Impact of COVID-19 lockdown in a biomedical research campus: A gender perspective analysis.

From March to September 2020, researchers working at a biomedical scientific campus in Spain faced two lockdowns and various mobility restrictions that affected their social and professional lifestyles. The working group "Women in Science," which acts as an independent observatory of scientific gender inequalities on campus launched an online survey to assess the impact of COVID-19 lockdowns on scientific activity, domestic and caregiving tasks, and psychological status. The survey revealed differences in scientific performance by gender: while male researchers participated in a larger number of scientific activities for career development, female researchers performed more invisible scientific tasks, including peer review or outreach activities. Mental impact was greater in researchers caring for children or dependents, and this was aggravated for women. Results spot a disproportionate impact of COVID-19 lockdowns on female scientific career development, and urges for equity measures to mitigate the consequences of an increase in the gender gap in biomedical sciences for current and future pandemics.

Delineating transitions during the evolution of specialised peroxisomes: Glycosome formation in kinetoplastid and diplonemid protists.

One peculiarity of protists belonging to classes Kinetoplastea and Diplonemea within the phylum Euglenozoa is compartmentalisation of most glycolytic enzymes within peroxisomes that are hence called glycosomes. This pathway is not sequestered in peroxisomes of the third Euglenozoan class, Euglenida. Previous analysis of well-studied kinetoplastids, the 'TriTryps' parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., identified within glycosomes other metabolic processes usually not present in peroxisomes. In addition, trypanosomatid peroxins, i.e. proteins involved in biogenesis of these organelles, are divergent from human and yeast orthologues. In recent years, genomes, transcriptomes and proteomes for a variety of euglenozoans have become available. Here, we track the possible evolution of glycosomes by querying these databases, as well as the genome of Naegleria gruberi, a non-euglenozoan, which belongs to the same protist supergroup Discoba. We searched for orthologues of TriTryps proteins involved in glycosomal metabolism and biogenesis. Predicted cellular location(s) of each metabolic enzyme identified was inferred from presence or absence of peroxisomal-targeting signals. Combined with a survey of relevant literature, we refine extensively our previously postulated hypothesis about glycosome evolution. The data agree glycolysis was compartmentalised in a common ancestor of the kinetoplastids and diplonemids, yet additionally indicates most other processes found in glycosomes of extant trypanosomatids, but not in peroxisomes of other eukaryotes were either sequestered in this ancestor or shortly after separation of the two lineages. In contrast, peroxin divergence is evident in all euglenozoans. Following their gain of pathway complexity, subsequent evolution of peroxisome/glycosome function is complex. We hypothesize compartmentalisation in glycosomes of glycolytic enzymes, their cofactors and subsequently other metabolic enzymes provided selective advantage to kinetoplastids and diplonemids during their evolution in changing marine environments. We contend two specific properties derived from the ancestral peroxisomes were key: existence of nonselective pores for small solutes and the possibility of high turnover by pexophagy. Critically, such pores and pexophagy are characterised in extant trypanosomatids. Increasing amenability of free-living kinetoplastids and recently isolated diplonemids to experimental study means our hypothesis and interpretation of bioinformatic data are suited to experimental interrogation.

Extracellular Vesicles in Trypanosoma cruzi Infection: Immunomodulatory Effects and Future Perspectives as Potential Control Tools against Chagas Disease.

Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is a neglected tropical disease and a major public health problem affecting more than 6 million people worldwide. Many challenges remain in the quest to control Chagas disease: the diagnosis presents several limitations and the two available treatments cause several side effects, presenting limited efficacy during the chronic phase of the disease. In addition, there are no preventive vaccines or biomarkers of therapeutic response or disease outcome. Trypomastigote form and T. cruzi-infected cells release extracellular vesicles (EVs), which are involved in cell-to-cell communication and can modulate the host immune response. Importantly, EVs have been described as promising tools for the development of new therapeutic strategies, such as vaccines, and for the discovery of new biomarkers. Here, we review and discuss the role of EVs secreted during T. cruzi infection and their immunomodulatory properties. Finally, we briefly describe their potential for biomarker discovery and future perspectives as vaccine development tools for Chagas Disease.

