The in vitro selection world.

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.

Deletion of the Candida albicans PIR32 results in increased virulence, stress response, and upregulation of cell wall chitin deposition.

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.

Characterisation of Pga1, a putative Candida albicans cell wall protein necessary for proper adhesion and biofilm formation.

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.

The parasite-derived rOv-ASP-1 is an effective antigen-sparing CD4+ T cell-dependent adjuvant for the trivalent inactivated influenza vaccine, and functions in the absence of MyD88 pathway.

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 4 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.

Systems level analysis of the Chlamydomonas reinhardtii metabolic network reveals variability in evolutionary co-conservation.

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.

Computational approaches for microalgal biofuel optimization: a review.

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.

The Candida albicans Dse1 Protein Is Essential and Plays a Role in Cell Wall Rigidity, Biofilm Formation, and Virulence.

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.