Tumoral Malignancy Decreases Coupled with Higher ROS and Lipid Peroxidation in HCT116 Colon Cancer Cells upon Loss of PRDX6.

Peroxiredoxin 6 (PRDX6) is an atypical member of the peroxiredoxin family that presents not only peroxidase but also phospholipase A2 and lysophosphatidylcholine acyl transferase activities able to act on lipid hydroperoxides of cell membranes. It has been associated with the proliferation and invasive capacity of different tumoral cells including colorectal cancer cells, although the effect of its removal in these cells has not been yet studied. Here, using CRISPR/Cas9 technology, we constructed an HCT116 colorectal cancer cell line knockout for PRDX6 to study whether the mechanisms described for other cancer cells in terms of proliferation, migration, and invasiveness also apply in this tumoral cell line. HCT116 cells lacking PRDX6 showed increased ROS and lipid peroxidation, a decrease in the antioxidant response regulator NRF2, mitochondrial dysfunction, and increased sensitivity to ferroptosis. All these alterations lead to a decrease in proliferation, migration, and invasiveness in these cells. Furthermore, the reduced migratory and invasive capacity of HCT116 cancer cells is consistent with the observed cadherin switch and decrease in pro-invasive proteins such as MMPs. Therefore, the mechanism behind the effects of loss of PRDX6 in HCT116 cells could differ from that in HepG2 cells which is coherent with the fact that the correlation of PRDX6 expression with patient survival is different in hepatocellular carcinomas. Nonetheless, our results point to this protein as a good therapeutic target also for colorectal cancer.

Current practices and perceptions of misoprostol use for cervical preparation prior to outpatient hysteroscopy: a nationwide survey among gynecologists in Spain.

BACKGROUND: Although misoprostol has been used as a potential option for cervical priming prior to hysteroscopy, the available scientific evidence regarding its use is not always conclusive. The purpose of this article is to assess the different practices and criteria applied for the use of misoprostol use for cervical preparation in outpatient hysteroscopy without anesthesia among gynecologists across Spain, exploring the need for the corresponding evidence-based guidelines. METHODS: We carried out a cross-sectional study in which a two-step approach survey was conducted. Firstly, an online pilot survey was conducted with a limited sample of 10 professionals from 10 centers to identify initial patterns and gather feedback. Subsequently, a comprehensive online survey was carried out over a larger sample of respondents. The comprehensive survey covered aspects of misoprostol usage, including dosage, timing, indications, safety concerns, and overall perception. The survey design allowed for a thorough examination of existing practices and provided valuable insights to help address the disparities noted in misoprostol use for cervical preparation in outpatient hysteroscopies. RESULTS: Through the comprehensive online survey, we received response of 102 gynecologists from 54 centers across Spain. The overall results show a considerable variation in misoprostol use prior to hysteroscopy, with 21.57% of respondents not using misoprostol in any case, while a substantial majority (78.43%) use it selectively (68.83%) or consistently (9.8%). When asked about the type of patients, the vast majority (72.55%) use it in postmenopausal nulliparous patients, while its was used considerably less in premenopausal nulliparous patients (35.29%), postmenopausal multiparous patients (22.55%), and premenopausal multiparous patients (3.92%), whereas some choose not to use it in any patients (26.47%). Furthermore, 89.22% of respondents felt that there is a lack of clear criteria on misoprostol use, while 79.41% believed that it is necessary to establish such criteria. CONCLUSIONS: There was a diverse landscape of misoprostol use which underscores the complexity and individual approach to cervical preparation for an outpatient hysteroscopy. Indeed, the findings regarding its application, notably more prevalent among postmenopausal patients, contravene the prevailing body of published evidence. Moreover, a considerable proportion of respondents expressed the need for clear criteria, which emphasizes the importance of evidence-based protocols to guide the optimal use of misoprostol.

Promastigote EPS secretion and haptomonad biofilm formation as evolutionary adaptations of trypanosomatid parasites for colonizing honeybee hosts.

