RESUMO
The modern semiconductor industry relies heavily on a process known as chemical mechanical planarization, which uses physical and chemical processes to remove excess material from the surface of silicon wafers during microchip fabrication. This process results in large volumes of wastewater containing dissolved metals including copper (Cu(2+)), which must then be filtered and treated before release into municipal waste systems. We have investigated the potential use of bacterial and fungal biomass as an alternative to the currently used ion-exchange resins for the adsorption of dissolved Cu(2+) from high-throughput industrial waste streams. A library of candidate microorganisms, including Lactobacillus casei and Pichia pastoris, was screened for ability to bind Cu(2+) from solution and to form static biofilm communities within packed-bed adsorption columns. The binding efficiency of these biomass-based adsorption columns was assessed under various flow conditions and compared to that of industrially used ion-exchange resins. We demonstrated the potential to regenerate the biomass within the adsorption columns through the use of a hydrochloric acid wash, and subsequently reuse the columns for additional copper binding. While the binding efficiency and capacity of the developed L. casei/P. pastoris biomass filters was inferior to ion-exchange resin, the potential for repeated reuse of these filters, coupled with the advantages of a more sustainable "green" adsorption process, make this technique an attractive candidate for use in industrial-scale CMP wastewater treatment.
Assuntos
Cobre/química , Manufaturas , Silício , Poluentes Químicos da Água/química , Adsorção , Biodegradação Ambiental , Biomassa , Cátions/química , Humanos , Resíduos IndustriaisRESUMO
Modern microbiological research has increasingly focused on the interactions between bacterial cells and the surfaces that they inhabit. To this end, microfluidic devices have played a large role in enabling research of cell-surface interactions, especially surface attachment and biofilm formation. This review provides background on microfluidic devices and their use in biological systems, as well specific examples from current literature. Methods to observe and interrogate cells within microfluidic devices are described, as well as the analytical techniques that are used to collect these data.
Assuntos
Aderência Bacteriana , Microfluídica , BiofilmesRESUMO
Due to extensive use in consumer goods, it is important to understand the genotoxicity of silver nanoparticles (AgNPs) and identify susceptible populations. 8-Oxoguanine DNA glycosylase 1 (OGG1) excises 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), a pro-mutagenic lesion induced by oxidative stress. To understand whether defects in OGG1 is a possible genetic factor increasing an individual's susceptibly to AgNPs, we determined DNA damage, genome rearrangements, and expression of DNA repair genes in Ogg1-deficient and wild type mice exposed orally to 4 mg/kg of citrate-coated AgNPs over a period of 7 d. DNA damage was examined at 3 and 7 d of exposure and 7 and 14 d post-exposure. AgNPs induced 8-oxoG, double strand breaks (DSBs), chromosomal damage, and DNA deletions in both genotypes. However, 8-oxoG was induced earlier in Ogg1-deficient mice and 8-oxoG levels were higher after 7-d treatment and persisted longer after exposure termination. AgNPs downregulated DNA glycosylases Ogg1, Neil1, and Neil2 in wild type mice, but upregulated Myh, Neil1, and Neil2 glycosylases in Ogg1-deficient mice. Neil1 and Neil2 can repair 8-oxoG. Thus, AgNP-mediated downregulation of DNA glycosylases in wild type mice may contribute to genotoxicity, while upregulation thereof in Ogg1-deficient mice could serve as an adaptive response to AgNP-induced DNA damage. However, our data show that Ogg1 is indispensable for the efficient repair of AgNP-induced damage. In summary, citrate-coated AgNPs are genotoxic in both genotypes and Ogg1 deficiency exacerbates the effect. These data suggest that humans with genetic polymorphisms and mutations in OGG1 may have increased susceptibility to AgNP-mediated DNA damage.
