RESUMO
The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (Ki of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency.
Assuntos
3-Fosfoshikimato 1-Carboxiviniltransferase , Glicina , Glifosato , Saccharomyces cerevisiae , 3-Fosfoshikimato 1-Carboxiviniltransferase/genética , 3-Fosfoshikimato 1-Carboxiviniltransferase/metabolismo , Glicina/análogos & derivados , Glicina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Herbicidas/farmacologia , Herbicidas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Resistência a Herbicidas/genéticaRESUMO
OBJECTIVE: Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. METHODS: Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. RESULTS: VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. DISCUSSION: The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20-30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. CONCLUSION: These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids' therapeutic potential against toxicant-triggered autoimmune responses.
Assuntos
Ácidos Graxos Ômega-3 , Glomerulonefrite , Pneumonia , Feminino , Camundongos , Humanos , Animais , Ácidos Graxos Ômega-3/toxicidade , Autoimunidade , Dióxido de Silício/toxicidade , Pneumonia/induzido quimicamente , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Ácidos Docosa-Hexaenoicos/toxicidade , Quimiocinas/toxicidade , Autoanticorpos , Imunoglobulina GRESUMO
Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Microfluídica/métodos , Técnicas Biossensoriais , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorescência , Levodopa/biossíntese , Mutagênese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Yarrowia/genética , Yarrowia/metabolismoRESUMO
Responsive survey design is a technique aimed at improving the efficiency or quality of surveys by using incoming data from the field to make design changes. The technique was pioneered on large national surveys, but the tools can also be applied on the smaller-scale surveys most commonly used by sociologists. We demonstrate responsive survey design in a small-scale, list-based sample survey of students on the topic of sexual misconduct. We investigate the impact of individual incentive levels and a two-phase responsive design with changes to mode of contact as approaches for limiting the potential of nonresponse bias in data from such surveys. Our analyses demonstrate that a two-phase design introducing telephone and face-to-face reminders to complete the survey can produce stronger change in response rates and characteristics of those who respond than higher incentive levels. These findings offer tools for sociologists designing smaller-scale surveys of special populations or sensitive topics.
RESUMO
Thousands of abandoned uranium mines (AUMs) exist in the western United States. Due to improper remediation, windblown dusts generated from AUMs are of significant community concern. A mobile inhalation lab was sited near an AUM of high community concern ("Claim 28") with three primary objectives: to (1) determine the composition of the regional ambient particulate matter (PM), (2) assess meteorological characteristics (wind speed and direction), and (3) assess immunological and physiological responses of mice after exposures to concentrated ambient PM (or CAPs). C57BL/6 and apolipoprotein E-null (ApoE-/-) mice were exposed to CAPs in AirCARE1 located approximately 1 km to the SW of Claim 28, for 1 or 28 days for 4 hr/day at approximately 80 µg/m3 CAPs. Bronchoalveolar lavage fluid (BALF) analysis revealed a significant influx of neutrophils after a single-day exposure in C57BL/6 mice (average PM2.5 concentration = 68 µg/m3). Lungs from mice exposed for 1 day exhibited modest increases in Tnfa and Tgfb mRNA levels in the CAPs exposure group compared to filtered air (FA). Lungs from mice exposed for 28 days exhibited reduced Tgfb (C57BL/6) and Tnfa (ApoE-/-) mRNA levels. Wind direction was typically moving from SW to NE (away from the community) and, while detectable in all samples, uranium concentrations in the PM2.5 fraction were not markedly different from published-reported values. Overall, exposure to CAPs in the region of the Blue GAP Tachee's Claim-28 uranium mine demonstrated little evidence of overt pulmonary injury or inflammation or ambient air contamination attributed to uranium or vanadium.
