ST8Sia2 polysialyltransferase protects against infection by Trypanosoma cruzi.

Glycosylation is one of the most structurally and functionally diverse co- and post-translational modifications in a cell. Addition and removal of glycans, especially to proteins and lipids, characterize this process which has important implications in several biological processes. In mammals, the repeated enzymatic addition of a sialic acid unit to underlying sialic acids (Sia) by polysialyltransferases, including ST8Sia2, leads to the formation of a sugar polymer called polysialic acid (polySia). The functional relevance of polySia has been extensively demonstrated in the nervous system. However, the role of polysialylation in infection is still poorly explored. Previous reports have shown that Trypanosoma cruzi (T. cruzi), a flagellated parasite that causes Chagas disease (CD), changes host sialylation of glycoproteins. To understand the role of host polySia during T. cruzi infection, we used a combination of in silico and experimental tools. We observed that T. cruzi reduces both the expression of the ST8Sia2 and the polysialylation of target substrates. We also found that chemical and genetic inhibition of host ST8Sia2 increased the parasite load in mammalian cells. We found that modulating host polysialylation may induce oxidative stress, creating a microenvironment that favors T. cruzi survival and infection. These findings suggest a novel approach to interfere with parasite infections through modulation of host polysialylation.

Pseudonormal Morphology of Salivary Gland Adenoid Cystic Carcinoma Cells Subverts the Antitumor Reactivity of Immune Cells: A Tumour-Cell-Based Initiation of Immune Evasion.

INTRODUCTION: Salivary gland adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC) and oral squamous cell carcinoma (OSCC) occurs within the head and neck region. So far immune check point inhibitors failed in ACC. Gipie (CCDC88B) is a microtubule linker protein that activates immune cells. Gipie expressions found in head and neck cancer cells. We hypothesised that the presence of Gipie diminishes anti-tumour reactivity of immune cells towards head and neck cancer. METHOD: To determine the effect of Gipie in oral and salivary gland cancer cells, Gipie was silenced in cancer cells in cancer-immune cells co-culture models and we performed 3D Z series confocal imaging, annexin V and immune activation flow cytometry, proteome profiler and discovery phase proteomics. RESULTS: ACC cells morphed into pseudonormal morphology in immune co-culture models. Silencing Gipie in ACC cells showed significant increase of apoptotic cells and activated natural killer cells, and lowering of regulatory T cells. Other salivary and oral cancer cells showed negligible effect of Gipie. Proteome profiler and proteomics assay confirmed Gipie affecting proliferation mechanism and immune activated proteins in ACC immune co-culture models. CONCLUSION: Overall, we conclude that the presence of Gipie has a confounding role during the ACC-immune cell interaction.

Aspergillus fumigatus conidial surface-associated proteome reveals factors for fungal evasion and host immunity modulation.

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.

Proteomic profiling of the extracellular matrix in the human adrenal cortex.

The extracellular matrix (ECM) comprises macromolecules that shape a complex three-dimensional network. Filling the intercellular space and playing a crucial role in the structure and function of tissues, ECM regulates essential cellular processes such as adhesion, differentiation, and cell signaling. In the human adrenal gland, composed of cortex and medulla surrounded by a capsule, the ECM has not yet been directly described, although its impact on the processes of proliferation and steroidogenesis of the adrenal cortex is recognized. This study analyzes the ECM of the adult human adrenal cortex, which was separated into outer fraction (OF) and inner fraction (IF), by comparing their proteomic profiles. The study discusses the composition, spatial distribution, and relevance of differentially expressed ECM signatures of the adrenal cortex matrisome on adrenal structure and function. The findings were validated through database analysis (cross-validation), histochemical, and immunohistochemical approaches. A total of 121 ECM proteins were identified and categorized into glycoproteins, collagens, ECM regulators, proteoglycans, ECM-affiliated proteins, and secreted factors. Thirty-one ECM proteins were identified only in OF, nine only in IF, and 81 were identified in common with both fractions. Additionally, 106 ECM proteins were reported in the Human matrisome DB 2.0, and the proteins differentially expressed in OF and IF, were identified. This study provides significant insights into the composition and regulation of the ECM in the human adrenal cortex, shedding light on the adrenal microenvironment and its role in the functioning, maintenance, and renewal of the adrenal gland.

Extracellular Matrix Protein Signatures of the Outer and Inner Zones of the Rat Adrenal Cortex.

This study analyzes the extracellular matrix (ECM) signatures of the outer (OF = capsule + subcapsular + zona glomerulosa cells) and inner fractions (IF = zona fasciculata cells) of the rat adrenal cortex, which comprise two distinct microenvironment niches. Proteomic profiles of decellularized OF and IF samples, male and female rats, identified 252 proteins, with 32 classified as ECM-component and ECM-related. Among these, 25 proteins were differentially regulated: 17 more abundant in OF, including Col1a1, Col1a2, Col6a1, Col6a2, Col6a3, Col12a1, Col14a1, Lama5, Lamb2, Lamc1, Eln, Emilin, Fbln5, Fbn1, Fbn2, Nid1, and Ltbp4, and eight more abundant in IF, including Col4a1, Col4a2, Lama2, Lama4, Lamb1, Fn1, Hspg2, and Ecm1. Eln, Tnc, and Nid2 were abundant in the female OF, while Lama2, Lama5, Lamb2, and Lamc1 were more abundant in the male IF. The complex protein signature of the OF suggests areas of tissue stress, stiffness, and regulatory proteins for growth factor signaling. The higher concentrations of Col4a1 and Col4a2 and their role in steroidogenesis should be further investigated in IF. These findings could significantly enhance our understanding of adrenal cortex functionality and its implications for human health and disease. Key findings were validated, and data are available in ProteomeXchange (PXD046828).

Leishmaniinae: Evolutionary inferences based on protein expression profiles (PhyloQuant) congruent with phylogenetic relationships among Leishmania, Endotrypanum, Porcisia, Zelonia, Crithidia, and Leptomonas.

Evolutionary relationships among parasites of the subfamily Leishmaniinae, which comprises pathogen agents of leishmaniasis, were inferred based on differential protein expression profiles from mass spectrometry-based quantitative data using the PhyloQuant method. Evolutionary distances following identification and quantification of protein and peptide abundances using Proteome Discoverer and MaxQuant software were estimated for 11 species from six Leishmaniinae genera. Results clustered all dixenous species of the genus Leishmania, subgenera L. (Leishmania), L. (Viannia), and L. (Mundinia), sister to the dixenous species of genera Endotrypanum and Porcisia. Placed basal to the assemblage formed by all these parasites were the species of genera Zelonia, Crithidia, and Leptomonas, so far described as monoxenous of insects although eventually reported from humans. Inferences based on protein expression profiles were congruent with currently established phylogeny using DNA sequences. Our results reinforce PhyloQuant as a valuable approach to infer evolutionary relationships within Leishmaniinae, which is comprised of very tightly related trypanosomatids that are just beginning to be phylogenetically unraveled. In addition to evolutionary history, mapping of species-specific protein expression is paramount to understand differences in infection processes, tissue tropisms, potential to jump from insects to vertebrates including humans, and targets for species-specific diagnostic and drug development.

Functional characterization of a cystatin A from the bat Myotis davidii.

Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73-79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.

Molecular tools to regulate gene expression in Trypanosoma cruzi.

Developing molecular strategies to manipulate gene expression in trypanosomatids is challenging, particularly with respect to the unique gene expression mechanisms adopted by these unicellular parasites, such as polycistronic mRNA transcription and multi-gene families. In the case of Trypanosoma cruzi (T. cruzi), the causative agent of Chagas Disease, the lack of RNA interference machinery further complicated functional genetic studies important for understanding parasitic biology and developing biomarkers and potential therapeutic targets. Therefore, alternative methods of performing knockout and/or endogenous labelling experiments were developed to identify and understand the function of proteins for survival and interaction with the host. In this review, we present the main tools for the genetic manipulation of T. cruzi, focusing on the Clustered Regularly Interspaced Short Palindromic Repeats Cas9-associated system technique widely used in this organism. Moreover, we highlight the importance of using these tools to elucidate the function of uncharacterized and glycosylated proteins. Further developments of these technologies will allow the identification of new biomarkers, therapeutic targets and potential vaccines against Chagas disease with greater efficiency and speed.

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.

Mass Spectrometry-Based Characterization of Protein Aggregates in Tissues and Biofluids.

Protein aggregation is a common mechanism in multiple neurodegenerative and heart diseases and the accumulation of proteins in aggregates is toxic to cells, causing injury and death. The degree of protein aggregation directly correlates with the severity of the disease. Misfolded proteins present thermodynamic barriers that culminate in the loss of structure and function and the exposure of hydrophobic residues. The exposure of hydrophobic residues is the driving force behind protein aggregation, as it reduces surface free energy and increases the propensity for the formation of large insoluble aggregates. Exploring the protein content of aggregates is fundamental to understanding their formation mechanism and pathophysiological effects. We demonstrate here a method for isolating aggregated protein content in human plasma and mouse brain samples. The samples were characterized by mass spectrometry analysis, transmission electron microscopy, and western blotting. We report the identification of proteins associated with neurodegenerative diseases in the isolated pellets. The western blotting analyses of the isolated pellet showed the positivity for CD89 and CD63, consolidated markers of exosomes, confirming the presence of exosomes within the pellet but not in the supernatant in human plasma. Notably, the concomitant isolation of exosomes together with the protein aggregates was feasible starting from 200 µL of human plasma. Moreover, the presented methodology separated albumin from the aggregated pellet, allowing identification of larger diversity of proteins through mass spectrometry analysis.

The protein map of the protozoan parasite Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis and Leishmania (Leishmania) infantum during growth phase transition and temperature stress.

Leishmania parasites cause a spectrum of diseases termed leishmaniasis, which manifests in two main clinical forms, cutaneous and visceral leishmaniasis. Leishmania promastigotes transit from proliferative exponential to quiescent stationary phases inside the insect vector, a relevant step that recapitulates early molecular events of metacyclogenesis. During the insect blood meal of the mammalian hosts, the released parasites interact initially with the skin, an event marked by temperature changes. Deep knowledge on the molecular events activated during Leishmania-host interactions in each step is crucial to develop better therapies and to understand the pathogenesis. In this study, the proteomes of Leishmania (Leishmania) amazonensis (La), Leishmania (Viannia) braziliensis (Lb), and Leishmania (Leishmania) infantum (syn L. L. chagasi) (Lc) were analyzed using quantitative proteomics to uncover the proteome modulation in three different conditions related to growth phases and temperature shifts: 1) exponential phase (Exp); 2) stationary phase (Sta25) and; 3) stationary phase subjected to heat stress (Sta34). Functional validations were performed using orthogonal techniques, focusing on α-tubulin, gp63 and heat shock proteins (HSPs). Species-specific and condition-specific modulation highlights the plasticity of the Leishmania proteome, showing that pathways related to metabolism and cytoskeleton are significantly modulated from exponential to stationary growth phases, while protein folding, unfolded protein binding, signaling and microtubule-based movement were differentially altered during temperature shifts. This study provides an in-depth proteome analysis of three Leishmania spp., and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts. SIGNIFICANCE: Leishmaniasis disease manifests in two main clinical forms according to the infecting Leishmania species and host immune responses, cutaneous and visceral leishmaniasis. In Brazil, cutaneous leishmaniasis (CL) is associated with L. braziliensis and L. amazonensis, while visceral leishmaniasis, also called kala-azar, is caused by L. infantum. Leishmania parasites remodel their proteomes during growth phase transition and changes in their mileu imposed by the host, including temperature. In this study, we performed a quantitative mass spectrometry-based proteomics to compare the proteome of three New world Leishmania species, L. amazonensis (La), L. braziliensis (Lb) and L. infantum (syn L. chagasi) (Lc) in three conditions: a) exponential phase at 25 °C (Exp); b) stationary phase at 25 °C (Sta25) and; c) stationary phase subjected to temperature stress at 34 °C (Sta34). This study provides an in-depth proteome analysis of three Leishmania spp. with varying pathophysiological outcomes, and contributes compelling evidence of the molecular alterations of these parasites in conditions mimicking the interaction of the parasites with the insect vector and vertebrate hosts.

A computational pipeline elucidating functions of conserved hypothetical Trypanosoma cruzi proteins based on public proteomic data.