Assuntos
Dano ao DNA/genética , DNA Glicosilases/genética , Reparo do DNA/genética , Expressão Gênica/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Animais , Regulação para Baixo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The ability to characterize the microscale mechanical properties of biological materials has the potential for great utility in the field of tissue engineering. The development and morphogenesis of mammalian tissues are known to be guided in part by mechanical stimuli received from the local environment, and tissues frequently develop to match the physical characteristics (i.e., elasticity) of their environment. Quantification of these material properties at the microscale may provide valuable information to guide researchers. Presented here is a microfluidic platform for the non-destructive ex vivo microscale mechanical characterization of mammalian tissue samples by atomic force microscopy (AFM). The device was designed to physically hold a tissue sample in a dynamically controllable fluid environment while allowing access by an AFM probe operating in force spectroscopy mode to perform mechanical testing. Results of measurements performed on mouse submandibular gland samples demonstrate the ability of the analysis platform to quantify sample elasticity at the microscale, and observe chemically-induced changes in elasticity.
RESUMO
Substrate compliance is reported to alter cell phenotype, but little is known about the effects of compliance on cell development within the context of a complex tissue. In this study, we used 0.48 and 19.66 kPa polyacrylamide gels to test the effects of the substrate modulus on submandibular salivary gland development in culture and found a significant decrease in branching morphogenesis in explants grown on the stiff 19.66 kPa gels relative to those grown on the more physiologically compliant 0.48 kPa gels. While proliferation and apoptosis were not affected by the substrate modulus, tissue architecture and epithelial acinar cell differentiation were profoundly perturbed by aberrant, high stiffness. The glands cultured on 0.48 kPa gels were similar to developing glands in morphology and expression of the differentiation markers smooth muscle alpha-actin (SM α-actin) in developing myoepithelial cells and aquaporin 5 (AQP5) in proacinar cells. At 19.66 kPa, however, tissue morphology and the expression and distribution of SM α-actin and AQP5 were disrupted. Significantly, aberrant gland development at 19.66 kPa could be rescued by both mechanical and chemical stimuli. Transfer of glands from 19.66 to 0.48 kPa gels resulted in substantial recovery of acinar structure and differentiation, and addition of exogenous transforming growth factor beta 1 at 19.66 kPa resulted in a partial rescue of morphology and differentiation within the proacinar buds. These results indicate that environmental compliance is critical for organogenesis, and suggest that both mechanical and chemical stimuli can be exploited to promote organ development in the contexts of tissue engineering and organ regeneration.
Assuntos
Materiais Biocompatíveis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Morfogênese/efeitos dos fármacos , Glândulas Salivares/crescimento & desenvolvimento , Alicerces Teciduais/química , Células Acinares/citologia , Células Acinares/efeitos dos fármacos , Células Acinares/metabolismo , Resinas Acrílicas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Complacência (Medida de Distensibilidade)/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Camundongos , Fenótipo , Glândulas Salivares/citologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Viable methods for bacterial biofilm remediation require a fundamental understanding of biofilm mechanical properties and their dependence on dynamic environmental conditions. Mechanical test data, such as elasticity or adhesion, can be used to perform physical modelling of biofilm behaviour, thus enabling the development of novel remediation strategies. To achieve real-time, dynamic measurements of these properties, a novel microfluidic flowcell device has been designed and fabricated for in situ analysis using atomic force microscopy (AFM). The flowcell consists of microfluidic channels for biofilm establishment that are then converted into an open architecture, laminar flow channel for AFM measurement in a liquid environment. Finite element analysis (FEA) was used to profile fluid conditions within the flowcell during biofilm establishment. Force-mode AFM was used to measure the elastic properties of mature Pseudomonas aeruginosa PAO1 biofilms as well as polyacrylamide hydrogels. Elastic moduli ranging from 0.58 to 2.61kPa were determined for the mature biofilm, which fall within the range of moduli previously reported by optical, rheometric, and microindentation techniques. These results demonstrate the validity of the microfluidic flowcell system as an effective platform for future investigations of biofilm mechanical and morphological response to dynamic environmental conditions.
Assuntos
Biofilmes/crescimento & desenvolvimento , Técnicas Analíticas Microfluídicas/métodos , Pseudomonas aeruginosa/fisiologia , Aderência Bacteriana , Fenômenos Biomecânicos , Elasticidade , Microscopia de Força Atômica/métodosRESUMO
Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.
Assuntos
Biofilmes/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/fisiologia , Compostos de Enxofre/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Drosophila melanogaster , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/metabolismo , Compostos de Enxofre/farmacologia , Transativadores/genética , Transativadores/metabolismoRESUMO
Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-l-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland.