Genetic selection scheme for isolation of signal transduction pathway mutants

Genetic characterization of a signal transduction pathway requires the isolation of mutations in the pathway. Characterization of these mutated genes and their loci enumerates the components of the pathway and leads to an understanding of the role of each gene locus in the pathway under study. We have designed and developed a strategy based on resistance to the chemical flucytosine for the identification of mutations in a given pathway. In this study, the Escherichia coli codA gene, which encodes the enzyme cytosine deaminase, was fused to the light-intensity-regulated gene promoter psbDII. Cytosine deaminase converts 5'-fluorocytosine to the toxic product 5-fluorouracil. Wild-type cells containing an intact signal transduction pathway that regulates the psbDII promoter will die in the presence of this chemical. Cells that carry mutations in the pathway that inactivate the psbDII promoter will not express the codA gene and, consequently, will live on 5'-fluorocytosine, allowing the isolation and subsequent characterization of mutations in this signaling pathway. Utilizing this selection method, we have successfully isolated and characterized mutations in the psbDII pathway. This selection scheme can be used with a tissue-specific or phase-specific promoter fused to the codA gene to direct the timing of expression of codA to obtain mutants defective in temporal or cell-specific expression of a particular pathway. This scheme also allows the isolation of mutants even when a clearly identifiable phenotype is not available. The selection scheme presented here extends the molecular tools available for the genetic dissection of signal transduction pathways.     

Microbial recognition via Toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.     

Genetic Selection Scheme for Isolation of Signal Transduction Pathway Mutants

Genetic characterization of a signal transduction pathway requires the isolation of mutations in the pathway. Characterization of these mutated genes and their loci enumerates the components of the pathway and leads to an understanding of the role of each gene locus in the pathway under study. We have designed and developed a strategy based on resistance to the chemical flucytosine for the identification of mutations in a given pathway. In this study, the Escherichia coli codA gene, which encodes the enzyme cytosine deaminase, was fused to the light-intensity-regulated gene promoter psbDII. Cytosine deaminase converts 5′-fluorocytosine to the toxic product 5-fluorouracil. Wild-type cells containing an intact signal transduction pathway that regulates the psbDII promoter will die in the presence of this chemical. Cells that carry mutations in the pathway that inactivate the psbDII promoter will not express the codA gene and, consequently, will live on 5′-fluorocytosine, allowing the isolation and subsequent characterization of mutations in this signaling pathway. Utilizing this selection method, we have successfully isolated and characterized mutations in the psbDII pathway. This selection scheme can be used with a tissue-specific or phase-specific promoter fused to the codA gene to direct the timing of expression of codA to obtain mutants defective in temporal or cell-specific expression of a particular pathway. This scheme also allows the isolation of mutants even when a clearly identifiable phenotype is not available. The selection scheme presented here extends the molecular tools available for the genetic dissection of signal transduction pathways.     

Indoor-Biofilter Growth and Exposure to Airborne Chemicals Drive Similar Changes in Plant Root Bacterial Communities

Due to the long durations spent inside by many humans, indoor air quality has become a growing concern. Biofiltration has emerged as a potential mechanism to clean indoor air of harmful volatile organic compounds (VOCs), which are typically found at concentrations higher indoors than outdoors. Root-associated microbes are thought to drive the functioning of plant-based biofilters, or biowalls, converting VOCs into biomass, energy, and carbon dioxide, but little is known about the root microbial communities of such artificially grown plants, how or whether they differ from those of plants grown in soil, and whether any changes in composition are driven by VOCs. In this study, we investigated how bacterial communities on biofilter plant roots change over time and in response to VOC exposure. Through 16S rRNA amplicon sequencing, we compared root bacterial communities from soil-grown plants with those from two biowalls, while also comparing communities from roots exposed to clean versus VOC-laden air in a laboratory biofiltration system. The results showed differences in bacterial communities between soil-grown and biowall-grown plants and between bacterial communities from plant roots exposed to clean air and those from VOC-exposed plant roots. Both biowall-grown and VOC-exposed roots harbored enriched levels of bacteria from the genus Hyphomicrobium. Given their known capacities to break down aromatic and halogenated compounds, we hypothesize that these bacteria are important VOC degraders. While different strains of Hyphomicrobium proliferated in the two studied biowalls and our lab experiment, strains were shared across plant species, suggesting that a wide range of ornamental houseplants harbor similar microbes of potential use in living biofilters.     

Characterization of a novel collagen‐like protein TrpA in the cyanobacterium Trichodesmium erythraeum IMS101

The collagen protein family is diverse and its membership is continually expanding as new collagen‐like molecules are identified. Identification of collagen in unicellular eukaryotes and prokaryotes has opened discussion on the function of these collagens and their role in the emergence of multicellularity. The previous identification of a collagen gene in Trichodesmium erythraeum raises the question of function of this structural protein in a prokaryote. In this study, we show that this gene is expressed during all phases of growth, indicating that it may be required for all phases of growth. Using immunofluorescence techniques, we demonstrate that the collagen‐like protein is localized in a specific manner between adjacent cells along the trichome of T. erythraeum. Trichomes treated with the enzyme collagenase exhibited fragmentation, supporting our immunofluorescence localization data that this collagen‐like protein is found between adjacent cells. Our data strongly suggest that the collagen‐like protein found in T. erythraeum functions to maintain the structural integrity of the trichome through the adhesion of adjacent cells.     

Susceptibility of mouse minute virus to inactivation by heat in two cell culture media types

Viral contaminations of biopharmaceutical manufacturing cell culture facilities are a significant threat and one for which having a risk mitigation strategy is highly desirable. High temperature, short time (HTST) mammalian cell media treatment may potentially safeguard manufacturing facilities from such contaminations. HTST is thought to inactivate virions by denaturing proteins of the viral capsid, and there is evidence that HTST provides ample virucidal efficacy against nonenveloped or naked viruses such as mouse minute virus (MMV), a parvovirus. The aim of the studies presented herein was to further delineate the susceptibility of MMV, known to have contaminated mammalian cell manufacturing facilities, to heat by exposing virus‐spiked cell culture media to a broad range of temperatures and for various times of exposure. The results of these studies show that HTST is capable of inactivating MMV by three orders of magnitude or more. Thus, we believe that HTST is a useful technology for the purposes of providing a barrier to adventitious contamination of mammalian cell culture processes in the biopharmaceutical industry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non‐infectious retrovirus‐like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO^? and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV. Biotechnol. Bioeng. 2012;109: 157–165. © 2011 Wiley Periodicals, Inc.