Bees are major pollinators involved in the maintenance of all terrestrial ecosystems. Biotic and abiotic factors placing these insects at risk is a research priority for ecological and agricultural sustainability. Parasites are one of the key players of this global decline and the study of their mechanisms of action is essential to control honeybee colony losses. Trypanosomatid parasites and particularly the Lotmaria passim are widely spread in honeybees, however their lifestyle is poorly understood. In this work, we show how these parasites are able to differentiate into a new parasitic lifestyle: the trypanosomatid biofilms. Using different microscopic techniques, we demonstrated that the secretion of Extracellular Polymeric Substances by free-swimming unicellular promastigote forms is a prerequisite for the generation and adherence of multicellular biofilms to solid surfaces in vitro and in vivo. Moreover, compared to human-infective trypanosomatid parasites our study shows how trypanosomatid parasites of honeybees increases their resistance and thus resilience to drastic changes in environmental conditions such as ultralow temperatures and hypoosmotic shock, which would explain their success thriving within or outside their hosts. These results set up the basis for the understanding of the success of this group of parasites in nature and to unveil the impact of such pathogens in honeybees, a keystones species in most terrestrial ecosystems.

Local terrestrial snails as natural intermediate hosts of the zoonotic parasite Angiostrongylus cantonensis in the new European endemic area of Valencia, Spain.

AIM: The rat lungworm, Angiostrongylus cantonensis, has recently been found in the city of Valencia, parasitizing rats, Rattus norvegicus and Rattus rattus, its natural definitive hosts. This is the first finding of this zoonotic nematode in continental Europe. After informing local and national health authorities, the collection of local terrestrial snails took place with the aim of elucidating their potential role as intermediate hosts of A. cantonensis. METHODS AND RESULTS: A total of 145 terrestrial snails, belonging to the species Cernuella virgata, Cornu aspersum, Eobania vermiculata, Otala punctata, Pseudotachea splendida, Rumina decollata and Theba pisana, were randomly collected between May and December 2022 in public gardens, parks and orchards in six districts of Valencia, in five of which A. cantonensis had been reported previously in rats. Once collected and identified, the snails were frozen at -20°C. Subsequently, the DNA was isolated and screened by PCR using specific primers targeting the A. cantonensis COI gene. Seven individual snails, belonging to the species C. virgata, C. aspersum and T. pisana, were positive, for an overall prevalence of 4.8%. The PCR product from one of them was sequenced by Sanger sequencing. CONCLUSIONS: The three positive terrestrial snail species are among the edible species that are frequently included in various dishes in Spain. C. virgata is reported as a previously unrecorded intermediate host and should be added to the list of more than 200 species of terrestrial snails that have been reported worldwide as intermediate hosts of the rat lungworm. Considering that these terrestrial snails may release infective larvae of A. cantonensis on leafy green vegetables on which they feed and during their handling and preparation for consumption, prophylactic measures to prevent human neuroangiostrongyliasis in Valencia and other regions to which this zoonotic parasite may spread are recommended.

Providing an in vitro depiction of microglial cells challenged with immunostimulatory extracellular vesicles of Naegleria fowleri.

Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis, a rapid and acute infection of the central nervous system with a fatal outcome in >97% of cases. Due to the infrequent report of cases and diagnostic gaps that hinder the possibility of recovering clinic isolates, studies related to pathogenesis of the disease are scarce. However, the secretion of cytolytic molecules has been proposed as a factor involved in the progression of the infection. Several of these molecules could be included in extracellular vesicles (EVs), making them potential virulence factors and even modulators of the immune response in this infection. In this work, we evaluated the immunomodulatory effect of EVs secreted by two clinic isolates of Naegleria fowleri using in vitro models. For this purpose, characterization analyses between EVs produced by both isolates were first performed, for subsequent gene transcription analyses post incubation of these vesicles with primary cultures from mouse cell microglia and BV-2 cells. Analyses of morphological changes induced in primary culture microglia cells by the vesicles were also included, as well as the determination of the presence of nucleic acids of N. fowleri in the EV fractions. Results revealed increased expression of NOS, proinflammatory cytokines IL-6, TNF-α, and IL-23, and the regulatory cytokine IL-10 in primary cultures of microglia, as well as increased expression of NOS and IL-13 in BV-2 cells. Morphologic changes from homeostatic microglia, with small cellular body and long processes to a more amoeboid morphology were also observed after the incubation of these cells with EVs. Regarding the presence of nucleic acids, specific Naegleria fowleri DNA that could be amplified using both conventional and qPCR was confirmed in the EV fractions. Altogether, these results confirm the immunomodulatory effects of EVs of Naegleria fowleri over microglial cells and suggest a potential role of these vesicles as biomarkers of primary acute meningoencephalitis.

Coping and anxiety symptoms in family carers of dependent older people: Mediation and moderation effects of subjective caregiver burden.

INTRODUCTION: Coping responses influence anxiety symptoms experienced by informal carers. However, only a few studies have investigated the longitudinal association between coping responses and anxiety symptoms in family carers. We also currently have limited knowledge on the mediating or moderating influence of subjective caregiver burden on this relationship over time. The aim of the present study was to investigate the longitudinal relationship between coping and anxiety symptoms in family carers of dependent older people, and examine the mediating or moderating role of subjective caregiver burden over time. DESIGN: Prospective longitudinal study. METHODS: We recruited and enrolled participants from a probability sample of 132 family carers of older dependent relatives. We measured coping strategies, anxiety symptoms, subjective caregiver burden, and several covariates (sex and intensity of care) at baseline and at 1-year follow-up. We used generalized estimating equations with multiple imputations to examine associations over time. RESULTS: Considering both direct and indirect effects through subjective burden, anxiety symptoms were positively associated with proactive coping (B = 0.13), planning (B = 0.15), self-distraction (B = 0.24), denial (B = 1.15), venting (B = 0.94) and self-blame (B = 0.90), and negatively associated with positive reframing (B = -0.83) and acceptance (B = -0.75). Subjective caregiver burden moderated the relationship between anxiety symptoms and planning, and the use of denial as a form of coping. CONCLUSIONS: Our results show that subjective caregiver burden is an important moderator and mediator of the longitudinal association between coping responses and anxiety symptoms in carers. CLINICAL RELEVANCE: Proactive coping and planning when subjective burden is low, self-distraction, denial, venting, and self-blame significantly increase levels of anxiety and caregiver burden in carers over time. Acceptance and positive reframing however as coping responses are associated with lower levels of anxiety and caregiver burden long-term. Our findings highlight the need for a multi-dimensional approach in future caregiving interventions.

Use of sera cell free DNA (cfDNA) and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease.

Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other regions that were considered free of the disease, and where the epidemiological risk is limited to transplacental transmission or blood or organ donations from infected persons. Parasite detection in chronically ill patients is restricted to serological tests that only determine infection by previous infection and not the presence of the parasite, especially in patients undergoing treatment evaluation or in newborns. We have evaluated the use of nucleic acids from both circulating exovesicles and cell-free DNA (cfDNA) from 50 samples twice randomly selected from a total of 448 serum samples from immunologically diagnosed patients in whom the presence of the parasite was confirmed by nested PCR on amplicons resulting from amplification with kinetoplastid DNA-specific primers 121F-122R. Six samples were randomly selected to quantify the limit of detection by qPCR in serum exovesicles. When the nucleic acids thus purified were assayed as a template and amplified with kinetoplastid DNA and nuclear satellite DNA primers, a 100% positivity rate was obtained for all positive samples assayed with kDNA-specific primers and 96% when SAT primers were used. However, isolation of cfDNA for Trypanosoma cruzi and amplification with SAT also showed 100% positivity. The results demonstrate that serum exovesicles contain DNA of mitochondrial and nuclear origin, which can be considered a mixed population of exovesicles of parasitic origin. The results obtained with serum samples prove that both cfDNA and Exovesicle DNA can be used to confirm parasitaemia in chronically ill patients or in samples where it is necessary to demonstrate the active presence of the parasite. The results confirm for the first time the existence of exovesicles of mitochondrial origin of the parasite in the serum of those affected by Chagas disease.

Extracellular vesicles of Trypanosoma cruzi and immune complexes they form with sialylated and non-sialylated IgGs increase small peritoneal macrophage subpopulation and elicit different cytokines profiles.

American trypanosomiasis, or Chagas disease, is caused by the protozoan parasite Trypanosoma cruzi and is characterized by the presence of cardiac or gastrointestinal symptoms in a large number of patients during the chronic phase of the disease. Although the origin of the symptoms is not clear, several mechanisms have been described involving factors related to T. cruzi and the host immune response. In this sense, the extracellular vesicles (EVs) secreted by the parasite and the immune complexes (ICs) formed after their recognition by host IgGs (EVs-IgGs) may play an important role in the immune response during infection. The aim of the present work is to elucidate the modulation of the immune response exerted by EVs and the ICs they form by analyzing the variation in the subpopulations of small and large peritoneal macrophages after intraperitoneal inoculation in mice and to evaluate the role of the sialylation of the host IgGs in this immunomodulation. Both macrophage subpopulations were purified and subjected to cytokine expression analysis by RT-qPCR. The results showed an increase in the small peritoneal macrophage subpopulation after intraperitoneal injection of parasite EVs, but a greater increase in this subpopulation was observed when sialylated and non-sialylated ICs were injected, which was similar to inoculation with the trypomastigote stage of the parasite. The cytokine expression results showed the ability of both subpopulations to express inflammatory and non-inflammatory cytokines. These results suggest the role of free EVs in the acute phase of the disease and the possible role of immune complexes in the immune response in the chronic phase of the disease, when the levels of antibodies against the parasite allow the formation of immune complexes. The differential expression of interleukins showed after the inoculation of immune complexes formed with sialylated and non-sialylated IgGs and the interleukins expression induced by EVs, demonstrates that the IgG glycosilation is involved in the type of immune response that dominates in each of the phases of the Chagas disease.

Extracellular vesicles of trypomastigotes of Trypanosoma cruzi induce changes in ubiquitin-related processes, cell-signaling pathways and apoptosis.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. The disease has an acute and a chronic phase in which approximately 30% of the chronic patients suffer from heart disease and/or gastrointestinal symptoms. The pathogenesis of the disease is multifactorial and involves the virulence of the strains, immunological factors and extracellular vesicles (EV) shed by the parasite which participate in cell-cell communication and evasion of the immune response. In this work, we present a transcriptomic analysis of cells stimulated with EV of the trypomastigote stage of T. cruzi. Results after EV-cell incubation revealed 322 differentially expressed genes (168 were upregulated and 154 were downregulated). In this regard, the overexpression of genes related to ubiquitin-related processes (Ube2C, SUMO1 and SUMO2) is highlighted. Moreover, the expression of Rho-GTPases (RhoA, Rac1 and Cdc42) after the interaction was analyzed, revealing a downregulation of the analyzed genes after 4 h of interaction. Finally, a protective role of EV over apoptosis is suggested, as relative values of cells in early and late apoptosis were significantly lower in EV-treated cells, which also showed increased CSNK1G1 expression. These results contribute to a better understanding of the EV-cell interaction and support the role of EV as virulence factors.

Update on the First Finding of the Rat Lungworm, Angiostrongylus cantonensis, in Rattus spp. in Continental Europe, Valencia, Spain, 2022.

The rat lungworm, Angiostrongylus cantonensis, is an emerging parasite that can cause eosinophilic meningitis in humans. Over the past 60 years, the worm has greatly expanded its original Asian distribution to most tropical and subtropical areas of the world, mostly by traveling on ships with its definitive hosts, rats. Angiostrongylus cantonensis was recently found for the first time in Continental Europe, specifically in 3 (2 Rattus norvegicus and 1 Rattus rattus) out of 27 rats trapped in the sewer system of the city of Valencia, Spain. Updating the investigation, the parasite has subsequently been found in 8 (5 R. norvegicus and 3 R. rattus) out of 94 rats analyzed. The highest prevalence of infection (20%) was obtained in rats trapped in the orchards that surround the city, where both snails and slugs (intermediate hosts) abound, and where vegetables consumed in Valencia, other parts of Spain, and abroad, are produced. The presence of the parasite in rats does not necessarily mean that the disease it causes becomes a relevant public health concern since it strongly depends on the food habits of the population at risk. If proper precautions are taken, the risk of acquiring neuroangiostrongylosis should be minimal.

Development of a TaqMan qPCR assay for trypanosomatid multi-species detection and quantification in insects.

BACKGROUND: Trypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample. METHODS: A TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major. RESULTS: The assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host. CONCLUSIONS: These results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.

Zurbarán attribution hypothesis supported by pigment analysis and multiband images observation of four paintings by his workshop.

Francisco de Zurbarán was one of the greatest painters of the Spanish Golden Age, with artworks scattered all over the world. Unfortunately, there are hardly any exhaustive studies on the pigments that he used. In this work, four canvas paintings attributed to the Zurbarán Workshop were studied. Each of them presents the figure of a different saint in a particular isolation scene: Saint Bruno, Saint Francis of Assisi, Saint Dominic of Guzman, and Saint Peter Martyr. Nevertheless, the painting of Saint Peter Martyr shows superior quality in both technique and colours, so it is not clear whether this canvas was also made by the Workshop or by Zurbarán himself, as some art historians claim. Coinciding with conservation and restoration works, the paintings were initially analysed with non-invasive techniques such as ultraviolet photography (UV), infrared reflectography (IRR) and X-ray fluorescence (XRF) to determine the characterization of the pigments. Those studies were complemented by stratigraphic analysis of some extracted samples with optical microscopy (OM) and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). Among a number of results obtained, we found significant differences between the pigment palette used in Saint Peter Martyr and the other paintings, supporting the hypothesis that this painting was done by Zurbarán himself instead of his workshop. These results could also help to distinguish other authentic paintings by the master's hand from very similar paintings by his workshop or by other artists.

Post-transplant diabetes mellitus and renal cell cancer after renal transplantation.

BACKGROUND: Diabetes is a risk factor for cancer in the general population. However, few data are available on the association between post-transplant diabetes mellitus (PTDM) and cancer after transplantation. METHODS: We analyzed this issue in a Spanish cohort of patients without diabetes before transplantation. PTDM was diagnosed with consensus criteria at 12 months after transplantation and 12 months before the diagnosis of cancer. The association between PTDM and cancer (overall and specific types) was evaluated with regression analysis. RESULTS: During a follow-up of 12 years (interquartile range 8-14), 85 cases of 603 developed cancer (829/100 000/year) and 164 (27%) PTDM. The most frequent cancers were renal cell cancer (RCC) n = 15, 146/cases/100 000/year), lung (n = 12, 117/cases/100 000/year), colon (n = 9, 88/cases/100 000/year) and prostate (n = 9, 88/cases/100 000/year). In logistic regression, PTDM was not associated with cancer. Eight of the 164 patients with PTDM (4.9%) vs 7 of the 439 without PTDM developed RCC (1.6%) (P = .027). In multivariate analysis, PTDM was independently associated with RCC [odds ratio (OR) 2.92, confidence interval (CI) 1.03-8.27], adjusting for smoking (OR 4.020, 95% CI 1.34-12.02) and other covariates. PTDM was not associated with other types of cancer. CONCLUSIONS: Patients with PTDM must be considered a population at risk for RCC and accordingly, the subject of active surveillance.

Parasite Fauna and Coinfections in Urban Rats Naturally Infected by the Zoonotic Parasite Angiostrongylus cantonensis.

When the zoonotic parasite of rodents that can cause human neuroangiostrongyliasis, i.e., Angiostrongylus cantonensis, is found in its natural definitive hosts, it is usually reported in isolation, as if the rat lungworm were the only component of its parasite community. In this study, we report the coinfections found in rats naturally infected by A. cantonensis in urban populations of Rattus norvegicus and Rattus rattus in Valencia, Spain. In addition to the rat lungworms, which were found in 14 of the 125 rats studied (a prevalence of 11.20%), 18 other parasite species (intestinal and tissular protists, microsporidia and helminths) were found, some of them with high burdens. Fourteen of these nineteen species found are potential zoonotic parasites, namely Blastocystis, Giardia duodenalis, Cryptosporidium spp., Enterocytozoon bieneusi, Encephalitozoon hellem, Toxoplasma gondii, Brachylaima spp., Hydatigera taeniaeformis s.l. larvae, Hymenolepis nana, Hymenolepis diminuta, Angiostrongylus cantonensis, Calodium hepaticum, Gongylonema neoplasticum and Moniliformis moniliformis. The total predominance of coinfected rats as well as their high parasite loads seem to indicate a trend towards parasite tolerance.

Perceptions, Motivations, and Empowerment Strategies of Midwives in Rural and Remote Areas of Northern Morocco.

