Isolation of Extracellular Vesicles Using Titanium Dioxide Microspheres.

Extracellular vesicles (EVs) are bilayer membrane particles released from several cell types to the extracellular environment. EVs have a crucial role in cell-cell communication, involving different biological processes in health and diseases. Due to the potential of biomarkers for several diseases as diagnostic and therapeutic tools, it is relevant to understand the biology of the EVs and their content. One of the current challenges involving EVs is regarding the purification method, which is a critical step for EV's functional and characterization studies. Ultracentrifugation is the most used method for EV isolation, where the nanoparticles are separated in sequential centrifugation to isolate the EVs based on their size. However, for viscous biofluids such as plasma, there is a co-isolation of the most abundant proteins, which can impair the EV's protein identification due to the low abundance of these proteins and signal suppression by the most abundant plasma proteins. Emerging techniques have gained attention in recent years. Titanium dioxide (TiO2) is one of the most promising techniques due to its property for selective isolation based on the interaction with phospholipids in the EV membrane. Using a small amount of TiO2 beads and a low volume of plasma, it is possible to isolate EVs with reduced plasma protein co-isolation. This study describes a comprehensive workflow for the isolation and characterization of plasma extracellular vesicles (EVs) using mass spectrometry-based proteomics techniques. The aim of this chapter is describe the EV isolation using TiO2 beads enrichment and high-throughput mass spectrometry techniques to efficiently identify the protein composition of EVs in a fast and straightforward manner.

Glycosort: A Computational Solution to Post-process Quantitative Large-Scale Intact Glycopeptide Analyses.

Protein glycosylation is a post-translational modification involving the addition of carbohydrates to proteins and plays a crucial role in protein folding and various biological processes such as cell recognition, differentiation, and immune response. The vast array of natural sugars available allows the generation of plenty of unique glycan structures in proteins, adding complexity to the regulation and biological functions of glycans. The diversity is further increased by enzymatic site preferences and stereochemical conjugation, leading to an immense amount of different glycan structures. Understanding glycosylation heterogeneity is vital for unraveling the impact of glycans on different biological functions. Evaluating site occupancies and structural heterogeneity aids in comprehending glycan-related alterations in biological processes. Several software tools are available for large-scale glycoproteomics studies; however, integrating identification and quantitative data to assess heterogeneity complexity often requires extensive manual data processing. To address this challenge, we present a python script that automates the integration of Byonic and MaxQuant outputs for glycoproteomic data analysis. The script enables the calculation of site occupancy percentages by glycans and facilitates the comparison of glycan structures and site occupancies between two groups. This automated tool offers researchers a means to organize and interpret their high-throughput quantitative glycoproteomic data effectively.

A phylogenetic approach to explore the Aspergillus fumigatus conidial surface-associated proteome and its role in pathogenesis.

Aspergillus fumigatus , an important pulmonary fungal pathogen causing several diseases collectively called aspergillosis, relies on asexual spores or conidia for initiating host infection. Here, we used a phylogenomic approach to compare proteins in the conidial surface of A. fumigatus , two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis , and the cryptic pathogen Aspergillus lentulus . After identifying 62 proteins uniquely expressed on the A. fumigatus conidial surface, we deleted 42 genes encoding conidial proteins. We found deletion of 33 of these genes altered susceptibility to macrophage killing, penetration and damage to epithelial cells, and cytokine production. Notably, a gene that encodes glycosylasparaginase, which modulates levels of the host pro-inflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins and effectors are important for evasion and modulation of the immune response at the onset of fungal infection.

MALDI-TOF mass spectrometry of saliva samples as a prognostic tool for COVID-19.

BACKGROUND: The SARS-CoV-2 infections are still imposing a great public health challenge despite the recent developments in vaccines and therapy. Searching for diagnostic and prognostic methods that are fast, low-cost and accurate are essential for disease control and patient recovery. The MALDI-TOF mass spectrometry technique is rapid, low cost and accurate when compared to other MS methods, thus its use is already reported in the literature for various applications, including microorganism identification, diagnosis and prognosis of diseases. METHODS: Here we developed a prognostic method for COVID-19 using the proteomic profile of saliva samples submitted to MALDI-TOF and machine learning algorithms to train models for COVID-19 severity assessment. RESULTS: We achieved an accuracy of 88.5%, specificity of 85% and sensitivity of 91.5% for classification between mild/moderate and severe conditions. When we tested the model performance in an independent dataset, we achieved an accuracy, sensitivity and specificity of 67.18, 52.17 and 75.60% respectively. CONCLUSION: Saliva is already reported to have high inter-sample variation; however, our results demonstrates that this approach has the potential to be a prognostic method for COVID-19. Additionally, the technology used is already available in several clinics, facilitating the implementation of the method. Further investigation using a larger dataset is necessary to consolidate the technique.

Extracellular Matrix Proteome Remodeling in Human Glioblastoma and Medulloblastoma.

Medulloblastomas (MBs) and glioblastomas (GBMs) are high-incidence central nervous system tumors. Different origin sites and changes in the tissue microenvironment have been associated with the onset and progression. Here, we describe differences between the extracellular matrix (ECM) signatures of these tumors. We compared the proteomic profiles of MB and GBM decellularized tumor samples between each other and their normal decellularized brain site counterparts. Our analysis revealed that 19, 28, and 11 ECM proteins were differentially expressed in MBs, GBMs, and in both MBs and GBMs, respectively. Next, we validated key findings by using a protein tissue array with 53 MB and 55 GBM cases and evaluated the clinical relevance of the identified differentially expressed proteins through their analysis on publicly available datasets, 763 MB samples from the GSE50161 and GSE85217 studies, and 115 GBM samples from RNAseq-TCGA. We report a shift toward a denser fibrillary ECM as well as a clear alteration in the glycoprotein signature, which influences the tumor pathophysiology. MS data have been submitted to the PRIDE repository, project accession: PXD023350.

Prognostic accuracy of MALDI-TOF mass spectrometric analysis of plasma in COVID-19.

SARS-CoV-2 infection poses a global health crisis. In parallel with the ongoing world effort to identify therapeutic solutions, there is a critical need for improvement in the prognosis of COVID-19. Here, we report plasma proteome fingerprinting that predict high (hospitalized) and low-risk (outpatients) cases of COVID-19 identified by a platform that combines machine learning with matrix-assisted laser desorption ionization mass spectrometry analysis. Sample preparation, MS, and data analysis parameters were optimized to achieve an overall accuracy of 92%, sensitivity of 93%, and specificity of 92% in dataset without feature selection. We identified two distinct regions in the MALDI-TOF profile belonging to the same proteoforms. A combination of SDS-PAGE and quantitative bottom-up proteomic analysis allowed the identification of intact and truncated forms of serum amyloid A-1 and A-2 proteins, both already described as biomarkers for viral infections in the acute phase. Unbiased discrimination of high- and low-risk COVID-19 patients using a technology that is currently in clinical use may have a prompt application in the noninvasive prognosis of COVID-19. Further validation will consolidate its clinical utility.

Serum Proteomics Reveals Alterations in Protease Activity, Axon Guidance, and Visual Phototransduction Pathways in Infants With In Utero Exposure to Zika Virus Without Congenital Zika Syndrome.

In 2015, ZIKV infection attracted international attention during an epidemic in the Americas, when neurological disorders were reported in infants who had their mothers exposed to ZIKV during pregnancy. World Health Organization (WHO) epidemiological data show that 5 to 15% of neonates exposed to ZIKV in the uterus have complications included in abnormalities related to Congenital Zika Syndrome (CZS). The risk of complications after birth is not well documented, however, clinical evidence shows that 6% of infants exposed to ZIKV during pregnancy have complications present at birth, and this rate rises to 14% when medical monitoring is performed in all exposed infants, regardless of birth condition. Thus, the evaluation and monitoring of all exposed infants are of foremost importance as the development of late complications has been increasingly supported by clinical evidence. The identification of changes in protein profile of infants exposed to ZIKV without CZS could provide valuable findings to better understand molecular changes in this cohort. Here, we use a shotgun-proteomics approach to investigate alterations in the serum of infants without CZS symptoms but exposed to intrauterine ZIKV (ZIKV) compared to unexposed controls (CTRL). A complex pattern of differentially expressed proteins was identified, highlighting the dysregulation of proteins involved in axon orientation, visual phototransduction, and global protease activity in children exposed to ZIKV without CZS. These data support the importance of monitoring children exposed to ZIKV during gestation and without early CZS symptoms. Our study is the first to assess molecular evidence of possible late disorders in children victims of the ZIKV outbreak in the Americas. We emphasize the importance of medical monitoring of symptomatic and asymptomatic children, as apparently unexplained late neurological and eye disorders may be due to intrauterine ZIKV exposure.

Cellular Imprinting Proteomics Assay: A Novel Method for Detection of Neural and Ocular Disorders Applied to Congenital Zika Virus Syndrome.

Congenital Zika syndrome was first described due to increased incidence of congenital abnormalities associated with Zika virus (ZIKV) infection. Since the eye develops as part of the embryo central nervous system (CNS) structure, it becomes a specialized compartment able to display symptoms of neurodegenerative diseases and has been proposed as a noninvasive approach to the early diagnosis of neurological diseases. Ocular lesions result from defects that occurred during embryogenesis and can become apparent in newborns exposed to ZIKV. Furthermore, the absence of microcephaly cannot exclude the occurrence of ocular lesions and other CNS manifestations. Considering the need for surveillance of newborns and infants with possible congenital exposure, we developed a method termed cellular imprinting proteomic assay (CImPA) to evaluate the ocular surface proteome specific to infants exposed to ZIKV during gestation compared to nonexposure. CImPA combines surface cells and fluid capture using membrane disks and a large-scale quantitative proteomics approach, which allowed the first-time report of molecular alterations such as neutrophil degranulation, cell death signaling, ocular and neurological pathways, which are associated with ZIKV infection with and without the development of congenital Zika syndrome, CZS. Particularly, infants exposed to ZIKV during gestation and without early clinical symptoms could be detected using the CImPA method. Lastly, this methodology has broad applicability as it could be translated in the study of several neurological diseases to identify novel diagnostic biomarkers. Data are available via ProteomeXchange with identifier PXD014038.

Inhibition of histone methyltransferase EZH2 in Schistosoma mansoni in vitro by GSK343 reduces egg laying and decreases the expression of genes implicated in DNA replication and noncoding RNA metabolism.

BACKGROUND: The possibility of emergence of praziquantel-resistant Schistosoma parasites and the lack of other effective drugs demand the discovery of new schistosomicidal agents. In this context the study of compounds that target histone-modifying enzymes is extremely promising. Our aim was to investigate the effect of inhibition of EZH2, a histone methyltransferase that is involved in chromatin remodeling processes and gene expression control; we tested different developmental forms of Schistosoma mansoni using GKS343, a selective inhibitor of EZH2 in human cells. METHODOLOGY/PRINCIPAL FINDINGS: Adult male and female worms and schistosomula were treated with different concentrations of GSK343 for up to two days in vitro. Western blotting showed a decrease in the H3K27me3 histone mark in all three developmental forms. Motility, mortality, pairing and egg laying were employed as schistosomicidal parameters for adult worms. Schistosomula viability was evaluated with propidium iodide staining and ATP quantification. Adult worms showed decreased motility when exposed to GSK343. Also, an approximate 40% reduction of egg laying by GSK343-treated females was observed when compared with controls (0.1% DMSO). Scanning electron microscopy showed the formation of bulges and bubbles throughout the dorsal region of GSK343-treated adult worms. In schistosomula the body was extremely contracted with the presence of numerous folds, and growth was markedly slowed. RNA-seq was applied to identify the metabolic pathways affected by GSK343 sublethal doses. GSK343-treated adult worms showed significantly altered expression of genes related to transmembrane transport, cellular homeostasis and egg development. In females, genes related to DNA replication and noncoding RNA metabolism processes were downregulated. Schistosomula showed altered expression of genes related to cell adhesion and membrane synthesis pathways. CONCLUSIONS/SIGNIFICANCE: The results indicated that GSK343 presents in vitro activities against S. mansoni, and the characterization of EZH2 as a new potential molecular target establishes EZH2 inhibitors as part of a promising new group of compounds that could be used for the development of schistosomicidal agents.

Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum.

BACKGROUND: Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. METHODS: The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate. RESULTS: The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 µM, 7.8 ± 1.3 µM and 2.06 ± 0.4 µM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. CONCLUSIONS: The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.

