An Improved Method to Enrich Large Extracellular Vesicles Derived from Giardia intestinalis through Differential Centrifugation.

Giardia intestinalis is a flagellated unicellular protozoan that colonizes the small intestine, causing the diarrheal disease called giardiasis. The production of extracellular vesicles (EVs) by G. intestinalis and the role of these EVs in the parasite's interaction with the host have been described. According to biogenesis, EVs are grouped mainly into large (microvesicles-derived from the plasma membrane) and small (exosomes-derived from multivesicular bodies). Populations of EVs are heterogeneous, and improved methods to separate and study them are needed to understand their roles in cell physiology and pathologies. This work aimed to enrich the large extracellular vesicles (LEVs) of G. intestinalis in order to better understand the roles of these vesicles in the interaction of the parasite with the host. To achieve the enrichment of the LEVs, we have modified our previously described method and compared it by protein dosage and using Nano tracking analysis. Giardia intestinalis vesiculation was induced by incubation in a TYI-S-33 medium without serum, to which 1 mM of CaCl2 was added at 37 °C for 1 h. Then, the supernatant was centrifuged at 15,000× g for 1 h (15 K 1 h pellet), 15,000× g for 4 h (15 K 4 h pellet) and 100,000× g for 1.5 h (100 K 1h30 pellet). The pellet (containing EVs) was resuspended in 1× PBS and stored at 4 °C for later analysis. The EVs were quantified based on their protein concentrations using the Pierce BCA assay, and by nanoparticle tracking analysis (NTA), which reports the concentration and size distribution of the particles. The NTA showed that direct ultracentrifugation at 100,000× g for 1.5 h and centrifugation at 15,000× g for 4 h concentrated more EVs compared to centrifugation at 15,000× g for 1 h. Additionally, it revealed that centrifugation at 15,000× g 4 h was able to concentrate at the same particle concentration levels as a direct ultracentrifugation at 100,000× g for 1.5 h. As for the enrichment of LEVs, the NTA has shown a higher concentration of LEVs in direct ultracentrifugation at 100,000× g for 1.5 h, and in centrifugation at 15,000× g for 4 h, compared to centrifugation at 15,000× g for 1 h. Our results have shown that the most used method at 15,000× g for 1 h is not enough to obtain a representative population of large EVs, and we suggest that LEVs released by G. intestinalis can be better enriched by direct ultracentrifugation at 100,000× g for 1.5 h, or by centrifugation at 15,000× g for 4 h.

Temperature influence on NiFeMo nanoparticles magnetic properties and their viability in biomedical applications.

NiFeMo alloy nanoparticles were synthesized by co-precipitation in the presence of organic additives. Nanoparticles thermal evolution shows that there is a significant increase in the average size (from 28 to 60 nm), consolidating a crystalline structure of the same type as the Ni3 Fe phase but with lattice parameter a = 0.362 nm. Measurements of magnetic properties follow this morphological and structural evolution increasing saturation magnetization (Ms) by 578% and reducing remanence magnetization (Mr) by 29%. Cell viability assays on as-synthesized revealed that nanoparticles (NPs) are not cytotoxic up to a concentration of 0.4 µg/mL for both non-tumorigenic (fibroblasts and macrophages) and tumor cells (melanoma).

Resensitization of Fosfomycin-Resistant Escherichia coli Using the CRISPR System.

Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria.

In vitro anti-Leishmania activity of triclabendazole and its synergic effect with amphotericin B.

Introduction: Leishmaniasis is a neglected tropical disease, with approximately 1 million new cases and 30,000 deaths reported every year worldwide. Given the lack of adequate medication for treating leishmaniasis, drug repositioning is essential to save time and money when searching for new therapeutic approaches. This is particularly important given leishmaniasis's status as a neglected disease. Available treatments are still far from being fully effective for treating the different clinical forms of the disease. They are also administered parenterally, making it challenging to ensure complete treatment, and they are extremely toxic, in some cases, causing death. Triclabendazole (TCBZ) is a benzimidazole used to treat fasciolosis in adults and children. It presents a lower toxicity profile than amphotericin B (AmpB) and is administered orally, making it an attractive candidate for treating other parasitoses. The mechanism of action for TCBZ is not yet well understood, although microtubules or polyamines could potentially act as a pharmacological target. TCBZ has already shown antiproliferative activity against T. cruzi, T. brucei, and L. infantum. However, further investigations are still necessary to elucidate the mechanisms of action of TCBZ. Methods: Cytotoxicity assay was performed by MTT assay. Cell inhibition (CI) values were obtained according to the equation CI = (O.D treatment x 100/O.D. negative control). For Infection evaluation, fixated cells were stained with Hoechst and read at Operetta High Content Imaging System (Perkin Elmer). For growth curves, cell culture absorbance was measured daily at 600 nm. For the synergism effect, Fractional Inhibitory Concentrations (FICs) were calculated for the IC50 of the drugs alone or combined. Mitochondrial membrane potential (DYm), cell cycle, and cell death analysis were evaluated by flow cytometry. Reactive oxygen species (ROS) and lipid quantification were also determined by fluorimetry. Treated parasites morphology and ultrastructure were analyzed by electron microscopy. Results: The selectivity index (SI = CC50/IC50) of TCBZ was comparable with AmpB in promastigotes and amastigotes of Leishmania amazonensis. Evaluation of the cell cycle showed an increase of up to 13% of cells concentrated in S and G2, and morphological analysis with scanning electron microscopy showed a high frequency of dividing cells. The ultrastructural analysis demonstrated large cytoplasmic lipid accumulation, which could suggest alterations in lipid metabolism. Combined administration of TCBZ and AmpB demonstrated a synergistic effect in vitro against intracellular amastigote forms with cSFICs of 0.25. Conclusions: Considering that TCBZ has the advantage of being inexpensive and administrated orally, our results suggest that TCBZ, combined with AmpB, is a promising candidate for treating leishmaniasis with reduced toxicity.

Increased Leishmania infantum resistance to miltefosine and amphotericin B after treatment of a dog with miltefosine and allopurinol.

BACKGROUND: Leishmania infantum is the most important etiological agent of visceral leishmaniasis in the Americas and Mediterranean region, and the dog is the main host. Miltefosine was authorized to treat canine leishmaniasis (CanL) in Brazil in 2017, but there is a persistent fear of the emergence of parasites resistant not only to this drug but, through cross-resistance mechanisms, also to meglumine antimoniate and amphotericin B. Additionally, the literature shows that acquisition of resistance is followed by increased parasite fitness, with higher rates of proliferation, infectivity and metacyclogenesis, which are drivers of parasite virulence. In this context, the aim of this study was to analyze the impact of treating a dog with miltefosine and allopurinol on the generation of parasites resistant to miltefosine, amphotericin B and meglumine antimoniate. METHODS: In vitro susceptibility tests were conducted against miltefosine, amphotericin B and meglumine antimoniate with T0 (parasites isolated from a dog before treatment with miltefosine plus allopurinol), T1 (after 1 course of treatment) and T2 (after 2 courses of treatment) isolates. The rates of cell proliferation, infectivity and metacyclogenesis of the isolates were also evaluated. RESULTS: The results indicate a gradual increase in parasite resistance to miltefosine and amphotericin B with increasing the number of treatment courses. An increasing trend in the metacyclogenesis rate of the parasites was also observed as drug resistance increased. CONCLUSION: The data indicates an increased L. infantum resistance to miltefosine and amphotericin B after the treatment of a dog with miltefosine plus allopurinol. Further studies with a larger number of L. infantum strains isolated from dogs with varied immune response profiles and undergoing different treatment regimes, are advocated.

Biocompatible gum arabic-gold nanorod composite as an effective therapy for mistreated melanomas.

Advanced melanoma patients that are not included in common genetic classificatory groups lack effective and safe therapeutic options. Chemotherapy and immunotherapy show unsatisfactory results and devastating adverse effects for these called triple wild-type patients. New approaches exploring the intrinsic antitumor properties of gold nanoparticles might reverse this scenario as a safer and more effective alternative. Therefore, we investigated the efficacy and safety of a composite made of gum arabic-functionalized gold nanorods (GA-AuNRs) against triple wild-type melanoma. The natural polymer gum arabic successfully stabilized the nanorods in the biological environment and was essential to improve their biocompatibility. In vivo results obtained from treating triple wild-type melanoma-bearing mice showed that GA-AuNRs remarkably reduced primary tumor growth by 45%. Furthermore, GA-AuNRs induced tumor histological features associated with better prognosis while also reducing superficial lung metastasis depth and the incidence of intrapulmonary metastasis. GA-AuNRs' efficacy comes from their capacity to reduce melanoma cells ability to invade the extracellular matrix and grow into colonies, in addition to a likely immunomodulatory effect induced by gum arabic. Additionally, a broad safety investigation found no evidence of adverse effects after GA-AuNRs treatment. Therefore, this study unprecedentedly reports GA-AuNRs as a potential nanomedicine for advanced triple wild-type melanomas.

Pathological findings and morphologic correlation of the lungs of autopsied patients with SARS-CoV-2 infection in the Brazilian Amazon using transmission electron microscopy.

INTRODUCTION: Electron microscopy (EM) is a rapid and effective tool that can be used to create images of a whole spectrum of virus-host interactions and, as such, has long been used in the discovery and description of viral mechanisms. METHODS: Electron microscopy was used to evaluate the pulmonary pathologies of postmortem lung sections from three patients who died from infection with SARS-associated coronavirus 2 (SARS-CoV-2), a new member of the Coronaviridae family. RESULTS: Diffuse alveolar damage (DAD) was predominant in all three patients. The early exudative stage was characterized principally by edema and extravasation of red blood cells into the alveolar space with injury to the alveolar epithelial cells; this was followed by detachment, apoptosis, and necrosis of type I and II pneumocytes. The capillaries exhibited congestion, exposure of the basement membrane from denuded endothelial cells, platelet adhesion, fibrin thrombi, and rupture of the capillary walls. The proliferative stage was characterized by pronounced proliferation of type II alveolar pneumocytes and multinucleated giant cells. The cytopathic effect of SARS-CoV-2 was observed both in degenerated type II pneumocytes and freely circulating in the alveoli, with components from virions, macrophages, lymphocytes, and cellular debris. CONCLUSIONS: Viral particles consistent with the characteristics of SARS-CoV-2 were observed mainly in degenerated pneumocytes, in the endothelium, or freely circulating in the alveoli. In the final stage of illness, the alveolar spaces were replaced by fibrosis.

Synergistic effect and ultrastructural changes in Trypanosoma cruzi caused by isoobtusilactone A in short exposure of time.

Butanolides have shown a variety of biological effects including anti-inflammatory, antibacterial, and antiprotozoal effects against certain strains of Trypanosoma cruzi. Considering the lack of an effective drug to treat T. cruzi infections and the prominent results obtained in literature with this class of lactones, we investigated the anti-T. cruzi activity of five butanolides isolated from two species of Lauraceae, Aiouea trinervis and Mezilaurus crassiramea. Initially, the activity of these compounds was evaluated on epimastigote forms of the parasite, after a treatment period of 4 h, followed by testing on amastigotes, trypomastigotes, and mammalian cells. Next, the synergistic effect of active butanolides against amastigotes was evaluated. Further, metacyclogenesis inhibition and infectivity assays were performed for the most active compound, followed by ultrastructural analysis of the treated amastigotes and trypomastigotes. Among the five butanolides studied, majoranolide and isoobtusilactone A were active against all forms of the parasite, with good selectivity indexes in Vero cells. Both butanolides were more active than the control drug against trypomastigote and epimastigote forms and also had a synergic effect on amastigotes. The most active compound, isoobtusilactone A, which showed activity against all tested strains inhibited metacyclogenesis and infection of new host cells. In addition, ultrastructural analysis revealed that this butanolide caused extensive damage to the mitochondria of both amastigotes and trypomastigotes, resulting in severe morphological changes in the infective forms of the parasite. Altogether, our results highlight the potential of butanolides against the etiologic agent of Chagas disease and the relevance of isoobtusilactone A as a strong anti-T. cruzi drug, affecting different events of the life cycle and all evolutionary forms of parasite after a short period of exposure.

Pirahy virus: Identification of a new and potential emerging arbovirus in South Brazil.

Genomic and epidemiological surveillance are paramount for the discovery of new viruses with the potential to cross species barriers. Here, we present a new member of the genus Alphavirus found in Trichoprosopon and Wyeomia mosquitoes, tentatively named Pirahy virus (PIRAV). PIRAV was isolated from mosquito pools collected in a rural area of Piraí do Sul, South Brazil. In vitro assays revealed that PIRAV replicates and causes cytopathic effects in vertebrate cell lines such as Vero E6, SH-SY5Y, BHK-21 and UMNSAH/DF-1. Genomic signature analysis supports these results showing a dinucleotide and codon usage balance compatible with several hosts. Phylogenetic analyses placed PIRAV basal to the Venezuelan equine encephalitis complex. Genome analyses, electron microscopy, and biological characterization show findings that may alert for the emergence of a new arbovirus in South America.

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi.

The flagellum of Trypanosomatids is an organelle that contributes to multiple functions, including motility, cell division, and host-pathogen interaction. Trypanin was first described in Trypanosoma brucei and is part of the dynein regulatory complex. TbTrypanin knockdown parasites showed motility defects in procyclic forms; however, silencing in bloodstream forms was lethal. Since TbTrypanin mutants show drastic phenotypic changes in mammalian stages, we decided to evaluate if the Trypanosoma cruzi ortholog plays a similar role by using the CRISPR-Cas9 system to generate null mutants. A ribonucleoprotein complex of SaCas9 and sgRNA plus donor oligonucleotide were used to edit both alleles of TcTrypanin without any selectable marker. TcTrypanin -/- epimastigotes showed a lower growth rate, partially detached flagella, normal numbers of nuclei and kinetoplasts, and motility defects such as reduced displacement and speed and increased tumbling propensity. The epimastigote mutant also showed decreased efficiency of in-vitro metacyclogenesis. Mutant parasites were able to complete the entire life cycle in vitro; however, they showed a reduction in their infection capacity compared with WT and addback cultures. Our data show that T. cruzi life cycle stages have differing sensitivities to TcTrypanin deletion. In conclusion, additional work is needed to dissect the motility components of T. cruzi and to identify essential molecules for mammalian stages.

Pathological findings and morphologic correlation of the lungs of autopsied patients with SARS-CoV-2 infection in the Brazilian Amazon using transmission electron microscopy

Abstract INTRODUCTION: Electron microscopy (EM) is a rapid and effective tool that can be used to create images of a whole spectrum of virus-host interactions and, as such, has long been used in the discovery and description of viral mechanisms. METHODS: Electron microscopy was used to evaluate the pulmonary pathologies of postmortem lung sections from three patients who died from infection with SARS-associated coronavirus 2 (SARS-CoV-2), a new member of the Coronaviridae family. RESULTS: Diffuse alveolar damage (DAD) was predominant in all three patients. The early exudative stage was characterized principally by edema and extravasation of red blood cells into the alveolar space with injury to the alveolar epithelial cells; this was followed by detachment, apoptosis, and necrosis of type I and II pneumocytes. The capillaries exhibited congestion, exposure of the basement membrane from denuded endothelial cells, platelet adhesion, fibrin thrombi, and rupture of the capillary walls. The proliferative stage was characterized by pronounced proliferation of type II alveolar pneumocytes and multinucleated giant cells. The cytopathic effect of SARS-CoV-2 was observed both in degenerated type II pneumocytes and freely circulating in the alveoli, with components from virions, macrophages, lymphocytes, and cellular debris. CONCLUSIONS: Viral particles consistent with the characteristics of SARS-CoV-2 were observed mainly in degenerated pneumocytes, in the endothelium, or freely circulating in the alveoli. In the final stage of illness, the alveolar spaces were replaced by fibrosis.

Beyond gold nanoparticles cytotoxicity: Potential to impair metastasis hallmarks.

Gold nanoparticle (AuNP)-based systems have been extensively investigated as diagnostic and therapeutic agents due to their tunable properties and easy surface functionalization. Upon cell uptake, AuNPs present an inherent cell impairment potential based on organelle and macromolecules damage, leading to cell death. Such cytotoxicity is concentration-dependent and completely undesirable, especially if unspecific. However, under non-cytotoxic concentrations, internalized AuNPs could potentially weaken cells and act as antitumor agents. Therefore, this study aimed to investigate the antitumor effect of ultrasmall AuNPs (~3 nm) stabilized by the anionic polysaccharide gum arabic (GA-AuNPs). Other than intrinsic cytotoxicity, the focus was downregulation of cancer hallmarks of aggressive tumors, using a highly metastatic model of melanoma. We first demonstrated that GA-AuNPs showed excellent stability under biological environment. Non-cytotoxic concentrations to seven different cell lines, including tumorigenic and non-tumorigenic cells, were determined by standard 2D in vitro assays. Gold concentrations ≤ 2.4 mg L-1 (16.5 nM AuNPs) were non-cytotoxic and therefore chosen for further analyses. Cells exposed to GA-AuNPs were uptaken by melanoma cells through endocytic processes. Next we described remarkable biological properties using non-cytotoxic concentrations of this nanomaterial. Invasion through an extracellular matrix barrier as well as 3D growth capacity (anchorage-independent colony formation and spheroids growth) were negatively affected by 2.4 mg L-1 GA-AuNPs. Additionally, exposed spheroids showed morphological changes, suggesting that GA-AuNPs could penetrate into the preformed tumor and affect its integrity. All together these results demonstrate that side effects, such as cytotoxicity, can be avoided by choosing the right concentration, nevertheless, preserving desirable effects such as modulation of key tumor cell malignancy features.

Gravitational Test beyond the First Post-Newtonian Order with the Shadow of the M87 Black Hole.

The 2017 Event Horizon Telescope (EHT) observations of the central source in M87 have led to the first measurement of the size of a black-hole shadow. This observation offers a new and clean gravitational test of the black-hole metric in the strong-field regime. We show analytically that spacetimes that deviate from the Kerr metric but satisfy weak-field tests can lead to large deviations in the predicted black-hole shadows that are inconsistent with even the current EHT measurements. We use numerical calculations of regular, parametric, non-Kerr metrics to identify the common characteristic among these different parametrizations that control the predicted shadow size. We show that the shadow-size measurements place significant constraints on deviation parameters that control the second post-Newtonian and higher orders of each metric and are, therefore, inaccessible to weak-field tests. The new constraints are complementary to those imposed by observations of gravitational waves from stellar-mass sources.

Fenbendazole Controls In Vitro Growth, Virulence Potential, and Animal Infection in the Cryptococcus Model.

The human diseases caused by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are associated with high indices of mortality and toxic and/or cost-prohibitive therapeutic protocols. The need for affordable antifungals to combat cryptococcal disease is unquestionable. Previous studies suggested benzimidazoles as promising anticryptococcal agents combining low cost and high antifungal efficacy, but their therapeutic potential has not been demonstrated so far. In this study, we investigated the antifungal potential of fenbendazole, the most effective anticryptococcal benzimidazole. Fenbendazole was inhibitory against 17 different isolates of C. neoformans and C. gattii at a low concentration. The mechanism of anticryptococcal activity of fenbendazole involved microtubule disorganization, as previously described for human parasites. In combination with fenbendazole, the concentrations of the standard antifungal amphotericin B required to control cryptococcal growth were lower than those required when this antifungal was used alone. Fenbendazole was not toxic to mammalian cells. During macrophage infection, the anticryptococcal effects of fenbendazole included inhibition of intracellular proliferation rates and reduced phagocytic escape through vomocytosis. Fenbendazole deeply affected the cryptococcal capsule. In a mouse model of cryptococcosis, the efficacy of fenbendazole to control animal mortality was similar to that observed for amphotericin B. These results indicate that fenbendazole is a promising candidate for the future development of an efficient and affordable therapeutic tool to combat cryptococcosis.

