558 research outputs found

    Ultrastructure Expansion Microscopy (U-ExM) in Trypanosoma cruzi: localization of tubulin isoforms and isotypes

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    Ultrastructure Expansion Microscopy (U-ExM) is a recently developed technique that enables the increase of the spatial resolution within a cell or a tissue for microscopic imaging by physically expanding the sample. For the first time, I report a detailed protocol validating the use of U-ExM in Trypanosoma cruzi and quantifying the expansion factors of different subcellular compartments. I was able to determine the localization patterns of different tubulin isoforms, such as α-tubulin and β-tubulin. Also, I immunolocalized acetylated and tyrosinated α-tubulin isotypes in epimastigotes and use mitochondrial cell-permeable dyes to identify this organelle. Finally, U-ExM was also performed in trypomastigotes and amastigotes validating this technique in all life cycle stages of T. cruzi.Fil: Alonso, Victoria Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

    Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

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    We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).Fil: Alonso, Victoria Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Microbiología; ArgentinaFil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Serra, Esteban Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

    Overexpression of Cytoplasmic TcSIR2RP1 and Mitochondrial TcSIR2RP3 Impacts on Trypanosoma cruzi Growth and Cell Invasion

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    Background: Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi. Methodology: Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity. Conclusion: TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.Fil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Alonso, Victoria Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Manarin, Romina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cribb, Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Serra, Esteban Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

    The transcription factor LEF1 interacts with NFIX and switches isoforms during adult hippocampal neural stem cell quiescence

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    Stem cells in adult mammalian tissues are held in a reversible resting state, known as quiescence, for prolonged periods of time. Recent studies have greatly increased our understanding of the epigenetic and transcriptional landscapes that underlie stem cell quiescence. However, the transcription factor code that actively maintains the quiescence program remains poorly defined. Similarly, alternative splicing events affecting transcription factors in stem cell quiescence have been overlooked. Here we show that the transcription factor T-cell factor/lymphoid enhancer factor LEF1, a central player in canonical beta-catenin-dependent Wnt signalling, undergoes alternative splicing and switches isoforms in quiescent neural stem cells. We found that active beta-catenin and its partner LEF1 accumulated in quiescent hippocampal neural stem and progenitor cell (Q-NSPC) cultures. Accordingly, Q-NSPCs showed enhanced TCF/LEF1-driven transcription and a basal Wnt activity that conferred a functional advantage to the cultured cells in a Wnt-dependent assay. At a mechanistic level, we found a fine regulation of Lef1 gene expression. The coordinate upregulation of Lef1 transcription and retention of alternative spliced exon 6 (E6) led to the accumulation of a full-length protein isoform (LEF1-FL) that displayed increased stability in the quiescent state. Prospectively isolated GLAST + cells from the postnatal hippocampus also underwent E6 retention at the time quiescence is established in vivo. Interestingly, LEF1 motif was enriched in quiescence-associated enhancers of genes upregulated in Q-NSPCs and quiescence-related NFIX transcription factor motifs flanked the LEF1 binding sites. We further show that LEF1 interacts with NFIX and identify putative LEF1/NFIX targets. Together, our results uncover an unexpected role for LEF1 in gene regulation in quiescent NSPCs, and highlight alternative splicing as a post-transcriptional regulatory mechanism in the transition from stem cell activation to quiescence.Peer reviewe

    Gemini Observations of Galaxies in Rich Early Environments (GOGREEN) I : survey description

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    We describe a new Large Program in progress on the Gemini North and South telescopes: Gemini Observations of Galaxies in Rich Early Environments (GOGREEN). This is an imaging and deep spectroscopic survey of 21 galaxy systems at 1 10 in halo mass. The scientific objectives include measuring the role of environment in the evolution of low-mass galaxies, and measuring the dynamics and stellar contents of their host haloes. The targets are selected from the SpARCS, SPT, COSMOS, and SXDS surveys, to be the evolutionary counterparts of today's clusters and groups. The new red-sensitive Hamamatsu detectors on GMOS, coupled with the nod-and-shuffle sky subtraction, allow simultaneous wavelength coverage over lambda similar to 0.6-1.05 mu m, and this enables a homogeneous and statistically complete redshift survey of galaxies of all types. The spectroscopic sample targets galaxies with AB magnitudes z' <24.25 and [3.6] mu m <22.5, and is therefore statistically complete for stellar masses M* greater than or similar to 10(10.3) M-circle dot, for all galaxy types and over the entire redshift range. Deep, multiwavelength imaging has been acquired over larger fields for most systems, spanning u through K, in addition to deep IRAC imaging at 3.6 mu m. The spectroscopy is similar to 50 per cent complete as of semester 17A, and we anticipate a final sample of similar to 500 new cluster members. Combined with existing spectroscopy on the brighter galaxies from GCLASS, SPT, and other sources, GOGREEN will be a large legacy cluster and field galaxy sample at this redshift that spectroscopically covers a wide range in stellar mass, halo mass, and clustercentric radius.Peer reviewe