Intraerythrocytic stages of Plasmodium falciparum biosynthesize vitamin E.

The 2-C-methyl-D-erythritol-4-phosphate and shikimate pathways were found to be active in Plasmodium falciparum and both can result in vitamin E biosynthesis in plants and algae. This study biochemically confirmed vitamin E biosynthesis in the malaria parasite, which can be inhibited by usnic acid. Furthermore, we found evidence pointing to a role of this vitamin in infected erythrocytes. These findings not only contribute to current understanding of P. falciparum biology but also reveal a pathway that could serve as a chemotherapeutic target.

Multilocus analyses of seven candidate genes suggest interacting pathways for obesity-related traits in Brazilian populations.

We investigated whether variants in major candidate genes for food intake and body weight regulation contribute to obesity-related traits under a multilocus perspective. We studied 375 Brazilian subjects from partially isolated African-derived populations (quilombos). Seven variants displaying conflicting results in previous reports and supposedly implicated in the susceptibility of obesity-related phenotypes were investigated: ß2-adrenergic receptor (ADRB2) (Arg16Gly), insulin induced gene 2 (INSIG2) (rs7566605), leptin (LEP) (A19G), LEP receptor (LEPR) (Gln223Arg), perilipin (PLIN) (6209T > C), peroxisome proliferator-activated receptor-γ (PPARG) (Pro12Ala), and resistin (RETN) (-420 C > G). Regression models as well as generalized multifactor dimensionality reduction (GMDR) were employed to test the contribution of individual effects and higher-order interactions to BMI and waist-hip ratio (WHR) variation and risk of overweight/obesity. The best multilocus association signal identified in the quilombos was further examined in an independent sample of 334 Brazilian subjects of European ancestry. In quilombos, only the PPARG polymorphism displayed significant individual effects (WHR variation, P = 0.028). No association was observed either with the risk of overweight/obesity (BMI ≥ 25 kg/m2), risk of obesity alone (BMI ≥ 30 kg/m2) or BMI variation. However, GMDR analyses revealed an interaction between the LEPR and ADRB2 polymorphisms (P = 0.009) as well as a third-order effect involving the latter two variants plus INSIG2 (P = 0.034) with overweight/obesity. Assessment of the LEPR-ADRB2 interaction in the second sample indicated a marginally significant association (P = 0.0724), which was further verified to be limited to men (P = 0.0118). Together, our findings suggest evidence for a two-locus interaction between the LEPR Gln223Arg and ADRB2 Arg16Gly variants in the risk of overweight/obesity, and highlight further the importance of multilocus effects in the genetic component of obesity.

Characterization of proteinases from the midgut of Rhipicephalus (Boophilus) microplus involved in the generation of antimicrobial peptides.

BACKGROUND: Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins). A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. RESULTS: An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus) microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. CONCLUSIONS: BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species.

In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.

Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species

In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.

Distribution of CGG repeats and FRAXAC1/DXS548 alleles in South American populations.

In order to assess the molecular variability related to fragile X (FMR1 locus), we investigated the distribution of CGG repeats and DXS548/FRAXAC1 haplotypes in normal South American populations of different ethnic backgrounds. Special attention was given to Amerindian Wai-Wai (Northern Brazil) and Ache (Paraguay), as well as to Brazilian isolated communities of African ancestry, the remnants of quilombos. Comparison of samples from quilombos, Amerindians, and the ethnically mixed, but mainly European-derived population of São Paulo revealed that the 30-copy allele of the fragile X gene is the most frequent in all groups. A second peak at 20 repeats was present in the population of São Paulo only, confirming this as a European peculiarity. The distribution of DXS548 and FRAXAC1 alleles led to a high expected heterozygosity in African Brazilians, followed by that observed in the population of São Paulo. Amerindians showed the lowest diversity in CGG repeats and DXS548/FRAXAC1 haplotypes. Some rare alleles, for example, the 148-bp (FRAXAC1) or 200-bp (DXS548) variants, which seem to be almost absent in Europe, occurred in higher frequencies among African Brazilians. This suggests a general trend for higher genetic diversity among Africans; these rarer alleles could be African in origin and would have been lost or possibly were not present in the groups that gave rise to the Europeans.