RESUMEN
Inherited bleeding disorders (IBDs) are the most frequent congenital diseases in the Colombian population; three of them are hemophilia A (HA), hemophilia B (HB), and von Willebrand Disease (VWD). Currently, diagnosis relies on multiple clinical laboratory assays to assign a phenotype. Due to the lack of accessibility to these tests, patients can receive an incomplete diagnosis. In these cases, genetic studies reinforce the clinical diagnosis. The present study characterized the molecular genetic basis of 11 HA, three HB, and five VWD patients by sequencing the F8, F9, or the VWF gene. Twelve variations were found in HA patients, four in HB patients, and 19 in WVD patients. From these variations a total of 25 novel variations were found. Disease-causing variations were used as positive controls for validation of the high-resolution melting (HRM) variant-scanning technique. This approach is a low-cost genetic diagnostic method proposed to be incorporated in developing countries. For the data analysis, we developed an accessible open-source code in Python that improves HRM data analysis with better sensitivity of 95% and without bias when using different HRM equipment and software. Analysis of amplicons with a length greater than 300 bp can be performed by implementing an analysis by denaturation domains.
Asunto(s)
Trastornos de la Coagulación Sanguínea Heredados/diagnóstico , Biología Computacional/métodos , Factor IX/genética , Pruebas Genéticas/métodos , Hemofilia A/genética , Factor de von Willebrand/genética , Trastornos de la Coagulación Sanguínea Heredados/genética , Colombia , Biología Computacional/economía , Biología Computacional/normas , Costos y Análisis de Costo , Factor IX/química , Pruebas Genéticas/economía , Pruebas Genéticas/normas , Hemofilia A/diagnóstico , Humanos , Dominios Proteicos , Sensibilidad y Especificidad , Factor de von Willebrand/químicaRESUMEN
The global demand for fine-flavour cocoa has increased worldwide during the last years. Fine-flavour cocoa offers exceptional quality and unique fruity and floral flavour attributes of high demand by the world's elite chocolatiers. Several studies have highlighted the relevance of cocoa fermentation to produce such attributes. Nevertheless, little is known regarding the microbial interactions and biochemistry that lead to the production of these attributes on farms of industrial relevance, where traditional fermentation methods have been pre-standardized and scaled up. In this study, we have used metagenomic approaches to dissect on-farm industrial fermentations of fine-flavour cocoa. Our results revealed the presence of a shared core of nine dominant microorganisms (i.e. Limosilactobacillus fermentum, Saccharomyces cerevisiae, Pestalotiopsis rhododendri, Acetobacter aceti group, Bacillus subtilis group, Weissella ghanensis group, Lactobacillus_uc, Malassezia restricta and Malassezia globosa) between two farms located at completely different agro-ecological zones. Moreover, a community metabolic model was reconstructed and proposed as a tool to further elucidate the interactions among microorganisms and flavour biochemistry. Our work is the first to reveal a core of microorganisms shared among industrial farms, which is an essential step to process engineering aimed to design starter cultures, reducing fermentation times, and controlling the expression of undesirable phenotypes.
Asunto(s)
Cacao/química , Cacao/microbiología , Fermentación/genética , Metagenoma/genética , Chocolate/microbiología , Aromatizantes/química , Microbiología de Alimentos/métodosRESUMEN
The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/genética , Enzimas/genética , Biblioteca de Genes , Metagenómica/métodos , Suelo/química , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Enzimas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Microbiología del SueloRESUMEN
Topological and stereo-electron characteristics are essential in major histocompability class II-peptide-T-cell receptor (MHC-p-TCR) complex formation for inducing an appropriate immune response. Modified high activity binding peptides (mHABPs) were synthesised for complete full protection antimalarial vaccine development producing a large panel of individually fully protection-inducing protein structures (FPIPS) and very high long-lasting antibody-inducing (VHLLAI) mHABPs. Most of those which did not interfere, compete, inhibit or suppress their individual VHLLAI or FPIPS activity contained or displayed a polyproline II-like (PPIIL) structure when mixed. Here we show that amino acid side-chains located in peptide binding region (PBR) positions p3 and p7 displayed specific electron charges and side-chain gauche(+) orientation for interacting with the TCR. Based on the above, and previously described physicochemical principles, non-interfering, long-lasting, full protection-inducing, multi-epitope, multistage, minimal subunit-based chemically synthesised mHABP mixtures can be designed for developing vaccines against diseases scourging humankind, malaria being one of them.
Asunto(s)
Vacunas contra la Malaria/química , Oligopéptidos/inmunología , Conformación Proteica , Adyuvantes Inmunológicos/administración & dosificación , Secuencia de Aminoácidos , Animales , Anticuerpos Antiprotozoarios/sangre , Formación de Anticuerpos , Aotus trivirgatus , Sitios de Unión , Cadenas beta de HLA-DR/inmunología , Malaria Falciparum/prevención & control , Datos de Secuencia Molecular , Oligopéptidos/síntesis químicaRESUMEN
Malaria caused by Plasmodium vivax continues being a public health problem in tropical and subtropical areas throughout the whole world. In spite of this species' epidemiological importance, its biological complexity has hampered advances being made in the field of vaccine development. Few antigens have been described and analyzed to date in preclinical and clinical studies, thereby highlighting the great challenge facing groups currently working on this parasite species. This review summarizes the most representative work done during the last few years and discusses the approaches adopted in making progress towards an anti-Plasmodium vivax vaccine.
Asunto(s)
Vacunas contra la Malaria/inmunología , Malaria Vivax/prevención & control , Plasmodium vivax/inmunología , Investigación Biomédica/tendencias , Humanos , Malaria Vivax/epidemiologíaRESUMEN
Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs' specific binding was sensitive to HeLa and HepG2 cells' pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.
Asunto(s)
Plasmodium falciparum/inmunología , Proteínas Protozoarias , Trombospondinas , Secuencia de Aminoácidos , Animales , Aotus trivirgatus , Sitios de Unión , Línea Celular Tumoral , Condroitina ABC Liasa/farmacología , Células HeLa , Células Hep G2 , Liasa de Heparina/farmacología , Hepatocitos/inmunología , Hepatocitos/metabolismo , Hepatocitos/parasitología , Humanos , Vacunas contra la Malaria/inmunología , Péptidos/análisis , Péptidos/inmunología , Péptidos/aislamiento & purificación , Plasmodium falciparum/citología , Plasmodium falciparum/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Esporozoítos/citología , Esporozoítos/inmunología , Esporozoítos/metabolismo , Trombospondinas/química , Trombospondinas/inmunología , Trombospondinas/aislamiento & purificaciónRESUMEN
SIAP-1 and SIAP-2 are proteins which are implicated in early events involving Plasmodium falciparum infection of the Anopheles mosquito vector and the human host. High affinity HeLa and HepG2 cell binding conserved peptides have been previously identified in these proteins, i.e. SIAP-1 34893 ((421)KVQGLSYLLRRKNGTKHPVY(440)) and SIAP-1 34899 ((541)YVLNSKLLNSRSFDKFKWIQ(560)) and SIAP-2 36879 ((181)LLLYSTNSEDNLDISFGELQ(200)). When amino acid sequences have been properly modified (replacements shown in bold) they have induced high antibody titres against sporozoites in Aotus monkeys (assessed by IFA) and in the corresponding recombinant proteins (determined by ELISA and Western blot). (1)H NMR studies of these conserved native and modified high activity binding peptides (HABPs) revealed that all had α-helical structures in different locations and lengths. Conserved and corresponding modified HABPs displayed different lengths between the residues fitting into MHCII molecule pockets 1-9 and different amino acid orientation based on their different HLA-DRß1(∗) binding motifs and binding registers, suggesting that such modifications were associated with making them immunogenic. The results suggested that these modified HAPBs could be potential targets for inclusion as components of a fully-effective, minimal sub-unit based, multi-epitope, and multistage anti-malarial vaccine.