Assuntos
Poluentes Atmosféricos/toxicidade , Exposição por Inalação/efeitos adversos , Mineração , Material Particulado/toxicidade , Urânio , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Toxicidade Aguda , Testes de Toxicidade SubcrônicaRESUMO
Polyketides represent an extremely diverse class of secondary metabolites often explored for their bioactive traits. These molecules are also attractive building blocks for chemical catalysis and polymerization. However, the use of polyketides in larger scale chemistry applications is stymied by limited titers and yields from both microbial and chemical production. Here, we demonstrate that an oleaginous organism (specifically, Yarrowia lipolytica) can overcome such production limitations owing to a natural propensity for high flux through acetyl-CoA. By exploring three distinct metabolic engineering strategies for acetyl-CoA precursor formation, we demonstrate that a previously uncharacterized pyruvate bypass pathway supports increased production of the polyketide triacetic acid lactone (TAL). Ultimately, we establish a strain capable of producing over 35% of the theoretical conversion yield to TAL in an unoptimized tube culture. This strain also obtained an averaged maximum titer of 35.9 ± 3.9 g/L with an achieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bioreactor fermentation. Additionally, we illustrate that a ß-oxidation-related overexpression (PEX10) can support high TAL production and is capable of achieving over 43% of the theoretical conversion yield under nitrogen starvation in a test tube. Next, through use of this bioproduct, we demonstrate the utility of polyketides like TAL to modify commodity materials such as poly(epichlorohydrin), resulting in an increased molecular weight and shift in glass transition temperature. Collectively, these findings establish an engineering strategy enabling unprecedented production from a type III polyketide synthase as well as establish a route through O-functionalization for converting polyketides into new materials.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Engenharia Genética/métodos , Pironas/metabolismo , Yarrowia/metabolismo , Estrutura Molecular , Oxirredução , Pironas/química , Piruvatos/metabolismo , Yarrowia/genéticaRESUMO
Mice exposed to the air pollutant ozone develop eosinophilic rhinitis that is mediated by group 2, GATA-3+, innate lymphoid cells (ILC2s). In the present study, we determined the influx, persistence, and recall of nasal ILC2s and eosinophils in ozone-exposed mice. C57BL/6 (T/B cell sufficient, ILC sufficient), Rag2-/- (T/B cell deficient, ILC sufficient), and Rag2-/-Il2rg-/- (T/B cell deficient, ILC deficient) mice were exposed to 0 or 0.8 ppm ozone for 1 or 9 weekdays and killed 1 or 17 days postexposure. GATA-3+ lymphocytes were sparse in nasal tissue of air-exposed ILC-sufficient mice and absent in ILC-deficient mice. Nine-day, but not 1-day, ozone exposures induced nasal influxes of eosinophils and GATA-3+ lymphocytes in C57BL/6 and Rag2-/- mice but not in Rag2-/-Il2rg-/- mice. Eosinophils waned 17 days postexposure in ILC-sufficient strains of mice. GATA-3+ lymphocytes in C57BL/6 mice also attenuated after exposure but not in ILC-sufficient Rag2-/- mice. Eosinophils, but not GATA-3+ cells, increased rapidly with reexposure in ILC-sufficient mice. Type 2 immune-related messenger RNA expression correlated with cellular responses to ozone. These new findings in mice further elucidate the role of ILC2s in ozone-induced eosinophilic rhinitis and support epidemiologic associations between ozone exposure and eosinophilic inflammation in children.
Assuntos
Poluentes Atmosféricos/toxicidade , Eosinófilos/imunologia , Linfócitos/imunologia , Mucosa Nasal/efeitos dos fármacos , Ozônio/toxicidade , Animais , Fator de Transcrição GATA3 , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Nasal/imunologia , Oxidantes Fotoquímicos/toxicidade , Rinite/induzido quimicamente , Rinite/imunologiaRESUMO
Rats repeatedly exposed to high airborne concentrations of ethylene develop eosinophilic rhinitis and mucous cell hyperplasia/hypertrophy (MCH) in nasal respiratory epithelium. Mechanisms underlying these lesions are not well understood to inform occupational exposure guidelines. In this study, we determined (1) the nasal histopathology in rats episodically exposed to ethylene, (2) the ethylene-induced nasal histopathology in similarly exposed mice, and (3) how innate lymphoid cells (ILCs) play a role in ethylene-induced MCH. Animals were exposed to 0 or 10,000 ppm ethylene, 6 h/d, 5 d/wk, for 2 weeks and sacrificed 1 day or 2 weeks postexposure. Others received three 2-week exposure blocks separated by 2-week intervals of no exposure. Episodic exposure was chosen to aid in distinguishing irritant from immune responses. Mucous cell hyperplasia/hypertrophy was induced by ethylene in both species. Rats developed a mild, but transient, eosinophilic rhinitis. Mucous cell hyperplasia/hypertrophy was transient in mice, but persistent in rats. Increases in epithelial mucosubstances after 2 weeks of exposure were only present in ILC-sufficient mice, but not in ILC-deficient mice suggesting that ILCs play a role in MCH and overexpression of genes associated with mucus production/secretion. These findings in animals suggest that inhaled ethylene does not act as a sensitizing agent and will not induce allergen-like nasal airway disease.
