Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4+CD25+ T Cells by Phage Display

Thymic CD4+CD25+ cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4+CD25+ T regulatory cells we targeted thymic CD4+CD25+ cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4+CD25+ T cells. After four rounds of selection on CD4+CD25+ enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4+CD25+ cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells

Predicting the current distribution of the chacoan peccary (catagonus wagneri) in the gran Chaco

The Chacoan peccary (Catagonus wagneri), or Tagua, an endemic species living in the Chaco eco¬region, is endangered by highly increasing deforestation rates across the region, particularly in the last decade. This situation highlights the need to better understand the current distribution of the species, as well as how environmental conditions affect habitat suitability. This study predicts the distribution of the Chacoan peccary and evaluates the current environmental conditions in the Chaco for this species. Using six environmental variables and 177 confirmed occurrence records (from 2000 to 2015) provided by researchers, we developed a Species Distribution Model (SDM) applying the Maxent algorithm. The final model was highly accurate and significant (p < 0.001; AUC 0.860 ± 0.0268; omission error 1.82 %; post¬hoc validation of omission error using independent presence¬only records 1.33 %), predicting that 46.24 % of the Chaco is suitable habitat for the Chacoan peccary, with the most important areas concentrated in the middle of Paraguay and northern Argentina. Land cover, isothermality and elevation were the variables that better explained the habitat suitability for the Chacoan peccary. Despite some portions of suitable areas occurring inside protected areas, the borders and the central portions of suitable areas have recently suffered from intensive deforestation and development, and most of the highly suitable areas for the species are not under protection. The results provide fundamental insights for the establishment of priority Chacoan peccary conservation areas within its rangeFil: Paschoaletto Micchi, Katia Maria. Universidade Do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz Esalq; Brasil. Conservation Breeding Specialist Group Brazilian network; BrasilFil: Silva Angelieri, Cintia Camila. Universidade Do Sao Paulo. Escola Superior de Agricultura Luiz de Queiroz Esalq; BrasilFil: Altrichter, Mariana. Prescott College; Estados UnidosFil: Desbiez, Arnaud. Royal Zoological Society of Scotland. Edimburgo; Reino Unido. Conservation Breeding Specialist Group Brazilian network; BrasilFil: Yanosky, Alberto. Asociación Guyra Paraguay. Asunción; ParaguayFil: Campos Krauer, Juan Manuel. Centro Chaqueño para la Conservación y la Investigación; ParaguayFil: Torres, Ricardo Jose. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; ArgentinaFil: Camino, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; ArgentinaFil: Cabral, Hugo. Asociación Guyra Paraguay. Asunción; ParaguayFil: Cartés, José. Asociación Guyra Paraguay. Asunción; ParaguayFil: Cuellar, Rosa Leny. Fundación Kaa Iya; BoliviaFil: Gallegos, Marcelo. Secretaría de Ambiente de la Provincia de Salta. Programa Guardaparques; ArgentinaFil: Giordano, Anthony J.. No especifica;Fil: Decarre, Julieta. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Maffei, Leonardo. Wildlife Conservation Society. Lima; PerúFil: Neris, Nora. Universidad Nacional de Asunción; ParaguayFil: Saldivar Bellassai, Silvia. Itaipu Binacional; ParaguayFil: Wallace, Robert. Wildlife Conservation Society. New York; Estados UnidosFil: Lizarraga, Leónidas. Delegación Regional Noroeste. Sistema de Información de Biodiversidad de la Administración de Parques Nacionales. Salta; ArgentinaFil: Thompson, Jeffrey. Universidad Nacional de Asunción; ParaguayFil: Velilla, Mariela. Universidad Nacional de Asunción; Paragua

Next-Generation Phage Display: Integrating and Comparing Available Molecular Tools to Enable Cost-Effective High-Throughput Analysis