    Tecnologías ganaderas en rodeos de cría del este del Chaco, Argentina

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    Es cada vez mayor el interés por mejorar los parámetros productivos de la ganadería bovina, para lo que resulta imprescindible identificar los problemas que dificultan la adopción de las tecnologías que permiten incrementar su productividad. Esta publicación contiene la información relevada, sistematizada y analizada proveniente de encuestas realizadas a productores en el área de influencia de la EEA Colonia Benítez de INTA, ubicada en el este de la provincia del Chaco. El relevamiento se realizó de forma simultánea en las Agencias de Extensión Rural de Las Palmas, Basail, Makallé y en la Oficina de Colonia Benítez con la participación de distintos estratos de productores. Se evaluaron tecnologías relacionadas al manejo del rodeo, reproducción, sanidad, nutrición, bienestar animal, así como la gestión integral de la empresa ganadera. Los resultados logrados se presentan de manera ordenada definiendo las tecnologías consultadas, los valores obtenidos en cada estrato de productores, junto a relatos breves en primera persona del “conocimiento y uso” de las tecnologías analizadas. La información resulta de utilidad para construir propuestas de intervención teniendo en cuenta.EEA Colonia BenítezFil: Verdoljak, Juan Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Gomez, Viviana Daniela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Rossner, Maria Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Pellerano, Liliana Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benítez; ArgentinaFil: Famin, Lucia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez. Agencia de Extensión Rural Las Palmas; ArgentinaFil: Vagabculov, Javier. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Monteros, Diego Ezequiel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Lestani Sablich, Mariano. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benítez. Agencia de Extensión Rural Makallé; ArgentinaFil: Geijo, Angel Ruben. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez. Agencia de Extensión Rural Basail; ArgentinaFil: Fernandez, Abel Leopoldo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez. Agencia de Extensión Rural Las Palmas; ArgentinaFil: Pamies, Marcelo Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benítez; ArgentinaFil: Monicault, Luis Ademar. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Davalos, Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez. Agencia de Extensión Rural Basail; ArgentinaFil: Saez, Roberto Alonso. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Vagabculow, Jorge Leandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez. Agencia de Extensión Rural Las Palmas; ArgentinaFil: Di Lorenzo, Elio Luis. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benitez; ArgentinaFil: Rosello Brajovich, José Emilio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Colonia Benítez; Argentin

    Long-term follow-up of IPEX syndrome patients after different therapeutic strategies : an international multicenter retrospective study

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    Background: Immunodysregulation polyendocrinopathy enteropathy x-linked(IPEX) syndrome is a monogenic autoimmune disease caused by FOXP3 mutations. Because it is a rare disease, the natural history and response to treatments, including allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppression (IS), have not been thoroughly examined. Objective: This analysis sought to evaluate disease onset, progression, and long-term outcome of the 2 main treatments in long-term IPEX survivors. Methods: Clinical histories of 96 patients with a genetically proven IPEX syndrome were collected from 38 institutions worldwide and retrospectively analyzed. To investigate possible factors suitable to predict the outcome, an organ involvement (OI) scoring system was developed. Results: We confirm neonatal onset with enteropathy, type 1 diabetes, and eczema. In addition, we found less common manifestations in delayed onset patients or during disease evolution. There is no correlation between the site of mutation and the disease course or outcome, and the same genotype can present with variable phenotypes. HSCT patients (n = 58) had a median follow-up of 2.7 years (range, 1 week-15 years). Patients receiving chronic IS (n 5 34) had a median follow-up of 4 years (range, 2 months-25 years). The overall survival after HSCT was 73.2% (95% CI, 59.4-83.0) and after IS was 65.1% (95% CI, 62.8-95.8). The pretreatment OI score was the only significant predictor of overall survival after transplant (P = .035) but not under IS. Conclusions: Patients receiving chronic IS were hampered by disease recurrence or complications, impacting long-term.disease-free survival. When performed in patients with a low OI score, HSCT resulted in disease resolution with better quality of life, independent of age, donor source, or conditioning regimen

    Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

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    Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio
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