaluation of the Crystal Structure of C-terminal Truncated Apolipoprotein A-I in Solution Reveals Structural Dynamics Related to Lipid Binding.

Apolipoprotein (apo) A-I mediates many of the anti-atherogenic functions attributed to high density lipoprotein. Unfortunately, efforts toward a high resolution structure of full-length apoA-I have not been fruitful, although there have been successes with deletion mutants. Recently, a C-terminal truncation (apoA-I(Δ185-243)) was crystallized as a dimer. The structure showed two helical bundles connected by a long, curved pair of swapped helical domains. To compare this structure to that existing under solution conditions, we applied small angle x-ray scattering and isotope-assisted chemical cross-linking to apoA-I(Δ185-243) in its dimeric and monomeric forms. For the dimer, we found evidence for the shared domains and aspects of the N-terminal bundles, but not the molecular curvature seen in the crystal. We also found that the N-terminal bundles equilibrate between open and closed states. Interestingly, this movement is one of the transitions proposed during lipid binding. The monomer was consistent with a model in which the long shared helix doubles back onto the helical bundle. Combined with the crystal structure, these data offer an important starting point to understand the molecular details of high density lipoprotein biogenesis.

Mechanisms and implications of a type IV functional response for short-term intake rate of dry matter in large mammalian herbivores.

The functional response (i.e. the relationship between consumers' intake rate and resource density) is central in plant-herbivore interactions. Its shape and the biological processes leading to it have significant implications for both foraging theory and ecology of grazing systems. A type IV functional response (i.e. dome-shaped relationship) of short-term intake rate of dry matter (intake while grazing) has rarely been reported for large herbivores and the conditions that can lead to it are poorly understood. We report a type IV functional response observed in heifers grazing monocultures of Cynodon sp. and Avena strigosa. The mechanisms and consequences of this type of functional response for grazed system dynamics are discussed. Intake rate was higher at intermediate than at short or tall sward heights in both grass species. The type IV functional response resulted from changes in bite mass instead of a longer time needed to encounter and process bites. Thus, the decrease of intake rate of dry matter in tall swards is not explained by a shift from process 3 (potential bites are concentrated and apparent) to process 2 (potential bites are apparent but dispersed, Spalinger & Hobbs 1992). Bite mass was smaller in tall than in intermediate swards due to a reduction of bite volume possibly caused by the greater proportion of stem and sheath acting as a physical barrier to bite formation. It is generally accepted that potential bites are abundant and apparent in most grassland and meadow systems, as they were in the present experiments. Therefore, a type IV response of intake rate not directly related to digestive constraints may determine the dynamics of intake and defoliation under a much larger set of conditions than previously thought. These results have implications for foraging theory and stability of grazing systems. For example, if animals prefer patches of intermediate stature that yield the highest intake rate, grazing should lead to the widely observed bimodal distribution of plant mass per unit area, even when tall patches are not of significantly lower digestive quality than the pasture average.

Time-course proteome analysis of developing extrafloral nectaries of Ricinus communis.

Floral and extrafloral nectaries are unique organs that secrete energy rich chemical components, but their contribution for nectar production is largely unknown. Here, we present the first comparative proteome dataset of four developmental stages of the extrafloral nectaries from castor plant (Ricinus communis), an important biofuel crop. Respectively, from stage I-IV, we identified 626, 613, 449 and 356 proteins, respectively, summing up 882 nonredundant proteins. Surprisingly, we identified two isoforms of the potent toxin ricin, indicating that ricin expression is not limited to seeds, but it may serve a general defense purpose for the castor plant. To date, this is the most complete dataset of proteins either from floral or extrafloral nectaries, thus contributing to lay the foundations for investigations on their ecological and evolutionary importance.

PepExplorer: a similarity-driven tool for analyzing de novo sequencing results.

Peptide spectrum matching is the current gold standard for protein identification via mass-spectrometry-based proteomics. Peptide spectrum matching compares experimental mass spectra against theoretical spectra generated from a protein sequence database to perform identification, but protein sequences not present in a database cannot be identified unless their sequences are in part conserved. The alternative approach, de novo sequencing, can make it possible to infer a peptide sequence directly from a mass spectrum, but interpreting long lists of peptide sequences resulting from large-scale experiments is not trivial. With this as motivation, PepExplorer was developed to use rigorous pattern recognition to assemble a list of homologue proteins using de novo sequencing data coupled to sequence alignment to allow biological interpretation of the data. PepExplorer can read the output of various widely adopted de novo sequencing tools and converge to a list of proteins with a global false-discovery rate. To this end, it employs a radial basis function neural network that considers precursor charge states, de novo sequencing scores, peptide lengths, and alignment scores to select similar protein candidates, from a target-decoy database, usually obtained from phylogenetically related species. Alignments are performed using a modified Smith-Waterman algorithm tailored for the task at hand. We verified the effectiveness of our approach using a reference set of identifications generated by ProLuCID when searching for Pyrococcus furiosus mass spectra on the corresponding NCBI RefSeq database. We then modified the sequence database by swapping amino acids until ProLuCID was no longer capable of identifying any proteins. By searching the mass spectra using PepExplorer on the modified database, we were able to recover most of the identifications at a 1% false-discovery rate. Finally, we employed PepExplorer to disclose a comprehensive proteomic assessment of the Bothrops jararaca plasma, a known biological source of natural inhibitors of snake toxins. PepExplorer is integrated into the PatternLab for Proteomics environment, which makes available various tools for downstream data analysis, including resources for quantitative and differential proteomics.

Ryegrass pasture combined with partial total mixed ration reduces enteric methane emissions and maintains the performance of dairy cows during mid to late lactation.

The inclusion of grazed pasture in dairy feeding systems based on a total mixed ration (TMR) reduces feed costs, benefits herd health, and reduces environmental impact. The present study aimed to evaluate the effect of ryegrass pasture combined with a partial TMR on enteric methane emissions, dry matter intake (DMI), and performance of dairy cows from mid to late lactation. The experimental treatments included 100% TMR (control), partial TMR + 6h of continuous grazing (0900-1500 h), and partial TMR + 6h of grazing that was divided into 2 periods of 3h each that took place after milking (0900-1200 h; 1530-1830 h). Twelve F1 cows (Holstein × Jersey; 132±44 DIM) were divided into 6 lots and distributed in a 3×3 Latin square design with 3 periods of 21 d (15 d of adaptation and 6 d of evaluation). Ryegrass (Lolium multiflorum Lam.) pasture was used, and the TMR was composed of 80% corn silage, 18% soybean meal, and 2% mineral and vitamin mixture, based on dry matter. The same mixture was used for cows with access to pasture. The total DMI, milk production, and 4% fat-corrected milk were similar for all cows; however, the pasture DMI (7.4 vs. 6.0kg/d) and grazing period (+ 40 min/d) were higher in cows that had access to pasture for 2 periods of 3h compared with those that grazed for a continuous 6-h period. Methane emission was higher (656 vs. 547g/d) in confined cows than in those that received partial TMR + pasture. The inclusion of annual ryegrass pasture in the diet of dairy cows maintained animal performance and reduced enteric methane emissions. The percentage of grazed forage in the cows' diet increased when access to pasture was provided in 2 periods after the morning and afternoon milking.

Proteomic Analysis of the Endosperm Ontogeny of Jatropha curcas L. Seeds.

Seeds of Jatropha curcas L. represent a potential source of raw material for the production of biodiesel. However, this use is hampered by the lack of basic information on the biosynthetic pathways associated with synthesis of toxic diterpenes, fatty acids, and triacylglycerols, as well as the pattern of deposition of storage proteins during seed development. In this study, we performed an in-depth proteome analysis of the endosperm isolated from five developmental stages which resulted in the identification of 1517, 1256, 1033, 752, and 307 proteins, respectively, summing up 1760 different proteins. Proteins with similar label free quantitation expression pattern were grouped into five clusters. The biological significance of these identifications is discussed with special focus on the analysis of seed storage proteins, proteins involved in the metabolism of fatty acids, carbohydrates, toxic components and proteolytic processing. Although several enzymes belonging to the biosynthesis of diterpenoid precursors were identified, we were unable to find any terpene synthase/cyclase, indicating that the synthesis of phorbol esters, the main toxic diterpenes, does not occur in seeds. The strategy used enabled us to provide a first in depth proteome analysis of the developing endosperm of this biodiesel plant, providing an important glimpse into the enzymatic machinery devoted to the production of C and N sources to sustain seed development.

Deciphering the human brain proteome: characterization of the anterior temporal lobe and corpus callosum as part of the Chromosome 15-centric Human Proteome Project.

Defining the proteomes encoded by each chromosome and characterizing proteins related to human illnesses are among the goals of the Chromosome-centric Human Proteome Project (C-HPP) and the Biology and Disease-driven HPP. Following these objectives, we investigated the proteomes of the human anterior temporal lobe (ATL) and corpus callosum (CC) collected post-mortem from eight subjects. Using a label-free GeLC-MS/MS approach, we identified 2454 proteins in the ATL and 1887 in the CC through roughly 7500 and 5500 peptides, respectively. Considering that the ATL is a gray-matter region while the CC is a white-matter region, they presented proteomes specific to their functions. Besides, 38 proteins were found to be differentially expressed between the two regions. Furthermore, the proteome data sets were classified according to their chromosomal origin, and five proteins were evidenced at the MS level for the first time. We identified 70 proteins of the chromosome 15 - one of them for the first time by MS - which were submitted to an in silico pathway analysis. These revealed branch point proteins associated with Prader-Willi and Angelman syndromes and dyskeratosis congenita, which are chromosome-15-associated diseases. Data presented here can be a useful for brain disorder studies as well as for contributing to the C-HPP initiative. Our data are publicly available as resource data to C-HPP participant groups at http://yoda.iq.ufrj.br/Daniel/chpp2013. Additionally, the mass spectrometry proteomics data have been deposited to the ProteomeXchange with identifier PXD000547 for the corpus callosum and PXD000548 for the anterior temporal lobe.

Exploring the proteomic landscape of a gastric cancer biopsy with the shotgun imaging analyzer.

Accessing localized proteomic profiles has emerged as a fundamental strategy to understand the biology of diseases, as recently demonstrated, for example, in the context of determining cancer resection margins with improved precision. Here, we analyze a gastric cancer biopsy sectioned into 10 parts, each one subjected to MudPIT analysis. We introduce a software tool, named Shotgun Imaging Analyzer and inspired in MALDI imaging, to enable the overlaying of a protein's expression heat map on a tissue picture. The software is tightly integrated with the NeXtProt database, so it enables the browsing of identified proteins according to chromosomes, quickly listing human proteins never identified by mass spectrometry (i.e., the so-called missing proteins), and the automatic search for proteins that are more expressed over a specific region of interest on the biopsy, all of which constitute goals that are clearly well-aligned with those of the C-HPP. Our software has been able to highlight an intense expression of proteins previously known to be correlated with cancers (e.g., glutathione S-transferase Mu 3), and in particular, we draw attention to Gastrokine-2, a "missing protein" identified in this work of which we were able to clearly delineate the tumoral region from the "healthy" with our approach. Data are available via ProteomeXchange with identifier PXD000584.

Effectively addressing complex proteomic search spaces with peptide spectrum matching.

SUMMARY: Protein identification by mass spectrometry is commonly accomplished using a peptide sequence matching search algorithm, whose sensitivity varies inversely with the size of the sequence database and the number of post-translational modifications considered. We present the Spectrum Identification Machine, a peptide sequence matching tool that capitalizes on the high-intensity b1-fragment ion of tandem mass spectra of peptides coupled in solution with phenylisotiocyanate to confidently sequence the first amino acid and ultimately reduce the search space. We demonstrate that in complex search spaces, a gain of some 120% in sensitivity can be achieved. AVAILABILITY: All data generated and the software are freely available for academic use at http://proteomics.fiocruz.br/software/sim. CONTACT: paulo@pcarvalho.com SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.