Mass Spectrometry Identification of Biomarkers in Extracellular Vesicles From Plasmodium vivax Liver Hypnozoite Infections.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.

Biogenesis and metabolic homeostasis of trypanosomatid glycosomes: New insights and new questions.

Kinetoplastea and Diplonemea possess peroxisome-related organelles that, uniquely, contain most of the enzymes of the glycolytic pathway and are hence called glycosomes. Enzymes of several other core metabolic pathways have also been located in glycosomes, in addition to some characteristic peroxisomal systems such as pathways of lipid metabolism. A considerable amount of research has been performed on glycosomes of trypanosomes since their discovery four decades ago. Not only the role of the glycosomal enzyme systems in the overall cell metabolism appeared to be unique, but also the organelles display remarkable features regarding their biogenesis and structural properties. These features are similar to those of the well-studied peroxisomes of mammalian and plant cells and yeasts yet exhibit also differences reflecting the large evolutionary distance between these protists and the representatives of other major eukaryotic lineages. Despite all research performed, many questions remain about various properties and the biological roles of glycosomes and peroxisomes. Here, we review the current knowledge about glycosomes, often comparing it with information about peroxisomes. Furthermore, we highlight particularly many questions that remain about the biogenesis, and the heterogeneity in structure and content of these enigmatic organelles, and the properties of their boundary membrane.

SOCS2 expression in hematopoietic and non-hematopoietic cells during Trypanosoma cruzi infection: Correlation with immune response and cardiac dysfunction.

Chagas disease has a complex pathogenesis wherein the host immune response is essential for controlling its development. Suppressor of cytokine signaling(SOCS)2 is a crucial protein that regulates cytokine production. In this study, SOCS2 deficiency resulted in an initial imbalance of IL12- and IL-10-producing neutrophils and dendritic cells (DCs), which caused a long-lasting impact reducing inflammatory neutrophils and DCs, and tolerogenic DCs at the peak of acute disease. A reduced number of inflammatory and pro-resolving macrophages, and IL17A-producing CD4+ T cells, and increased lymphocyte apoptosis was found in SOCS2-deficient mice. Electrocardiogram analysis of chimeric mice showed that WT mice that received SOCS2 KO bone marrow transplantation presented increased heart dysfunction. Taken together, the results demonstrated that SOCS2 is a crucial regulator of the immune response during Trypanosoma cruzi infection, and suggest that a SOCS2 genetic polymorphism, or failure of its expression, may increase the susceptibility of cardiomyopathy development in Chagasic patients.

Pitting of malaria parasites in microfluidic devices mimicking spleen interendothelial slits.

The spleen is a hematopoietic organ that participates in cellular and humoral immunity. It also serves as a quality control mechanism for removing senescent and/or poorly deformable red blood cells (RBCs) from circulation. Pitting is a specialized process by which the spleen extracts particles, including malaria parasites, from within circulating RBCs during their passage through the interendothelial slits (IES) in the splenic cords. To study this physiological function in vitro, we have developed two microfluidic devices modeling the IES, according to the hypothesis that at a certain range of mechanical stress on the RBC, regulated through both slit size and blood flow, would force it undergo the pitting process without affecting the cell integrity. To prove its functionality in replicating pitting of malaria parasites, we have performed a characterization of P. falciparum-infected RBCs (P.f.-RBCs) after their passage through the devices, determining hemolysis and the proportion of once-infected RBCs (O-iRBCs), defined by the presence of a parasite antigen and absence of DAPI staining of parasite DNA using a flow cytometry-based approach. The passage of P.f.-RBCs through the devices at the physiological flow rate did not affect cell integrity and resulted in an increase of the frequency of O-iRBCs. Both microfluidic device models were capable to replicate the pitting of P.f.-RBCs ex vivo by means of mechanical constraints without cellular involvement, shedding new insights on the role of the spleen in the pathophysiology of malaria.

