¿Tips? Formulación de proyectos

La presentación brinda una serie de consejos para la formulación de proyectos de investigación, enfatizando aspectos a considerar para garantizar un buen desarrollo y gestión del proceso.UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de QuímicaUCR::Vicerrectoría de Investigació

Evaluation of three chroroplastic markers for barcoding and for phylogenetic reconstruction purposes in native plants of Costa Rica

DNA barcoding has been proposed as a practical and standardized tool for species identification. However, the determination of the appropriate marker DNA regions is still a major challenge. In this study, we extracted DNA from 27 plant species belonging to 27 different families native of Costa Rica, amplified and sequenced the plastid genes matK and rpoC1 and the intergenic spacer trnH-­psbA. Bioinformatic analyses were performed with the aim of determining the utility of these markers as possible barcodes to discriminate among species and for phylogenetic reconstruction. From the markers selected, the trnH-­psbA spacer was the most variable in terms of genetic distance and the most promising region for barcoding. However, it presented a limited use for constructing phylogenies due to the complexity of its alignment. The locus matK was less variable but was also useful for species discrimination and for phylogenetic tree generation. The rpoC1 region was highly conserved and suitable for phylogenetic studies, but presented a limited utility as a barcode. The marker combination matK and rpoC1 provided the best resolution for establishing valid phylogenetic relationships among the analyzed plant families. In conclusion, more than one marker should be used for plant barcoding purposes, to provide complementary and variable information for the interespecific species discrimination.Se ha propuesto el empleo de un código de barras de ADN como una herramienta práctica y estandarizada para la identificación de especies. Sin embargo, la determinación de los marcadores moleculares apropiados se constituye en todo un reto. En este estudio se ha extraído el ADN de 27 especies de plantas que pertenecen a 27 familias distintas, nativas de Costa Rica, de las cuales se amplificaron y secuenciaron los marcadores matK y rpoC1 del genoma del plastidio y el espaciador intergénico trnH-­psbA. Se realizaron análisis bioinformáticos con el propósito de determinar la utilidad de estos marcadores como posibles códigos de barra para discriminar entre especies y en la reconstrucción filogenética. De los marcadores seleccionados, el espaciador trnH-­psbA fue el más variable en términos de distancia genética y la región más prometedora como código de barras. Sin embargo, presenta limitaciones en la construcción de filogenias debido a la complejidad del alineamiento. El locus matK fue menos variable, pero más útil en la discriminación de especies y en la generación de árboles filogenéticos. La región rpoC1 fue altamente conservada y útil para estudios filogenéticos, pero de utilidad limitada para ser empleada como código de barras. La combinación de los marcadores matK y rpoC1 provee la mejor resolución para establecer relaciones filogenéticas dentro de las familias de plantas analizadas. En conclusión, se requiere más de un marcador molecular para aplicaciones de código de barras y para proveer información complementaria variable para la discriminación de especies inter-específicas.National Institutes of Health /[U01 TW007404­01 ICB]/NIH/Estados Unido

Identification of Anziaic Acid, a Lichen Depside from Hypotrachyna sp., as a New Topoisomerase Poison Inhibitor

Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestis topoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia coli topoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds

Comparación de metodologías de extracción para limoneno y carvona en lippia alba usando cromatografía de gases.

Abstract Different plants drying procedures, hydrodistillation and solvent extraction by sonication were evaluated for the essential oil extraction of Lippia alba. Quantitative analysis of the markers limonene and carvone was performed in the extracts by gas chromatograph with flame ionization detection (GC-­FID). Experimental precision, accuracy, specificity, linearity, limits of detection and quantitation were addressed. Lyophilization as a drying procedure and solvent extraction by sonication using ethyl acetate showed the best results regarding the highest concentration for marker compounds.Diferentes procedimientos de secado, extracción mediante disolventes e hidrodestilación se evaluaron para la extracción del aceite esencial de Lippia alba. Se utilizaron los compuestos limoneno y carvona como marcadores para el análisis cuantitativo mediante cromatografía de gases con ionización de llama (GC-­FID). Se definió la precisión, exactitud, especificidad, linealidad y los límites de detección y cuantificación para el análisis. La liofilización como método de secado y la extracción con acetato de etilo utilizando baño ultrasónico mostraron los mejores resultados en función de la obtención de la mayor cantidad de los compuestos marcadores