Assuntos
Exposição por Inalação , Rinite , Animais , Etilenos , Imunidade Inata , Exposição por Inalação/efeitos adversos , Linfócitos , Camundongos , Ratos , Ratos Endogâmicos F344 , Rinite/induzido quimicamenteRESUMO
BACKGROUND: Commercial uranium mining on the Navajo Nation has subjected communities on tribal lands in the Southwestern United States to exposures from residual environmental contamination. Vascular health effects from these ongoing exposures are an active area of study. There is an association between residential mine-site proximity and circulating biomarkers in residents, however, the contribution of mine-site derived wind-blown dusts on vascular and other health outcomes is unknown. To assess neurovascular effects of mine-site derived dusts, we exposed mice using a novel exposure paradigm, the AirCARE1 mobile inhalation laboratory, located 2 km from an abandoned uranium mine, Claim 28 in Blue Gap Tachee, AZ. Mice were exposed to filtered air (FA) (n = 6) or concentrated ambient particulate matter (CAPs) (n = 5) for 2 wks for 4 h per day. RESULTS: To assess miRNA differential expression in cultured mouse cerebrovascular cells following particulate matter (PM) exposure (average: 96.6 ± 60.4 µg/m3 for all 4 h exposures), the serum cumulative inflammatory potential (SCIP) assay was employed. MiRNA sequencing was then performed in cultured mouse cerebrovascular endothelial cells (mCECs) to evaluate transcriptional changes. Results indicated 27 highly differentially expressed (p < 0.01) murine miRNAs, as measured in the SCIP assay. Gene ontology (GO) pathway analysis revealed notable alterations in GO enrichment related to the cytoplasm, protein binding and the cytosol, while significant KEGG pathways involved pathways in cancer, axon guidance and Wnt signaling. Expression of these 27 identified, differentially expressed murine miRNAs were then evaluated in the serum. Nine of these miRNAs (~ 30%) were significantly altered in the serum and 8 of those miRNAs demonstrated the same directional change (either upregulation or downregulation) as cellular miRNAs, as measured in the SCIP assay. Significantly upregulated miRNAs in the CAPs exposure group included miRNAs in the let-7a family. Overexpression of mmu-let-7a via transfection experiments, suggested that this miRNA may mediate mCEC barrier integrity following dust exposure. CONCLUSIONS: Our data suggest that mCEC miRNAs as measured in the SCIP assay show similarity to serum-borne miRNAs, as approximately 30% of highly differentially expressed cellular miRNAs in the SCIP assay were also found in the serum. While translocation of miRNAs via exosomes or an alternative mechanism is certainly possible, other yet-to-be-identified factors in the serum may be responsible for significant miRNA differential expression in endothelium following inhaled exposures. Additionally, the most highly upregulated murine miRNAs in the CAPs exposure group were in the let-7a family. These miRNAs play a prominent role in cell growth and differentiation and based on our transfection experiments, mmu-let-7a may contribute to cerebrovascular mCEC alterations following inhaled dust exposure.
Assuntos
Poluentes Atmosféricos/toxicidade , Material Particulado/toxicidade , Animais , Biomarcadores/sangue , Diferenciação Celular , Proliferação de Células , Endotélio , Exposição por Inalação , Camundongos , MicroRNAs , Sudoeste dos Estados Unidos , UrânioRESUMO
Background: Inhalation exposure to biological particulate matter (BioPM) from livestock farms may provoke exacerbations in subjects suffering from allergy and asthma. The aim of this study was to use a murine model of allergic asthma to determine the effect of BioPM derived from goat farm on airway allergic responses.Methods: Fine (<2.5 µm) BioPM was collected from an indoor goat stable. Female BALB/c mice were ovalbumin (OVA) sensitized and challenged with OVA or saline as control. The OVA and saline groups were divided in sub-groups and exposed intranasally to different concentrations (0, 0.9, 3, or 9 µg) of goat farm BioPM. Bronchoalveolar lavage fluid (BALF), blood and lung tissues were collected.Results: In saline-challenged mice, goat farm BioPM induced 1) a dose-dependent increase in neutrophils in BALF and 2) production of macrophage inflammatory protein-3a. In OVA-challenged mice, BioPM induced 1) inflammatory cells in BALF, 2) OVA-specific Immunoglobulin (Ig)G1, 3) airway mucus secretion-specific gene expression. RNAseq analysis of lungs indicates that neutrophil chemotaxis and oxidation-reduction processes were the representative genomic pathways in saline and OVA-challenged mice, respectively.Conclusions: A single exposure to goat farm BioPM enhanced airway inflammation in both saline and OVA-challenged allergic mice, with neutrophilic response as Th17 disorder and eosinophilic response as Th2 disorder indicative of the severity of allergic responses. Identification of the mode of action by which farm PM interacts with airway allergic pathways will be useful to design potential therapeutic approaches.
Assuntos
Poluentes Atmosféricos/toxicidade , Asma , Cabras , Material Particulado/toxicidade , Doença Aguda , Alérgenos , Animais , Asma/genética , Asma/imunologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Eosinófilos/imunologia , Fazendas , Feminino , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Pulmão/imunologia , Pulmão/patologia , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Ovalbumina , TranscriptomaRESUMO
BACKGROUND: Several instruments intend to measure clinical reasoning capability, yet we lack evidence contextualizing their scores. The authors compared three clinical reasoning instruments [Clinical Reasoning Task (CRT), Patient Note Scoring rubric (PNS), and Summary Statement Assessment Rubric (SSAR)] using Messick's convergent validity framework in pre-clinical medical students. Scores were compared to a validated clinical reasoning instrument, Clinical Data Interpretation (CDI). METHOD: Authors administered CDI and the first clinical case to 235 students. Sixteen randomly selected students (four from each CDI quartile) wrote a note on a second clinical case. Each note was scored with CRT, PNS, and SSAR. Final scores were compared to CDI. RESULTS: CDI scores did not significantly correlate with any other instrument. A large, significant correlation between PNS and CRT was seen (r = 0.71; p = 0.002). CONCLUSIONS: None of the tested instruments outperformed the others when using CDI as a standard measure of clinical reasoning. Differing strengths of association between clinical reasoning instruments suggest they each measure different components of the clinical reasoning construct. The large correlation between CRT and PNS scoring suggests areas of novice clinical reasoning capability, which may not be yet captured in CDI or SSAR, which are weighted toward knowledge synthesis and hypothesis testing.
Assuntos
Educação de Graduação em Medicina , Estudantes de Medicina , Competência Clínica , Raciocínio Clínico , Avaliação Educacional , Humanos , Resolução de Problemas , Reprodutibilidade dos TestesRESUMO
Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Biblioteca Gênica , Genes Fúngicos , Yarrowia/genéticaRESUMO
Epidemiological associations have been made between the new onset of childhood rhinitis/asthma and exposures to elevated ambient levels of ozone, a commonly encountered gaseous air pollutant. Our laboratory was the first to find that mice repeatedly exposed to ozone develop nasal type 2 immunity and eosinophilic rhinitis with mucous cell metaplasia. More recently, we have found that these ozone-induced upper airway alterations are mediated by group 2 innate lymphoid cells (ILC2s) and not by T and B cells that are important in adaptive immune responses typically associated with allergic rhinitis and asthma. Furthermore, repeated exposures of mice to ozone cause ILC2-mediated type 2 immunity and airway pathology in the lungs, like those found in the nasal airways. Our recent findings in ozone-exposed mice complement and extend previous reports of nonallergic nasal airway disease in ozone-exposed rats and nonhuman primates. Overall, these experimental results in laboratory animals suggest a plausible ILC2-dependent paradigm for the toxicologic pathobiology that underlies the development of nonallergic rhinitis/asthma in children who live in environments with repeated occurrences of high ambient concentrations of ozone.
Assuntos
Poluentes Atmosféricos/toxicidade , Imunidade Inata/efeitos dos fármacos , Exposição por Inalação/efeitos adversos , Linfócitos/efeitos dos fármacos , Ozônio/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Animais , Humanos , Linfócitos/imunologia , Linfócitos/patologia , Metaplasia , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Doenças Respiratórias/induzido quimicamente , Doenças Respiratórias/imunologia , Doenças Respiratórias/patologia , Especificidade da EspécieRESUMO
BACKGROUND: Engineered nanoparticles (NPs) have been shown to enhance allergic airways disease in mice. However, the influence of the different physicochemical properties of these particles on their adjuvant properties is largely unknown. Here we investigate the effects of chemical composition and redox activity of poorly soluble NPs on their adjuvant potency in a mouse model of airway hypersensitivity. RESULTS: NPs of roughly similar sizes with different chemical composition and redox activity, including CeO2, Zr-doped CeO2, Co3O4, Fe-doped Co3O4(using Fe2O3 or Fe3O4) and TiO2 NPs, all showed adjuvant activity. OVA induced immune responses following intranasal exposure of BALB/c mice to 0.02% OVA in combination with 200 µg NPs during sensitization (on day 1, 3, 6 and 8) and 0.5% OVA only during challenge (day 22, 23 and 24) were more pronounced compared to the same OVA treatment regime without NPs. Changes in OVA-specific IgE and IgG1 plasma levels, differential cell count and cytokines in bronchoalveolar lavage fluid (BALF), and histopathological detection of mucosa cell metaplasia and eosinophil density in the conducting airways were observed. Adjuvant activity of the CeO2 NPs was primarily mediated via the Th2 response, while that of the Co3O4 NPs was characterised by no or less marked increases in IgE plasma levels, BALF IL-4 and IL-5 concentrations and percentages of eosinophils in BALF and more pronounced increases in BALF IL-6 concentrations and percentages of lymphocytes in BALF. Co-exposure to Co3O4 NPs with OVA and subsequent OVA challenge also induced perivascular and peribronchiolar lymphoid cell accumulation and formation of ectopic lymphoid tissue in lungs. Responses to OVA combined with various NPs were not affected by the amount of doping or redox activity of the NPs. CONCLUSIONS: The findings indicate that chemical composition of NPs influences both the relative potency of NPs to exacerbate allergic airway sensitization and the type of immune response. However, no relation between the acellular redox activity and the observed adjuvant activity of the different NPs was found. Further research is needed to pinpoint the precise physiological properties of NPs and biological mechanisms determining adjuvant activity in order to facilitate a safe-by-design approach to NP development.
Assuntos
Pulmão/efeitos dos fármacos , Nanoestruturas/química , Nanoestruturas/toxicidade , Hipersensibilidade Respiratória/induzido quimicamente , Administração Intranasal , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interleucinas/análise , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Oxirredução , Hipersensibilidade Respiratória/sangue , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , SolubilidadeRESUMO
Ionic liquids show promise for deconstruction of lignocellulosic biomass prior to fermentation. Yet, imidazolium ionic liquids (IILs) can be toxic to microbes even at concentrations present after recovery. Here, we show that dominant overexpression of an Ilt1p homolog (encoded by YlILT1/YALI0C04884) from the IIL-tolerant yeast Yarrowia lipolytica confers an improvement in 1-ethyl-3-methylimidazolium acetate tolerance in Saccharomyces cerevisiae compared to the endogenous Ilt1p (ScILT1/YDR090C). We subsequently enhance tolerance in S. cerevisiae through directed evolution of YlILT1 using growth-based selection, leading to identification of mutants that grow in up to 3.5% v/v ionic liquid. Lastly, we demonstrate that strains expressing YlILT1 variants demonstrate improved growth rate and ethanol production in the presence of residual IIL. This shows that dominant overexpression of a heterologous protein (wild type or evolved) from an IIL-tolerant yeast can increase tolerance in S. cerevisiae at concentrations relevant to bioethanol production from IIL-treated biomass.
Assuntos
Imidazóis/farmacologia , Líquidos Iônicos/farmacologia , Saccharomyces cerevisiae/metabolismo , Yarrowia/metabolismo , Biomassa , Etanol/metabolismo , Evolução Molecular , Fermentação , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Yarrowia/genéticaRESUMO
Social processes that change quickly are difficult to study, because they require frequent survey measurement. Weekly, daily, or even hourly measurement may be needed depending on the topic. With more frequent measurement comes the prospect of more complex patterns of missing data. The mechanisms creating the missing data may be varied, ranging from technical issues such as lack of an Internet connection to refusal to complete a requested survey. We examine one approach to mitigating the damage of these missing data - a follow-up or closeout interview that is completed after the frequent measurement. The Relationship Dynamics and Social Life (RDSL) study used this approach. The study asked women weekly about their attitudes and behaviors related to sexual relationships and pregnancy. The surveys were carried out for 130 weeks and concluded with a closeout interview. We explore the patterns of missing data in the RDSL study and then examine associations between the data collected in the closeout survey and key variables collected in the weekly survey. We then assess the extent to which data from the closeout survey are useful in repairing the damage caused by missing data.
Assuntos
Confiabilidade dos Dados , Coleta de Dados/normas , Seguimentos , Entrevistas como Assunto/normas , Estudos Longitudinais , Projetos de Pesquisa/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Coleta de Dados/estatística & dados numéricos , Feminino , Humanos , Entrevistas como Assunto/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Projetos de Pesquisa/estatística & dados numéricosRESUMO
Evolutionary approaches to strain engineering inherently require the identification of suitable selection techniques for the product and phenotype of interest. In this work, we undertake a comparative analysis of two related but functionally distinct methods of high-throughput screening: traditional single cell fluorescence activated cell sorting (single cell FACS) and microdroplet-enabled FACS (droplet FACS) using water/oil/water (w/o/w) emulsions. To do so, we first engineer and evolve the non-conventional yeast Yarrowia lipolytica for high extracellular production of riboflavin (vitamin B2), an innately fluorescent product. Following mutagenesis and adaptive evolution, a direct parity-matched comparison of these two selection strategies was conducted. Both single cell FACS and droplet FACS led to significant increases in total riboflavin titer (32 and 54 fold relative to the parental PO1f strain, respectively). However, single cell FACS favored intracellular riboflavin accumulation (with only 70% of total riboflavin secreted) compared with droplet FACS that favored extracellular product accumulation (with 90% of total riboflavin secreted). We find that for the test case of riboflavin, the extent of secretion and total production were highly correlated. The resulting differences in production modes and levels clearly demonstrate the significant impact that selection approaches can exert on final evolutionary outcomes in strain engineering. Moreover, we note that these results provide a cautionary tale when intracellular read-outs of product concentration (including signals from biosensors) are used as surrogates for total production of potentially secreted products. In this regard, these results demonstrate that extracellular production is best assayed through an encapsulation technique when performing high throughput screening.
Assuntos
Citometria de Fluxo , Riboflavina , Yarrowia , Evolução Molecular Direcionada/métodos , Mutagênese , Riboflavina/biossíntese , Riboflavina/genética , Yarrowia/citologia , Yarrowia/genética , Yarrowia/metabolismoRESUMO
Challenges to survey data collection have increased the costs of social research via face-to-face surveys so much that it may become extremely difficult for social scientists to continue using these methods. A key drawback to less expensive Internet-based alternatives is the threat of biased results from coverage errors in survey data. The rise of Internet-enabled smartphones presents an opportunity to re-examine the issue of Internet coverage for surveys and its implications for coverage bias. Two questions (on Internet access and smartphone ownership) were added to the National Survey of Family Growth (NSFG), a U.S. national probability survey of women and men age 15-44, using a continuous sample design. We examine 16 quarters (4 years) of data, from September 2012 to August 2016. Overall, we estimate that 82.9% of the target NSFG population has Internet access, and 81.6% has a smartphone. Combined, this means that about 90.7% of U.S. residents age 15-44 have Internet access, via either traditional devices or a smartphone. We find some evidence of compensatory coverage when looking at key race/ethnicity and age subgroups. For instance, while Black teens (15-18) have the lowest estimated rate of Internet access (81.9%) and the lowest rate of smartphone usage (72.6%), an estimated 88.0% of this subgroup has some form of Internet access. We also examine the socio-demographic correlates of Internet and smartphone coverage, separately and combined, as indicators of technology access in this population. In addition, we look at the effect of differential coverage on key estimates produced by the NSFG, related to fertility, family formation, and sexual activity. While this does not address nonresponse or measurement biases that may differ for alternative modes, our paper has implications for possible coverage biases that may arise when switching to a Web-based mode of data collection, either for follow-up surveys or to replace the main face-to-face data collection.
RESUMO
Ozone is an irritating gas found in photochemical smog. Epidemiological associations have been made between the onset of asthma and childhood exposures to increasing levels of ambient ozone (i.e., air pollutant-induced nonatopic asthma). Individuals, however, vary in their susceptibility to this outdoor air pollutant, which may be due, in part, to their genetic makeup. The present study was designed to test the hypothesis that there are murine strain-dependent differences in pulmonary and nasal pathologic responses to repeated ozone exposures. C57BL/6NTac and BALB/cNTac mice were exposed to 0 or 0.8 ppm ozone, 4 hr/day, for 9 consecutive weekdays. In both strains of mice, ozone induced eosinophilic inflammation and mucous cell metaplasia in the nasal and pulmonary airways. Lungs of ozone-exposed C57BL/6NTac mice, however, had greater eosinophilic inflammation, mucous cell metaplasia, and expression of genes related to type 2 immunity and airway mucus hypersecretion, as compared to similarly exposed BALB/cNTac mice. Ozone-exposed C57BL/6NTac mice also had greater eosinophilic rhinitis but a similar degree of mucous cell metaplasia in nasal epithelium, as ozone-exposed BALB/cNTac mice. These findings suggest that nonatopic individuals may differ in their inflammatory and epithelial responses to repeated ozone exposures that are due, in part, to genetic factors.
Assuntos
Poluentes Atmosféricos/toxicidade , Asma/induzido quimicamente , Imunidade nas Mucosas/efeitos dos fármacos , Ozônio/toxicidade , Mucosa Respiratória/efeitos dos fármacos , Rinite/induzido quimicamente , Animais , Asma/imunologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Modelos Animais de Doenças , Eosinófilos/citologia , Expressão Gênica/efeitos dos fármacos , Imunidade nas Mucosas/genética , Masculino , Metaplasia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Rinite/imunologia , Rinite/patologia , Especificidade da EspécieRESUMO
Exposure to elevated levels of ambient ozone in photochemical smog is associated with eosinophilic airway inflammation and nonatopic asthma in children. In the present study, we determined the role of innate lymphoid cells (ILCs) in the pathogenesis of ozone-induced nonatopic asthma by using lymphoid cell-sufficient C57BL/6 mice, ILC-sufficient Rag2-/- mice (devoid of T and B cells), and ILC-deficient Rag2-/-Il2rg-/- mice (depleted of all lymphoid cells including ILCs). Mice were exposed to 0 or 0.8 parts per million ozone for 1 day or 9 consecutive weekdays (4 hr/day). A single exposure to ozone caused neutrophilic inflammation, airway epithelial injury, and reparative DNA synthesis in all strains of mice, irrespective of the presence or absence of ILCs. In contrast, 9-day exposures induced eosinophilic inflammation and mucous cell metaplasia only in the lungs of ILC-sufficient mice. Repeated ozone exposures also elicited increased messenger RNA expression of transcripts associated with type 2 immunity and airway mucus production in ILC-sufficient mice. ILC-deficient mice repeatedly exposed to ozone had no pulmonary pathology or increased gene expression related to type 2 immunity. These results suggest a new paradigm for the biologic mechanisms underlying the development of a phenotype of childhood nonatopic asthma that has been linked to ambient ozone exposures.