Tityus serrulatus scorpion venom as a potential drug source for Chagas' disease: Trypanocidal and immunomodulatory activity.

Current chemical therapies for Chagas Disease (CD) lack ability to clear Trypanosoma cruzi (Tc) parasites and cause severe side effects, making search for new strategies extremely necessary. We evaluated the action of Tityus serrulatus venom (TsV) components during Tc infection. TsV treatment increased nitric oxide and pro-inflammatory cytokine production by Tc-infected macrophages (MØ), decreased intracellular parasite replication and trypomastigotes release, also triggering ERK1/2, JNK1/2 and p38 activation. Ts7 demonstrated the highest anti-Tc activity, inducing high levels of TNF and IL-6 in infected MØ. TsV/Ts7 presented synergistic effect on p38 activation when incubated with Tc antigen. KPP-treatment of MØ also decreased trypomastigotes releasing, partially due to p38 activation. TsV/Ts7-pre-incubation of Tc demonstrated a direct effect on parasite decreasing MØ-trypomastigotes releasing. In vivo KPP-treatment of Tc-infected mice resulted in decreased parasitemia. Summarizing, this study opens perspectives for new bioactive molecules as CD-therapeutic treatment, demonstrating the TsV/Ts7/KPP-trypanocidal and immunomodulatory activity during Tc infection.

Multiparameter Flow Cytometry Analysis of the Human Spleen Applied to Studies of Plasma-Derived EVs From Plasmodium vivax Patients.

The spleen is a secondary lymphoid organ with multiple functions including the removal of senescent red blood cells and the coordination of immune responses against blood-borne pathogens, such as malaria parasites. Despite the major role of the spleen, the study of its function in humans is limited by ethical implications to access human tissues. Here, we employed multiparameter flow cytometry combined with cell purification techniques to determine human spleen cell populations from transplantation donors. Spleen immuno-phenotyping showed that CD45+ cells included B (30%), CD4+ T (16%), CD8+ T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented 70%, reticulocytes 0.7% and hematopoietic stem cells 0.02%. Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in intercellular communication and secreted by almost all cell types. EVs play several roles in malaria that range from modulation of immune responses to vascular alterations. To investigate interactions of plasma-derived EVs from Plasmodium vivax infected patients (PvEVs) with human spleen cells, we used size-exclusion chromatography (SEC) to separate EVs from the bulk of soluble plasma proteins and stained isolated EVs with fluorescent lipophilic dyes. The integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and EVs that showed an increased proportion of T cells (CD4+ 3 fold and CD8+ 4 fold), monocytes (1.51 fold), B cells (2.3 fold) and erythrocytes (3 fold) interacting with PvEVs as compared to plasma-derived EVs from healthy volunteers (hEVs). Future functional studies of these interactions can contribute to unveil pathophysiological processes involving the spleen in vivax malaria.

Antigen Discovery in Circulating Extracellular Vesicles From Plasmodium vivax Patients.

Plasmodium vivax is the most widely distributed human malaria parasite with 7 million annual clinical cases and 2.5 billion people living under risk of infection. There is an urgent need to discover new antigens for vaccination as only two vaccine candidates are currently in clinical trials. Extracellular vesicles (EVs) are small membrane-bound vesicles involved in intercellular communication and initially described in reticulocytes, the host cell of P. vivax, as a selective disposal mechanism of the transferrin receptor (CD71) in the maturation of reticulocytes to erythrocytes. We have recently reported the proteomics identification of P. vivax proteins associated to circulating EVs in P. vivax patients using size exclusion chromatography followed by mass spectrometry (MS). Parasite proteins were detected in only two out of ten patients. To increase the MS signal, we have implemented the direct immuno-affinity capture (DIC) technique to enrich in EVs derived from CD71-expressing cells. Remarkably, we identified parasite proteins in all patients totaling 48 proteins and including several previously identified P. vivax vaccine candidate antigens (MSP1, MSP3, MSP7, MSP9, Serine-repeat antigen 1, and HSP70) as well as membrane, cytosolic and exported proteins. Notably, a member of the Plasmodium helical interspersed sub-telomeric (PHIST-c) family and a member of the Plasmodium exported proteins, were detected in five out of six analyzed patients. Humoral immune response analysis using sera from vivax patients confirmed the antigenicity of the PHIST-c protein. Collectively, we showed that enrichment of EVs by CD71-DIC from plasma of patients, allows a robust identification of P. vivax immunogenic proteins. This study represents a significant advance in identifying new antigens for vaccination against this human malaria parasite.

Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence.

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.

Structure, Properties, and Function of Glycosomes in Trypanosoma cruzi.

Glycosomes are peroxisome-related organelles that have been identified in kinetoplastids and diplonemids. The hallmark of glycosomes is their harboring of the majority of the glycolytic enzymes. Our biochemical studies and proteome analysis of Trypanosoma cruzi glycosomes have located, in addition to enzymes of the glycolytic pathway, enzymes of several other metabolic processes in the organelles. These analyses revealed many aspects in common with glycosomes from other trypanosomatids as well as features that seem specific for T. cruzi. Their enzyme content indicates that T. cruzi glycosomes are multifunctional organelles, involved in both several catabolic processes such as glycolysis and anabolic ones. Specifically discussed in this minireview are the cross-talk between glycosomal metabolism and metabolic processes occurring in other cell compartments, and the importance of metabolite translocation systems in the glycosomal membrane to enable the coordination between the spatially separated processes. Possible mechanisms for metabolite translocation across the membrane are suggested by proteins identified in the organelle's membrane-homologs of the ABC and MCF transporter families-and the presence of channels as inferred previously from the detection of channel-forming proteins in glycosomal membrane preparations from the related parasite T. brucei. Together, these data provide insight in the way in which different parts of T. cruzi metabolism, although uniquely distributed over different compartments, are integrated and regulated. Moreover, this information reveals opportunities for the development of drugs against Chagas disease caused by these parasites and for which currently no adequate treatment is available.

Proteomic analysis of glycosomes from Trypanosoma cruzi epimastigotes.

In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as ß-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite's glycosomes.

Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice.

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax, responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.

Nuevo método de evaluación del efecto antiparasitario en modelos in vitro e in vivo, a través de la visualización de Trypanosoma cruzi-GFP / New method of evaluation of the antiparasitic effect models in in vitro and in vivo, through the viewing of Trypanosoma cruzi-GFP

Los tratamientos de primera línea para la enfermedad de Chagas generan importantes efectos adversos que acentúan el deterioro de la salud en los pacientes. La necesidad de generar fármacos alternativos ha permitido desarrollar estudios donde se emplean parásitos capaces de expresar una proteína fluorescente, a fin de correlacionar fluorescencia con población de protozoarios. En este sentido, ideamos una metodología para el seguimiento de la proliferación de Trypanosoma cruzi-GFP (Green Fluorescent Protein) en modelos in vitro e in vivo, empleando el equipo iBox- UVP. Los ensayos in vitro se iniciaron con una curva de calibración usando concentraciones entre 5x105 y 5x107 parásitos/mL. Seguidamente, con una curva de proliferación evidenciamos a través de la fluorescencia la susceptibilidad de los parásitos frente a la droga comercial Benznidazol (IC50= 5,3±1,3 μM). En el ensayo in vivo se corroboró cualitativamente el efecto quimioterapéutico del Benznidazol (100 mg/kg/día) en ratones C57BL/6, partiendo de un inóculo de 2,5x105 parásitos, haciendo captura de imágenes de fluorescencia cada dos días a partir del día 1, e inicio del tratamiento por vía oral el sexto día. El coeficiente de correlación cercano a 1 obtenido en la curva de calibración habla de un método de cuantificación parasitario sencillo y robusto; también los ensayos en modelos in vitro e in vivo permitieron monitorear el efecto dosis-dependiente de Benznidazol sobre T. cruzi-GFP. En síntesis, elaboramos una metodología novedosa, rápida, no invasiva y que sigue en tiempo real la respuesta quimioterapéutica de drogas anti-T. cruzi.

The first-line treatments for Chagas disease generate significant adverse effects that accentuate the health deterioration in patients. The need to generate alternative drugs has led to the development of studies in which parasites will express a fluorescent protein, and correlate this expression with protozoan population. We devised a methodology for monitoring the proliferation of Trypanosoma cruzi- GFP (Green Fluorescent Protein) in models in vitro and in vivo, using the equipment iBox-UVP. In vitro assays were initiated with a calibration curve using concentrations between 5x105 and 5x107 parasites/mL. Subsequently, with a proliferation curve, through fluorescence we determined the susceptibility of the parasites against the commercial drug Benznidazol (IC50= 5,3±1,3 μM). In vivo assays corroborated qualitatively the chemotherapeutic effect of Benznidazol (100 mg/kg/day) in C57BL/6 mice, starting from an inoculum of 2.5x105 parasites, making capture of fluorescence imaging every two days from day 1, and starting oral treatment on the sixth day. The correlation coefficient close to 1 obtained in the calibration curve showed that this quantification method of parasites is simple and robust; assays in vitro and in vivo allowed monitoring dose-dependent effects of Benznidazol agains T. cruzi-GFP. We have produced an innovative, rapid, non-invasive method that monitors in real time the chemotherapeutic response of anti-T. cruzi drugs.

The Aryl Hydrocarbon Receptor Modulates Production of Cytokines and Reactive Oxygen Species and Development of Myocarditis during Trypanosoma cruzi Infection.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor involved in controlling several aspects of immune responses, including the activation and differentiation of specific T cell subsets and antigen-presenting cells, thought to be relevant in the context of experimental Trypanosoma cruzi infection. The relevance of AhR for the outcome of T. cruzi infection is not known and was investigated here. We infected wild-type (WT) mice and AhR knockout (AhR KO) mice with T. cruzi (Y strain) and determined levels of parasitemia, myocardial inflammation and fibrosis, expression of AhR/cytokines/suppressor of cytokine signaling (SOCS) (spleen/heart), and production of nitric oxide (NO), reactive oxygen species (ROS), and peroxynitrite (ONOO(-)) (spleen). AhR expression was increased in the heart of infected WT mice. Infected AhR KO mice displayed significantly reduced parasitemia, inflammation, and fibrosis of the myocardium. This was associated with an anticipated increased immune response characterized by increased levels of inflammatory cytokines and reduced expression of SOCS2 and SOCS3 in the heart. In vitro, AhR deficiency caused impairment in parasite replication and decreased levels of ROS production. In conclusion, AhR influences the development of murine Chagas disease by modulating ROS production and regulating the expression of key physiological regulators of inflammation, SOCS1 to -3, associated with the production of cytokines during experimental T. cruzi infection.

Suppressor of cytokine signaling 2 modulates the immune response profile and development of experimental cerebral malaria.

Plasmodium falciparum infection results in severe malaria in humans, affecting various organs, including the liver, spleen and brain, and resulting in high morbidity and mortality. The Plasmodium berghei ANKA (PbA) infection in mice closely recapitulates many aspects of human cerebral malaria (CM); thus, this model has been used to investigate the pathogenesis of CM. Suppressor of cytokine signaling 2 (SOCS2), an intracellular protein induced by cytokines and hormones, modulates the immune response, neural development, neurogenesis and neurotrophic pathways. However, the role of SOCS2 during CM remains unknown. SOCS2 knockout (SOCS2(-/-)) mice infected with PbA show an initial resistance to infection with reduced parasitemia and production of TNF, TGF-ß, IL-12 and IL-17 in the brain. Interestingly, in the late phase of infection, SOCS2(-/-) mice display increased parasitemia and reduced Treg cell infiltration, associated with enhanced levels of Th1 and Th17 cells and related cytokines IL-17, IL-6, and TGF-ß in the brain. A significant reduction in protective neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), was also observed. Moreover, the molecular alterations in the brain of infected SOCS2(-/-) mice were associated with anxiety-related behaviors and cognition impairment. Mechanistically, these results revealed enhanced nitric oxide (NO) production in PbA-infected SOCS2(-/-) mice, and the inhibition of NO synthesis through l-NAME led to a marked decrease in survival, the disruption of parasitemia control and more pronounced anxiety-like behavior. Treatment with l-NAME also shifted the levels of Th1, Th7 and Treg cells in the brains of infected SOCS2(-/-) mice to the background levels observed in infected WT, with remarkable exception of increased CD8(+)IFN(+) T cells and inflammatory monocytes. These results indicate that SOCS2 plays a dual role during PbA infection, being detrimental in the control of the parasite replication but crucial in the regulation of the immune response and production of neurotrophic factors. Here, we provided strong evidence of a critical relationship between SOCS2 and NO in the orchestration of the immune response and development of CM during PbA infection.

Biogenesis, maintenance and dynamics of glycosomes in trypanosomatid parasites.

Peroxisomes of organisms belonging to the protist group Kinetoplastea, which include trypanosomatid parasites of the genera Trypanosoma and Leishmania, are unique in playing a crucial role in glycolysis and other parts of intermediary metabolism. They sequester the majority of the glycolytic enzymes and hence are called glycosomes. Their glycosomal enzyme content can vary strongly, particularly quantitatively, between different trypanosomatid species, and within each species during its life cycle. Turnover of glycosomes by autophagy of redundant ones and biogenesis of a new population of organelles play a pivotal role in the efficient adaptation of the glycosomal metabolic repertoire to the sudden, major nutritional changes encountered during the transitions in their life cycle. The overall mechanism of glycosome biogenesis is similar to that of peroxisomes in other organisms, but the homologous peroxins involved display low sequence conservation as well as variations in motifs mediating crucial protein-protein interactions in the process. The correct compartmentalisation of enzymes is essential for the regulation of the trypanosomatids' metabolism and consequently for their viability. For Trypanosoma brucei it was shown that glycosomes also play a crucial role in its life-cycle regulation: a crucial developmental control switch involves the translocation of a protein phosphatase from the cytosol into the organelles. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative of regulation of enzyme activities as an additional means to adapt the metabolic network to the different environmental conditions encountered.

Evolution, dynamics and specialized functions of glycosomes in metabolism and development of trypanosomatids.

Kinetoplastea such as trypanosomatid parasites contain specialized peroxisomes that uniquely contain enzymes of the glycolytic pathway and other parts of intermediary metabolism and hence are called glycosomes. Their specific enzyme content can vary strongly, quantitatively and qualitatively, between different species and during the parasites' life cycle. The correct sequestering of enzymes has great importance for the regulation of the trypanosomatids' metabolism and can, dependent on environmental conditions, even be essential. Glycosomes also play a pivotal role in life-cycle regulation of Trypanosoma brucei, as the translocation of a protein phosphatase from the cytosol forms part of a crucial developmental control switch. Many glycosomal proteins are differentially phosphorylated in different life-cycle stages, possibly indicative for unique forms of activity regulation, whereas many kinetic activity regulation mechanisms common for glycolytic enzymes are absent in these organisms. Glycosome turnover occurs by autophagic degradation of redundant organelles and assembly of new ones. This may provide the trypanosomatids with a manner to rapidly and efficiently adapt their metabolism to the sudden, major nutritional changes often encountered during the life cycle. This could also have helped facilitating successful adaptation of kinetoplastids, at multiple occasions during evolution, to their parasitic life style.

Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features.

Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ∆PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ∆PEX4 mutant.