Background: Combinatorial phage display has been used in the last 20 years in the identification of protein-ligands and protein-protein interactions, uncovering relevant molecular recognition events. Rate-limiting steps of combinatorial phage display library selection are (i) the counting of transducing units and (ii) the sequencing of the encoded displayed ligands. Here, we adapted emerging genomic technologies to minimize such challenges. Methodology/Principal Findings: We gained efficiency by applying in tandem real-time PCR for rapid quantification to enable bacteria-free phage display library screening, and added phage DNA next-generation sequencing for large-scale ligand analysis, reporting a fully integrated set of high-throughput quantitative and analytical tools. The approach is far less labor-intensive and allows rigorous quantification; for medical applications, including selections in patients, it also represents an advance for quantitative distribution analysis and ligand identification of hundreds of thousands of targeted particles from patient-derived biopsy or autopsy in a longer timeframe post library administration. Additional advantages over current methods include increased sensitivity, less variability, enhanced linearity, scalability, and accuracy at much lower cost. Sequences obtained by qPhage plus pyrosequencing were similar to a dataset produced from conventional Sanger-sequenced transducing-units (TU), with no biases due to GC content, codon usage, and amino acid or peptide frequency. These tools allow phage display selection and ligand analysis at.1,000-fold faster rate, and reduce costs,250fol

Role of the gp85/Trans-Sialidases in Trypanosoma cruzi Tissue Tropism: Preferential Binding of a Conserved Peptide Motif to the Vasculature in Vivo

Background: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. the reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature.Methods: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. for that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied.Findings and Conclusions: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv São Paulo, Inst Quim, Dept Bioquim, BR-01498 São Paulo, BrazilUniv Texas MD Anderson Canc Ctr, David H Koch Ctr, Houston, TX 77030 USAUniv Texas MD Anderson Canc Ctr, Dept Canc Biol, Houston, TX 77030 USAUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 2004/03303-5FAPESP: 2008/54.806-8Web of Scienc

Increasing synergistic effects of habitat destruction and hunting on mammals over three decades in the Gran Chaco

Habitat destruction and overexploitation are the main threats to biodiversity and where they co-occur, their combined impact is often larger than their individual one. Yet, detailed knowledge of the spatial footprints of these threats is lacking, including where they overlap and how they change over time. These knowledge gaps are real barriers for effective conservation planning. Here, we develop a novel approach to reconstruct the individual and combined footprints of both threats over time. We combine satellite-based land-cover change maps, habitat suitability models and hunting pressure models to demonstrate our approach for the community of larger mammals (48 species > 1 kg) across the 1.1 million km2 Gran Chaco region, a global deforestation hotspot covering parts of Argentina, Bolivia and Paraguay. This provides three key insights. First, we find that the footprints of habitat destruction and hunting pressure expanded considerably between 1985 and 2015, across ~40% of the entire Chaco – twice the area affected by deforestation. Second, both threats increasingly acted together within the ranges of larger mammals in the Chaco (17% increase on average, ± 20% SD, cumulative increase of co-occurring threats across 465 000 km2), suggesting large synergistic effects. Conversely, core areas of high-quality habitats declined on average by 38%. Third, we identified remaining priority areas for conservation in the northern and central Chaco, many of which are outside the protected area network. We also identify hotspots of high threat impacts in central Paraguay and northern Argentina, providing a spatial template for threat-specific conservation action. Overall, our findings suggest increasing synergistic effects between habitat destruction and hunting pressure in the Chaco, a situation likely common in many tropical deforestation frontiers. Our work highlights how threats can be traced in space and time to understand their individual and combined impact, even in situations where data are sparse.Fil: Romero-Muñoz, Alfredo. Humboldt-Universität zu Berlin; AlemaniaFil: Benítez-López, Ana. Consejo Superior de Investigaciones Científicas. Estación Biológica de Doñana; EspañaFil: Zurell, Damaris. Humboldt-Universität zu Berlin; AlemaniaFil: Baumann, Matthias. Humboldt-Universität zu Berlin; AlemaniaFil: Camino, Micaela. Proyecto Quimilero; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Centro de Ecología Aplicada del Litoral. Universidad Nacional del Nordeste. Centro de Ecología Aplicada del Litoral; ArgentinaFil: Decarre, Julieta. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; ArgentinaFil: Castillo, Hugo del. Guyra Paraguay; ParaguayFil: Giordano, Anthony J.. University of California; Estados UnidosFil: Gómez-Valencia, Bibiana. Instituto de Investigación de Recursos Biológicos Alexander Von Humboldt, Bogota; Colombia. Universidad de Buenos Aires; ArgentinaFil: Levers, Christian. Humboldt-Universität zu Berlin; AlemaniaFil: Noss, Andrew J.. University of Florida; Estados UnidosFil: Quiroga, Verónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Thompson, Jeffrey J.. Guyra Paraguay; ParaguayFil: Torres, Ricardo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Velilla, Marianela. Guyra Paraguay; ParaguayFil: Weiler, Andrea. Universidad Nacional de Asunción; ParaguayFil: Kuemmerle, Tobias. Humboldt-Universität zu Berlin; Alemani

Synchronous down-modulation of miR-17 family members is an early causative event in the retinal angiogenic switch

Six members of the microRNA-17 (miR-17) family were mapped to three different chromosomes, although they share the same seed sequence and are predicted to target common genes, among which are those encoding hypoxia-inducible factor-1α (HIF1A) and VEGFA. Here, we evaluated the in vivo expression profile of the miR-17 family in the murine retinopathy of prematurity (ROP) model, whereby Vegfa expression is highly enhanced at the early stage of retinal neovascularization, and we found simultaneous reduction of all miR-17 family members at this stage. Using gene reporter assays, we observed binding of these miRs to specific sites in the 3′ UTRs of Hif1a and Vegfa. Furthermore, overexpression of these miRs decreased HIF1A and VEGFA expression in vitro. Our data indicate that this miR-17 family elicits a regulatory synergistic down-regulation of Hif1a and Vegfa expression in this biological model. We propose the existence of a coordinated regulatory network, in which diverse miRs are synchronously regulated to target the Hif1a transcription factor, which in turn, potentiates and reinforces the regulatory effects of the miRs on Vegfa to trigger and sustain a significant physiological response

Wild dogs at stake: deforestation threatens the only Amazon endemic canid, the short-eared dog (Atelocynus microtis)

The persistent high deforestation rate and fragmentation of the Amazon forests are the main threats to their biodiversity. To anticipate and mitigate these threats, it is important to understand and predict how species respond to the rapidly changing landscape. The short-eared dog Atelocynus microtis is the only Amazon-endemic canid and one of the most understudied wild dogs worldwide. We investigated short-eared dog habitat associations on two spatial scales. First, we used the largest record database ever compiled for short-eared dogs in combination with species distribution models to map species habitat suitability, estimate its distribution range and predict shifts in species distribution in response to predicted deforestation across the entire Amazon (regional scale). Second, we used systematic camera trap surveys and occupancy models to investigate how forest cover and forest fragmentation affect the space use of this species in the Southern Brazilian Amazon (local scale). Species distribution models suggested that the short-eared dog potentially occurs over an extensive and continuous area, through most of the Amazon region south of the Amazon River. However, approximately 30% of the short-eared dog's current distribution is expected to be lost or suffer sharp declines in habitat suitability by 2027 (within three generations) due to forest loss. This proportion might reach 40% of the species distribution in unprotected areas and exceed 60% in some interfluves (i.e. portions of land separated by large rivers) of the Amazon basin. Our local-scale analysis indicated that the presence of forest positively affected short-eared dog space use, while the density of forest edges had a negative effect. Beyond shedding light on the ecology of the short-eared dog and refining its distribution range, our results stress that forest loss poses a serious threat to the conservation of the species in a short time frame. Hence, we propose a re-assessment of the short-eared dog's current IUCN Red List status (Near Threatened) based on findings presented here. Our study exemplifies how data can be integrated across sources and modelling procedures to improve our knowledge of relatively understudied species

A Ligand Peptide Motif Selected from a Cancer Patient Is a Receptor-Interacting Site within Human Interleukin-11

Interleukin-11 (IL-11) is a pleiotropic cytokine approved by the FDA against chemotherapy-induced thrombocytopenia. From a combinatorial selection in a cancer patient, we isolated an IL-11-like peptide mapping to domain I of the IL-11 (sequence CGRRAGGSC). Although this motif has ligand attributes, it is not within the previously characterized interacting sites. Here we design and validate in-tandem binding assays, site-directed mutagenesis and NMR spectroscopy to show (i) the peptide mimics a receptor-binding site within IL-11, (ii) the binding of CGRRAGGSC to the IL-11Rα is functionally relevant, (iii) Arg4 and Ser8 are the key residues mediating the interaction, and (iv) the IL-11-like motif induces cell proliferation through STAT3 activation. These structural and functional results uncover an as yet unrecognized receptor-binding site in human IL-11. Given that IL-11Rα has been proposed as a target in human cancer, our results provide clues for the rational design of targeted drugs