The proteome is complex, dynamic, and functionally diverse. Functional proteomics aims to characterize the functions of proteins in biological systems. However, there is a delay in annotating the function of proteins, even in model organisms. This gap is even greater in other organisms, including Trypanosoma cruzi, the causative agent of the parasitic, systemic, and sometimes fatal disease called Chagas disease. About 99.8% of Trypanosoma cruzi proteome is not manually annotated (unreviewed), among which>25% are conserved hypothetical proteins (CHPs), calling attention to the knowledge gap on the protein content of this organism. CHPs are conserved proteins among different species of various evolutionary lineages; however, they lack functional validation. This study describes a bioinformatics pipeline applied to public proteomic data to infer possible biological functions of conserved hypothetical Trypanosoma cruzi proteins. Here, the adopted strategy consisted of collecting differentially expressed proteins between the epimastigote and metacyclic trypomastigotes stages of Trypanosoma cruzi; followed by the functional characterization of these CHPs applying a manifold learning technique for dimension reduction and 3D structure homology analysis (Spalog). We found a panel of 25 and 26 upregulated proteins in the epimastigote and metacyclic trypomastigote stages, respectively; among these, 18 CHPs (8 in the epimastigote stage and 10 in the metacyclic stage) were characterized. The data generated corroborate the literature and complement the functional analyses of differentially regulated proteins at each stage, as they attribute potential functions to CHPs, which are frequently identified in Trypanosoma cruzi proteomics studies. However, it is important to point out that experimental validation is required to deepen our understanding of the CHPs.

Unveiling the multifaceted landscape of N-glycosylation in antibody variable domains: Insights and implications.

N-glycosylation at the antibody variable domain has emerged as an important modification influencing antibody function. Despite its significance, information regarding its role and regulation remains limited. To address this gap, we comprehensively explored antibody structures housing N-glycosylation within the Protein Data Bank, yielding fresh insights into this intricate landscape. Our findings revealed that among 208 structures, N-glycosylation was more prevalent in human and mouse antibodies containing IGHV1-8 and IGHV2-2 germline genes, respectively. Moreover, our research highlights the potential for somatic hypermutation to introduce N-glycosylation sites by substituting polar residues (Ser or Thr) in germline variable genes with asparagine. Notably, our study underscores the prevalence of N-glycosylation in antiviral antibodies, especially anti-HIV. Besides antigen-antibody interaction, our findings suggest that N-glycosylation may impact antibody specificity, affinity, and avidity by influencing Fab dimer formation and complementary-determining region orientation. We also identified different glycan structures in HIV and SARS-CoV-2 antibody proteomic datasets, highlighting disparities from the N-glycan structures between PDB antibodies and biological repertoires further highlighting the complexity of N-glycosylation patterns. Our findings significantly enrich our understanding of the N-glycosylation's multifaceted characteristics within the antibody variable domain. Additionally, they underscore the pressing imperative for a more comprehensive characterization of its impact on antibody function.

1,3-Diphenylureido hydroxamate as a promising scaffold for generation of potent antimalarial histone deacetylase inhibitors.

We report a series of 1,3-diphenylureido hydroxamate HDAC inhibitors evaluated against sensitive and drug-resistant P. falciparum strains. Compounds 8a-d show potent antiplasmodial activity, indicating that a phenyl spacer allows improved potency relative to cinnamyl and di-hydrocinnamyl linkers. In vitro, mechanistic studies demonstrated target activity for PfHDAC1 on a recombinant level, which agreed with cell quantification of the acetylated histone levels. Compounds 6c, 7c, and 8c, identified as the most active in phenotypic assays and PfHDAC1 enzymatic inhibition. Compound 8c stands out as a remarkable inhibitor, displaying an impressive 85% inhibition of PfHDAC1, with an IC50 value of 0.74 µM in the phenotypic screening on Pf3D7 and 0.8 µM against multidrug-resistant PfDd2 parasites. Despite its potent inhibition of PfHDAC1, 8c remains the least active on human HDAC1, displaying remarkable selectivity. In silico studies suggest that the phenyl linker has an ideal length in the series for permitting effective interactions of the hydroxamate with PfHDAC1 and that this compound series could bind as well as in HsHDAC1. Taken together, these results highlight the potential of diphenylurea hydroxamates as a privileged scaffold for the generation of potent antimalarial HDAC inhibitors with improved selectivity over human HDACs.

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 (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 assessed null mutants for 42 genes encoding conidial proteins. 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.

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.

Complement System Activation Is a Plasma Biomarker Signature during Malaria in Pregnancy.

Malaria in pregnancy (MiP) is a public health problem in malaria-endemic areas, contributing to detrimental outcomes for both mother and fetus. Primigravida and second-time mothers are most affected by severe anemia complications and babies with low birth weight compared to multigravida women. Infected erythrocytes (IE) reach the placenta, activating the immune response by placental monocyte infiltration and inflammation. However, specific markers of MiP result in poor outcomes, such as low birth weight, and intrauterine growth restriction for babies and maternal anemia in women infected with Plasmodium falciparum are limited. In this study, we identified the plasma proteome signature of a mouse model infected with Plasmodium berghei ANKA and pregnant women infected with Plasmodium falciparum infection using quantitative mass spectrometry-based proteomics. A total of 279 and 249 proteins were quantified in murine and human plasma samples, of which 28% and 30% were regulated proteins, respectively. Most of the regulated proteins in both organisms are involved in complement system activation during malaria in pregnancy. CBA anaphylatoxin assay confirmed the complement system activation by the increase in C3a and C4a anaphylatoxins in the infected plasma compared to non-infected plasma. Moreover, correlation analysis showed the association between complement system activation and reduced head circumference in newborns from Pf-infected mothers. The data obtained in this study highlight the correlation between the complement system and immune and newborn outcomes resulting from malaria in pregnancy.

Deciphering the Functional Status of Breast Cancers through the Analysis of Their Extracellular Vesicles.

Breast cancer (BC) accounts for the highest incidence of tumor-related mortality among women worldwide, justifying the growing search for molecular tools for the early diagnosis and follow-up of BC patients under treatment. Circulating extracellular vesicles (EVs) are membranous nanocompartments produced by all human cells, including tumor cells. Since minimally invasive methods collect EVs, which represent reservoirs of signals for cell communication, these particles have attracted the interest of many researchers aiming to improve BC screening and treatment. Here, we analyzed the cargoes of BC-derived EVs, both proteins and nucleic acids, which yielded a comprehensive list of potential markers divided into four distinct categories, namely, (i) modulation of aggressiveness and growth; (ii) preparation of the pre-metastatic niche; (iii) epithelial-to-mesenchymal transition; and (iv) drug resistance phenotype, further classified according to their specificity and sensitivity as vesicular BC biomarkers. We discuss the therapeutic potential of and barriers to the clinical implementation of EV-based tests, including the heterogeneity of EVs and the available technologies for analyzing their content, to present a consistent, reproducible, and affordable set of markers for further evaluation.

Leishmania (L.) amazonensis LaLRR17 increases parasite entry in macrophage by a mechanism dependent on GRP78.

Leishmaniases affect 12 million people worldwide. They are caused by Leishmania spp., protozoan parasites transmitted to mammals by female phlebotomine flies. During the life cycle, promastigote forms of the parasite live in the gut of infected sandflies and convert into amastigotes inside the vertebrate macrophages. The parasite evades macrophage's microbicidal responses due to virulence factors that affect parasite phagocytosis, survival and/or proliferation. The interaction between Leishmania and macrophage molecules is essential to phagocytosis and parasite survival. Proteins containing leucine-rich repeats (LRRs) are common in several organisms, and these motifs are usually involved in protein­protein interactions. We have identified the LRR17 gene, which encodes a protein with 6 LRR domains, in the genomes of several Leishmania species. We show here that promastigotes of Leishmania (L.) amazonensis overexpressing LaLRR17 are more infective in vitro. We produced recombinant LaLRR17 protein and identified macrophage 78 kDa glucose-regulated protein (GRP78) as a ligand for LaLRR17 employing affinity chromatography followed by mass spectrometry. We showed that GRP78 binds to LaLRR17 and that its blocking precludes the increase of infection conferred by LaLRR17. Our results are the first to report LRR17 gene and protein, and we hope they stimulate further studies on how this protein increases phagocytosis of Leishmania.