Visible-light reduced silver nanoparticles' toxicity in Allium cepa test system.

Silver nanoparticles (AgNPs) are widely used in consumer products due to their antibacterial property; however, their potential toxicity and release into the environment raises concern. Based on the limited understanding of AgNPs aggregation behavior, this study aimed to investigate the toxicity of uncoated (uc-AgNP) and coated with polyvinylpyrrolidone (PVP-AgNP), at low concentrations (0.5-100 ng/mL), under dark and visible-light exposure, using a plant test system. We exposed Allium cepa seeds to both types of AgNPs for 4-5 days to evaluate several toxicity endpoints. AgNPs did not cause acute toxicity (i.e., inhibition of seed germination and root development), but caused genotoxicity and biochemical alterations in oxidative stress parameters (lipid peroxidation) and activities of antioxidant enzymes (superoxide dismutase and catalase) in light and dark conditions. However, the light exposure decreased the rate of chromosomal aberration and micronuclei up to 5.60x in uc-AgNP and 2.01x in PVP-AgNP, and 2.69x in uc-AgNP and 3.70x in PVP-AgNP, respectively. Thus, light exposure reduced the overall genotoxicity of these AgNPs. In addition, mitotic index alterations and morphoanatomical changes in meristematic cells were observed only in the dark condition at the highest concentrations, demonstrating that light also reduces AgNPs cytotoxicity. The light-dependent aggregation of AgNPs may have reduced toxicity by reducing the uptake of these NPs by the cells. Our findings demonstrate that AgNPs can be genotoxic, cytotoxic and induce morphoanatomical and biochemical changes in A. cepa roots even at low concentrations, and that visible-light alters their aggregation state, and decreases their toxicity. We suggest that visible light can be an alternative treatment to remediate AgNP residues, minimizing their toxicity and environmental risks.

Identification of a novel alphavirus related to the encephalitis complexes circulating in southern Brazil.

In early 2017, an outbreak caused by an unknown and supposedly viral agent in the Marilena region of southern Brazil was investigated. Since the etiological agent causing the outbreak was not identified from human samples, mosquitoes from this region were collected. Three out of 121 mosquito pools collected from the region tested positive for alphavirus in molecular tests. Next generation sequencing results revealed the presence of a novel alphavirus, tentatively named here as Caainguá virus (CAAV). DNA barcoding analyses indicated that different species of Culex are hosts for CAAV. This new virus was basal to the New World encephalitic alphaviruses in a comprehensive and robust phylogenetic approach using complete genomes. Viral particles were observed in the cytosol and inside of intracellular compartments of cells in mosquito-derived cell cultures. Despite being noninfectious in vertebrate derived cell cultures, primary culturing of CAAV in human mononuclear cells suggests monocytes and lymphocytes as CAAV targets. However, the epidemiological link of CAAV on the human outbreak should be further explored.

Serological proteomic screening and evaluation of a recombinant egg antigen for the diagnosis of low-intensity Schistosoma mansoni infections in endemic area in Brazil.

BACKGROUND: Despite decades of use of control programs, schistosomiasis remains a global public health problem. To further reduce prevalence and intensity of infection, or to achieve the goal of elimination in low-endemic areas, there needs to be better diagnostic tools to detect low-intensity infections in low-endemic areas in Brazil. The rationale for development of new diagnostic tools is that the current standard test Kato-Katz (KK) is not sensitive enough to detect low-intensity infections in low-endemic areas. In order to develop new diagnostic tools, we employed a proteomics approach to identify biomarkers associated with schistosome-specific immune responses in hopes of developing sensitive and specific new methods for immunodiagnosis. METHODS AND FINDINGS: Immunoproteomic analyses were performed on egg extracts of Schistosoma mansoni using pooled sera from infected or non-infected individuals from a low-endemic area of Brazil. Cross reactivity with other soil-transmitted helminths (STH) was determined using pooled sera from individuals uniquely infected with different helminths. Using this approach, we identified 23 targets recognized by schistosome acute and chronic sera samples. To identify immunoreactive targets that were likely glycan epitopes, we compared these targets to the immunoreactivity of spots treated with sodium metaperiodate oxidation of egg extract. This treatment yielded 12/23 spots maintaining immunoreactivity, suggesting that they were protein epitopes. From these 12 spots, 11 spots cross-reacted with sera from individuals infected with other STH and 10 spots cross-reacted with the negative control group. Spot number 5 was exclusively immunoreactive with sera from S. mansoni-infected groups in native and deglycosylated conditions and corresponds to Major Egg Antigen (MEA). We expressed MEA as a recombinant protein and showed a similar recognition pattern to that of the native protein via western blot. IgG-ELISA gave a sensitivity of 87.10% and specificity of 89.09% represented by area under the ROC curve of 0.95. IgG-ELISA performed better than the conventional KK (2 slides), identifying 56/64 cases harboring 1-10 eggs per gram of feces that were undiagnosed by KK parasitological technique. CONCLUSIONS: The serological proteome approach was able to identify a new diagnostic candidate. The recombinant egg antigen provided good performance in IgG-ELISA to detect individuals with extreme low-intensity infections (1 egg per gram of feces). Therefore, the IgG-ELISA using this newly identified recombinant MEA can be a useful tool combined with other techniques in low-endemic areas to determine the true prevalence of schistosome infection that is underestimated by the KK method. Further, to overcome the complexity of ELISA in the field, a second generation of antibody-based rapid diagnostic tests (RDT) can be developed.

A Novel Protocol for the Isolation of Fungal Extracellular Vesicles Reveals the Participation of a Putative Scramblase in Polysaccharide Export and Capsule Construction in Cryptococcus gattii.

Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii and Cryptococcus neoformans on solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in solid media. The properties of cryptococcal vesicles varied according to the culture medium used and the EV-producing species. EV detection was reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces cerevisiae Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questions related to cryptococcal secretion. On the basis that polysaccharide export in Cryptococcus requires highly organized membrane traffic culminating with EV release, we analyzed the participation of a putative scramblase (Aim25; CNBG_3981) in EV-mediated GXM export and capsule formation in C. gattii EVs from a C. gattiiaim25Δ strain differed from those obtained from wild-type (WT) cells in physical-chemical properties and cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, EVs obtained from the aim25Δ mutant were more efficiently used as a source of capsular polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and increased capsular dimensions. These results associate the description of a novel protocol for the isolation of fungal EVs with the identification of a previously unknown regulator of polysaccharide release.IMPORTANCE Extracellular vesicles (EVs) are fundamental components of the physiology of cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of transfer of antifungal resistance and virulence, as well as in immune stimulation and prion transmission. However, studies on the functions of fungal EVs are still limited by the lack of efficient methods for isolation of these compartments. In this study, we developed an alternative protocol for isolation of fungal EVs and demonstrated an application of this new methodology in the study of the physiology of the fungal pathogen Cryptococcus gattii Our results describe a fast and reliable method for the study of fungal EVs and reveal the participation of scramblase, a phospholipid-translocating enzyme, in secretory processes of C. gattii.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins.

Trypanosomatids (order Kinetoplastida), including the human pathogens Trypanosoma cruzi (agent of Chagas disease), Trypanosoma brucei, (African sleeping sickness), and Leishmania (leishmaniasis), affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9), but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9), and in vitro-transcribed single guide RNAs (sgRNAs) results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.IMPORTANCE Protozoan parasites remain some of the highest-impact human and animal pathogens, with very limited treatment and prevention options. The development of improved therapeutics and vaccines depends on a better understanding of the unique biology of these organisms, and understanding their biology, in turn, requires the ability to track and manipulate the products of genes. In this work, we describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for easily and rapidly editing the genomes of kinetoplastid parasites. We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. This is all achieved without gene cloning or drug selection. We expect this advance to enable investigations, especially in Trypanosoma cruzi and Leishmania spp., that have eluded investigators for decades.