The shortage of midwives is a problem in rural and remote areas. This is mainly the consequence of job insecurity and difficult living conditions. The present study aimed to identify and analyse the perceptions and motivations of midwives in rural and remote areas of northern Morocco on the quality of their working life and the motivational factors and empowerment strategies they use to maintain and develop their work. It is a qualitative study that follows Van Manen's hermeneutic phenomenology approach. Three focus groups and in-depth interviews were conducted with 15 midwives from rural and remote areas. The results indicate that midwives in rural and remote areas have a negative perception of the quality of the work and their personal life because of the scarcity of basic resources, unfavourable working conditions, and the personal sacrifices they have to make to support themselves. However, some factors favour their efforts. Therefore, there is a need to promote intersectoral policies that focus on improving material and human resources, as well as the working and personal conditions of midwives and the factors that support and empower them.

Autochthonous Angiostrongylus cantonensis Lungworms in Urban Rats, Valencia, Spain, 2021.

To determine the role of rats as potential reservoirs of zoonotic parasites, we examined rats trapped in urban sewers of Valencia, Spain, in 2021. Morphologic and molecular identification and sequencing identified autochthonous Angiostrongylus cantonensis nematodes, the most common cause of human eosinophilic meningitis, in pulmonary arteries of Rattus norvegicus and R. rattus rats.

Trypanosoma cruzi pathogenicity involves virulence factor expression and upregulation of bioenergetic and biosynthetic pathways.

The molecular repertoire of Trypanosoma cruzi effects its virulence and impacts the clinical course of the resulting Chagas disease. This study aimed to determine the mechanism underlying the pathogenicity of T. cruzi. Two T. cruzi cell lines (C8C3hvir and C8C3lvir), obtained from the clone H510 C8C3 and exhibiting different virulence phenotypes, were used to evaluate the parasite's infectivity in mice. The organ parasite load was analysed by qPCR. The proteomes of both T. cruzi cell lines were compared using nLC-MS/MS. Cruzipain (Czp), complement regulatory protein (CRP), trans-sialidase (TS), Tc-85, and sialylated epitope expression levels were evaluated by immunoblotting. High-virulence C8C3hvir was highly infectious in mice and demonstrated three to five times higher infectivity in mouse myocardial cells than low-virulence C8C3lvir. qPCR revealed higher parasite loads in organs of acute as well as chronically C8C3hvir-infected mice than in those of C8C3lvir-infected mice. Comparative quantitative proteomics revealed that 390 of 1547 identified proteins were differentially regulated in C8C3hvir with respect to C8C3lvir. Amongst these, 174 proteins were upregulated in C8C3hvir and 216 were downregulated in C8C3lvir. The upregulated proteins in C8C3hvir were associated with the tricarboxylic acid cycle, ribosomal proteins, and redoxins. Higher levels of Czp, CRP, TS, Tc-85, and sialylated epitopes were expressed in C8C3hvir than in C8C3lvir. Thus, T. cruzi virulence may be related to virulence factor expression as well as upregulation of bioenergetic and biosynthetic pathways proteins.

Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo.

Extracellular vesicles (EVs) are small lipid vesicles released by both prokaryotic and eukaryotic cells, involved in intercellular communication, immunomodulation and pathogenesis. In this study, we performed a characterization of the EVs produced by trophozoites of a clinical isolate of the free-living amoeba Naegleria fowleri (N. fowleri). Size distribution, zeta potential, protein profile and protease activity were analyzed. Under our incubation conditions, EVs of different sizes were observed, with a predominant population ranging from 206 to 227 nm. SDS-PAGE revealed protein bands of 25 to 260 KDa. The presence of antigenic proteins was confirmed by Western blot, which evidenced strongest recognition by rat polyclonal antibodies raised against N. fowleri in the region close to 80 KDa and included peptidases, as revealed by zymography. Proteins in selected immunorecognized bands were further identified using nano-ESI-MS/MS. A preliminary proteomic profile of the EVs identified at least 184 proteins as part of the vesicles' cargo. Protease activity assays, in combination with the use of inhibitors, revealed the predominance of serine proteases. The present characterization uncovers the complexity of EVs produced by N. fowleri, suggesting their potential relevance in the release of virulence factors involved in pathogenicity. Owing to their cargo's diversity, further research on EVs could reveal new therapeutic targets or biomarkers for developing rapid and accurate diagnostic tools for lethal infections such as the one caused by this amoeba.