RESUMEN
Through iterative cycles of selection, amplification, and mutagenesis, in vitro selection provides the ability to isolate molecules of desired properties and function from large pools (libraries) of random molecules with as many as 10(16) distinct species. This review, in recognition of a quarter of century of scientific discoveries made through in vitro selection, starts with a brief overview of the method and its history. It further covers recent developments in in vitro selection with a focus on tools that enhance the capabilities of in vitro selection and its expansion from being purely a nucleic acids selection to that of polypeptides and proteins. In addition, we cover how next generation sequencing and modern biological computational tools are being used to complement in vitro selection experiments. On the very least, sequencing and computational tools can translate the large volume of information associated with in vitro selection experiments to manageable, analyzable, and exploitable information. Finally, in vivo selection is briefly compared and contrasted to in vitro selection to highlight the unique capabilities of each method.
Asunto(s)
Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Proteínas/aislamiento & purificación , Técnica SELEX de Producción de Aptámeros/métodos , Proteínas/química , Proteínas/genética , ARN/genéticaRESUMEN
Prochlorococcus, a cyanobacteria genus of the smallest and most abundant oceanic phototrophs, encompasses ecotype strains adapted to high-light (HL) and low-light (LL) niches. To elucidate the adaptive evolution of this genus, we analyzed 40 Prochlorococcus marinus ORFeomes, including two cornerstone strains, MED4 and NATL1A. Employing deep learning with robust statistical methods, we detected new protein family distributions in the strains and identified key genes differentiating the HL and LL strains. The HL strains harbor genes (ABC-2 transporters) related to stress resistance, such as DNA repair and RNA processing, while the LL strains exhibit unique chlorophyll adaptations (ion transport proteins, HEAT repeats). Additionally, we report the finding of variable, depth-dependent endogenous viral elements in the 40 strains. To generate biological resources to experimentally study the HL and LL adaptations, we constructed the ORFeomes of two representative strains, MED4 and NATL1A synthetically, covering 99% of the annotated protein-coding sequences of the two species, totaling 3976 cloned, sequence-verified open reading frames (ORFs). These comparative genomic analyses, paired with MED4 and NATL1A ORFeomes, will facilitate future genotype-to-phenotype mappings and the systems biology exploration of Prochlorococcus ecology.
RESUMEN
Candida albicans is a common opportunistic pathogen that causes a wide variety of diseases in a human immunocompromised host leading to death. In a pathogen, cell wall proteins are important for stability as well as for acting as antigenic determinants and virulence factors. Pir32 is a cell wall protein and member of the Pir protein family previously shown to be upregulated in response to macrophage contact and whose other member, Pir1, was found to be necessary for cell wall rigidity. The purpose of this study is to characterize Pir32 by generating a homozygous null strain and comparing the phenotype of the null with that of the wild-type parental strain as far as filamentation, virulence in a mouse model of disseminated candidiasis, resistance to oxidative stress and cell wall disrupting agents, in addition to adhesion, biofilm capacities, and cell wall chitin content. Our mutant was shown to be hyperfilamentous, resistant to sodium dodecyl sulfate, hydrogen peroxide, sodium chloride, and more virulent in a mouse model when compared to the wild type. These results were unexpected, considering that most cell wall mutations weaken the wall and render it more susceptible to external stress factors and suggests the possibility of a cell surface compensatory mechanism. As such, we measured cell wall chitin deposition and found a twofold increase in the mutant, possibly explaining the above-observed phenotypes.
Asunto(s)
Candida albicans/fisiología , Pared Celular/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/genética , Eliminación de Gen , Estrés Fisiológico , Animales , Candida albicans/citología , Candida albicans/metabolismo , Candida albicans/patogenicidad , Candidiasis/microbiología , Candidiasis/patología , Modelos Animales de Enfermedad , Peróxido de Hidrógeno/toxicidad , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana/efectos de los fármacos , Microscopía , Presión Osmótica , Estrés Oxidativo , Cloruro de Sodio/metabolismo , Dodecil Sulfato de Sodio/toxicidad , VirulenciaRESUMEN
The fungal pathogen Candida albicans is a leading causative agent of death in immunocompromised individuals. Many factors have been implicated in virulence including filamentation-inducing transcription factors, adhesins, lipases and proteases. Many of these factors are glycosylphosphatidylinositol-anchored cell surface antigenic determinant proteins. Pga1 is one such protein shown to be upregulated during cell wall regeneration. The purpose of this study was to characterise the role Pga1 plays by creating a homozygous pga1 null strain and comparing the phenotype with the parental strain. It was observed that the mutant strain showed less oxidative stress tolerance and an increased susceptibility to calcofluor white, a cell surface disrupting agent that inhibits chitin fibre assembly which translated as a 40% decrease in cell wall chitin content. Furthermore, the mutant exhibited a 50% reduction in adhesion and a 33% reduction in biofilm formation compared with the parental strain, which was reflected as a slight reduction in virulence. Our data suggest that Pga1 plays an important role in cell wall rigidity and stability. It was also observed that the pga1 null was over filamentous on both liquid and solid media and exhibited increased resistance to SDS suggesting upregulation of filamentation-inducing genes and cell surface components to partially compensate for the deletion.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida albicans/fisiología , Adhesión Celular , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Factores de Virulencia/metabolismo , Animales , Candida albicans/genética , Candidiasis/microbiología , Candidiasis/mortalidad , Candidiasis/patología , Línea Celular Tumoral , Pared Celular/química , Proteínas Fúngicas/genética , Eliminación de Gen , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Análisis de Supervivencia , Virulencia , Factores de Virulencia/genéticaRESUMEN
Vaccination remains the most cost-effective biomedical approach for controlling influenza disease. In times of pandemics, however, these vaccines cannot be produced in sufficient quantities for worldwide use by the current manufacturing capacities and practices. What is needed is the development of adjuvanted vaccines capable of inducing an adequate or better immune response at a decreased antigen dose. Previously we showed that the protein adjuvant rOv-ASP-1 augments influenza-specific antibody titers and survival after virus challenge in both young adult and old-age mice when administered with the trivalent inactivated influenza vaccine (IIV3). In this study we show that a reduced amount of rOv-ASP-1, with 40-times less IIV3 can also induce protection. Apparently the potency of the rOv-ASP-1 adjuvanted IIV3 vaccine is independent of the IIV3-specific Th1/Th2 associated antibody responses, and independent of the presence of HAI antibodies. However, CD4+ T helper cells were indispensable for the protection. Further, rOv-ASP-1 with or without IIV3 elicited the increased level of various chemokines, which are known chemoattractant for immune cells, into the muscle 4â¯h after immunization, and significantly induced the recruitment of monocytes, macrophages and neutrophils into the muscles. The recruited monocytes had higher expression of the activation marker MHCII on their surface as well as CXCR3 and CCR2; receptors for IP-10 and MCP-1, respectively. These results show that the rOv-ASP-1 adjuvant allows substantial antigen sparing of IIV3 by stimulating at the site of injection the accumulation of chemokines and the recruitment of immune cells that can augment the activation of CD4+ T cell immune responses, essential for the production of antibody responses. Protection elicited by the rOv-ASP-1 adjuvanted IIV3 vaccine also appears to function in the absence of MyD88-signaling. Future studies will attempt to delineate the precise mechanisms by which the rOv-ASP-1 adjuvanted IIV3 vaccine works.
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Adyuvantes Inmunológicos/administración & dosificación , Envejecimiento/inmunología , Anticuerpos Antivirales/biosíntesis , Antígenos Helmínticos/administración & dosificación , Proteínas del Helminto/administración & dosificación , Inmunización/métodos , Vacunas contra la Influenza/administración & dosificación , Infecciones por Orthomyxoviridae/prevención & control , Envejecimiento/genética , Animales , Femenino , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/virología , Ratones , Ratones Noqueados , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/virología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inmunología , Músculo Esquelético/virología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Neutrófilos/virología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/virología , Receptores CCR2/genética , Receptores CCR2/inmunología , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Análisis de Supervivencia , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/virología , Carga Viral/efectos de los fármacos , Carga Viral/inmunologíaRESUMEN
Metabolic networks, which are mathematical representations of organismal metabolism, are reconstructed to provide computational platforms to guide metabolic engineering experiments and explore fundamental questions on metabolism. Systems level analyses, such as interrogation of phylogenetic relationships within the network, can provide further guidance on the modification of metabolic circuitries. Chlamydomonas reinhardtii, a biofuel relevant green alga that has retained key genes with plant, animal, and protist affinities, serves as an ideal model organism to investigate the interplay between gene function and phylogenetic affinities at multiple organizational levels. Here, using detailed topological and functional analyses, coupled with transcriptomics studies on a metabolic network that we have reconstructed for C. reinhardtii, we show that network connectivity has a significant concordance with the co-conservation of genes; however, a distinction between topological and functional relationships is observable within the network. Dynamic and static modes of co-conservation were defined and observed in a subset of gene-pairs across the network topologically. In contrast, genes with predicted synthetic interactions, or genes involved in coupled reactions, show significant enrichment for both shorter and longer phylogenetic distances. Based on our results, we propose that the metabolic network of C. reinhardtii is assembled with an architecture to minimize phylogenetic profile distances topologically, while it includes an expansion of such distances for functionally interacting genes. This arrangement may increase the robustness of C. reinhardtii's network in dealing with varied environmental challenges that the species may face. The defined evolutionary constraints within the network, which identify important pairings of genes in metabolism, may offer guidance on synthetic biology approaches to optimize the production of desirable metabolites.
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Evolución Biológica , Chlamydomonas reinhardtii/metabolismo , Redes y Vías Metabólicas , Biología Sintética , Chlamydomonas reinhardtii/genética , Biología Computacional/métodos , Evolución Molecular , Ontología de Genes , Redes Reguladoras de Genes , Genómica/métodos , Sistemas de Lectura Abierta/genética , Biología Sintética/métodosRESUMEN
The increased demand and consumption of fossil fuels have raised interest in finding renewable energy sources throughout the globe. Much focus has been placed on optimizing microorganisms and primarily microalgae, to efficiently produce compounds that can substitute for fossil fuels. However, the path to achieving economic feasibility is likely to require strain optimization through using available tools and technologies in the fields of systems and synthetic biology. Such approaches invoke a deep understanding of the metabolic networks of the organisms and their genomic and proteomic profiles. The advent of next generation sequencing and other high throughput methods has led to a major increase in availability of biological data. Integration of such disparate data can help define the emergent metabolic system properties, which is of crucial importance in addressing biofuel production optimization. Herein, we review major computational tools and approaches developed and used in order to potentially identify target genes, pathways, and reactions of particular interest to biofuel production in algae. As the use of these tools and approaches has not been fully implemented in algal biofuel research, the aim of this review is to highlight the potential utility of these resources toward their future implementation in algal research.
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Biocombustibles/microbiología , Biología Computacional/métodos , Microalgas/metabolismo , Análisis de Flujos Metabólicos , Redes y Vías Metabólicas , Modelos TeóricosRESUMEN
The fungal pathogen Candida albicans is one of the leading causative agents of death in immunocompromised individuals. It harbors an arsenal of cell wall anchored factors that are implicated in virulence such as filamentation inducing factors, adhesins, lipases, proteases, and superoxide dismutases. Dse1 is a cell wall protein involved in cell wall metabolism. The purpose of this study is to characterize the role Dse1 plays in virulence. Dse1 appears to be an essential gene as no homozygous null mutant was possible. The heterozygote mutant exhibited increased susceptibility to calcofluor white, a cell wall disrupting agent, with a subsequent reduction in cell wall chitin content, decreased oxidative stress tolerance, a 30% reduction in biofilm formation, and a delay in adhesion that was mirrored by a reduction in virulence in a mouse model of infection. Dse1 thus appears to be an important protein involved in cell wall integrity and rigidity.