Recolecta de artrópodos para prospección de la biodiversidad en el Área de Conservación Guanacaste, Costa Rica

artículo -- Universidad de Costa Rica. Escuela de Biología, 2004This study describes the results and collection practices for obtaining arthropod samples to be studied as potential sources of new medicines in a bioprospecting effort. From 1994 to 1998, 1800 arthropod samples of 6-10 g were collected in 21 sites of the Área de Conservación Guancaste (A.C.G) in Northwestern Costa Rica. The samples corresponded to 642 species distributed in 21 orders and 95 families. Most of the collections were obtained in the rainy season and in the tropical rainforest and dry forest of the ACG. Samples were obtained from a diversity of arthropod orders: 49.72% of the samples collected corresponded to Lepidoptera, 15.75% to Coleoptera, 13.33% to Hymenoptera, 11.43% to Orthoptera, 6.75% to Hemiptera, 3.20% to Homoptera and 7.89% to other groups. Different life stages per arthropod species were obtained in most samples, 54.26% of them were adults, 19.90% corresponded to larvae, 6.46% to pupae, 6.12% to pre-pupae, 2.07% to nymphs and 3.74% to other stages. Other materials associated to insects like frass represented 11.20% of the samples collected. Several collecting methods were explored, based on the possibility of accessing the necessary amount of material causing the less impact. Most of the samples were obtained by manual collection (44.38%), followed by insects breeding (25.73%), light traps (18.80%), different types of nets (10.52%) and other methods (0.16%). In general, collecting methods and practices excluded the use of solvents, mixing different species or life stages in the same bag, which might have introduced undesirable effects in the screening systems for new compounds. Based on the possibility of finding new chemicals in similar samples associated to one arthropod species, the collecting strategy included the generation of several samples from same species, separated according to differences in life stages, collecting sites, ecosystems, seasons, feeding materials or behavioral aspects. This strategy allowed the generation a larger number of samples submitted to bioassays in different areas of pharmaceutical research.Desde 1994 hasta 1997, 1800 muestras de artrópodoscorrespondientes a 642 especies distribuidas en 21 órdenes y 95 familias fueron recolectadas en 21 localidades protegidas del Área de Conservación Guanacaste, para ser estudiadas como fuente potencial de nuevos medicamentos. Las localidades con más recolectas fueron Santa María (231 spp.; 421 muestras), Santa Rosa (110 spp.; 172 muestras), Cacao (98 spp.; 203 muestras) y Pitilla (67 spp.; 79 muestras), siendo la mayor cantidad recolectadas durante la estación lluviosa. El 49.72% de las muestras recolectadas correspondió al orden Lepidoptera, 15.75% a Coleoptera, 13.33% a Hymenoptera, 11.43% a Orthoptera, 6.75% a Hemiptera, 3.20% a Homoptera y 7.89% a otros grupos. Además, un 54.26% de las muestras correspondía a individuos adultos, 19.90% a larvas, 11.20% a estiércol, 6.46% a pupas, 6.12% a prepupas, 2.07% a ninfas y 3.74% a otros estadios. Un 44.38% de las muestras se recolectó mediante recolecta manual, seguido por crianza de insectos (25.73%), trampa de luz (18.80%), diferentes tipos de redes (10.52%) y otros métodos (0.16%). La alta diversidad de los trópicos y la gran abundancia de artrópodos hace de este grupo una fuente potencial de productos farmacéuticos. Sin embargo, se debe evitar la recolecta intensiva en la misma localidad durante años consecutivos (sobre todo hembras), minimizar la interferencia en procesos vitales de los organismos y la dinámica de sus poblaciones. Además se debe recolectar la menor cantidad posible de individuos y escoger la época del año adecuada, para lograr el reestablecimiento natural de las poblaciones.National Institute of Health (Fogarty International Center) No. 5U01TW/CA00312, facilitado por el proyecto NSF DEB 9400829 y DEB 9705072, D. H. JanzenUniversidad de Costa Rica, Vicerrectoría de Investigación, proyecto VI 801-96-582.INBioUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Biologí

Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors

The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography

Novel Lobophorins Inhibit Oral Cancer Cell Growth and Induce Atf4- and Chop-Dependent Cell Death in Murine Fibroblasts

As part of the International Cooperative Biodiversity Groups (ICBG) Program, we were interested in identifying biologically active unfolded protein response (UPR) inducing compounds from marine microorganisms isolated from Costa Rican biota. With this aim in mind we have now generated more than 33,000 unique prefractionated natural product extracts from marine and terrestrial organisms that have been submitted to the Center of Chemical Genomics (CCG) at the University of Michigan for high throughput screening (HTS). An effective complementary cell-based assay to identify novel modulators of UPR signaling was used for screening extracts. Active fractions were iteratively subjected to reverse-phase HPLC chromatographic analysis, and together with lobophorin A, B, E, and F (1–4), three new lobophorin congeners, designated as CR1 (5), CR2 (6), and CR3 (7) were isolated. Herein, we report that secondary assays revealed that the new lobophorins induced UPR-associated gene expression, inhibited oral squamous cell carcinoma cell growth, and led to UPR-dependent cell death in murine embryonic fibroblast (MEF) cells.International Cooperative Biodiversity Groups/[U01 TW007404]/ICBG/Estados UnidosNational Institutes of Health/[DK042394]/NIH/Estados UnidosNational Institutes of Health/[DK088227]/NIH/Estados UnidosNational Institutes of Health/[HL052173]/NIH/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis

Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells.National Institutes of Health/[DE019678]/NIH/Estados UnidosInternational Cooperative Biodiversity Groups/[U01 TW007404]/ICBG/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific

The world's oceans contain a complex mixture of micro-organisms that are for the most part, uncharacterized both genetically and biochemically. We report here a metagenomic study of the marine planktonic microbiota in which surface (mostly marine) water samples were analyzed as part of the Sorcerer II Global Ocean Sampling expedition. These samples, collected across a several-thousand km transect from the North Atlantic through the Panama Canal and ending in the South Pacific yielded an extensive dataset consisting of 7.7 million sequencing reads (6.3 billion bp). Though a few major microbial clades dominate the planktonic marine niche, the dataset contains great diversity with 85% of the assembled sequence and 57% of the unassembled data being unique at a 98% sequence identity cutoff. Using the metadata associated with each sample and sequencing library, we developed new comparative genomic and assembly methods. One comparative genomic method, termed “fragment recruitment,” addressed questions of genome structure, evolution, and taxonomic or phylogenetic diversity, as well as the biochemical diversity of genes and gene families. A second method, termed “extreme assembly,” made possible the assembly and reconstruction of large segments of abundant but clearly nonclonal organisms. Within all abundant populations analyzed, we found extensive intra-ribotype diversity in several forms: (1) extensive sequence variation within orthologous regions throughout a given genome; despite coverage of individual ribotypes approaching 500-fold, most individual sequencing reads are unique; (2) numerous changes in gene content some with direct adaptive implications; and (3) hypervariable genomic islands that are too variable to assemble. The intra-ribotype diversity is organized into genetically isolated populations that have overlapping but independent distributions, implying distinct environmental preference. We present novel methods for measuring the genomic similarity between metagenomic samples and show how they may be grouped into several community types. Specific functional adaptations can be identified both within individual ribotypes and across the entire community, including proteorhodopsin spectral tuning and the presence or absence of the phosphate-binding gene PstS

Study of the diversity of culturable actinomycetes in the North Pacific and Caribbean coasts of Costa Rica

In this study, 137 actinomycetes were isolated from subtidal marine sediments in the North Pacific and Caribbean coasts of Costa Rica. Bioinformatics analysis of the 16S rRNA gene sequences assigned the isolates to 15 families and 21 genera. Streptomyces was the dominant genus while the remaining 20 genera were poorly represented. Nearly 70% of the phylotypes presented a coastal-restricted distribution whereas the other 30% were common inhabitants of both shores. The coastal tropical waters of Costa Rica showed a high diversity of actinomycetes, both in terms of the number of species and phylogenetic composition, although significant differences were observed between and within shores. The observed pattern of species distribution might be the result of several factors including the characteristics of the ecosystems, presence of endemic species and the influence of terrestrial runoff.University of Aberdeen/[]//EscociaNational Institutes of Health/[U01 TW007404-01 ICBG]/NIH/Estados UnidosUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de BiologíaUCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic