RESUMO
In this study, we report an oral drug delivery system without any additional process using pH-sensitive biopolymer, alginate, and alginate oligosaccharide with lysozyme as a model drug. These biopolymers exhibited pH-sensitive characteristics such as shrinking at acidic pH and eroding with dissolution at alkaline pH. The incorporation of lysozyme and biopolymers was performed an artificial intestinal juice (pH 6.8). The immobilization efficiency and lysozyme stability in gastric juice (pH 1.2) has been tested by E coil antimicrobial activity. The lysozyme without biopolymer immobilization lost approximately 80-90% of antimicrobial activity than that of pure lysozyme. However, the pH-sensitive biopolymer-controlled lysozyme maintained similar antimicrobial activity compared to that of pure lysozyme (50-90% of cell mortality). Therefore, this simple, easy, and rapid system can be effectively and practically applied for pathogen treatment for in vivo oral drug delivery.
Assuntos
Alginatos , Anti-Infecciosos , Sistemas de Liberação de Medicamentos , Escherichia coli K12/crescimento & desenvolvimento , Muramidase , Oligossacarídeos , Alginatos/química , Alginatos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Suco Gástrico/química , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Concentração de Íons de Hidrogênio , Muramidase/química , Muramidase/farmacologia , Oligossacarídeos/química , Oligossacarídeos/farmacologiaRESUMO
Having the ability to coordinate the behavior of stem cells to induce regeneration of specific large-scale structures would have far-reaching consequences in the treatment of degenerative diseases, acute injury, and aging. Thus, identifying and learning to manipulate the sequential steps that determine the fate of new tissue within the overall morphogenetic program of the organism is fundamental. We identified novel early signals, mediated by the central nervous system and 3 innexin proteins, which determine the fate and axial polarity of regenerated tissue in planarians. Modulation of gap junction-dependent and neural signals specifically induces ectopic anterior regeneration blastemas in posterior and lateral wounds. These ectopic anterior blastemas differentiate new brains that establish permanent primary axes re-established during subsequent rounds of unperturbed regeneration. These data reveal powerful novel controls of pattern formation and suggest a constructive model linking nervous inputs and polarity determination in early stages of regeneration.
Assuntos
Polaridade Celular , Conexinas/fisiologia , Planárias/fisiologia , Regeneração , Animais , Sistema Nervoso Central/embriologia , Hibridização In SituRESUMO
We have successfully developed optically coded functional microbeads by co-encapsulating both bioluminescent reporter bacterial cells and fluorescent microspheres within a common alginate microbead. These microbeads harboring an individual self-identification code using fluorescent microspheres could be randomly scattered on any multi-well chip plate as long as the size of the microbeads are made to fit on it with the result that, since cell types are identified on the basis of fluorescent color, microbead arrays were fabricated without pre-designation of an individual well. As an example of this method, five different stress specific bioluminescent bacterial strains, each with a different optical code, were successfully implemented to make five different types of optically coded functional microbeads, with a speed of about 30 microbeads/min. Each functional microbead has a specific stress-specific bacterial strain and, as an identification optical code, one of five optical codes generated from fluorescence microspheres such as yellow, green, red, yellow + green, or no fluorescence. This final randomly scattered functional microbeads array biochip, with a fast fabrication of each chip at every 2 min, successfully demonstrated its ability in toxicity screening and monitoring for samples with a few examples for five different stress chemicals. This simple and fast, but not tedious and complicated procedure should be widely and practically used in making cell array chips for the monitoring of environmental toxicity, new-borne chemicals, pharmaceutical drugs and cosmic rays in the space station or spaceships in future.
Assuntos
Bioensaio/instrumentação , Citometria de Fluxo/instrumentação , Análise em Microsséries/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Dispositivos Ópticos , Espectrometria de Fluorescência/instrumentação , Testes de Toxicidade/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Microesferas , Coloração e RotulagemRESUMO
In this study, we aim to identify a common, general mode of toxic action in Escherichia coli when experiencing DNA damage, irrespective of the agents used. We conducted or collected 69 microarray data from seven different DNA damaging agents. In a quantitative manner, we constructed a probable DNA damage stress network, entitled the 'Functional Linked Network (FLN)', which consists of 399 significantly perturbed genes and the 1283 interactions among them. The SOS response related genes (LexA modules) were found to be dominantly activated by DNA damage, irrespective of the agents. Several minor, plausible modules were also implicated in this network, and appear to be related with the metabolic inhibition response to DNA damage or mediate the induction of SOS response. This systems and comparison approach across a variety of genotoxic agents may serve as a starting point to specify some of the unknown and common features of DNA damage responses in bacteria.
Assuntos
Dano ao DNA/fisiologia , Escherichia coli/fisiologia , Redes Reguladoras de Genes/genética , Mutagênicos/toxicidade , Resposta SOS em Genética/fisiologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Análise em Microsséries , Resposta SOS em Genética/efeitos dos fármacos , Resposta SOS em Genética/genéticaRESUMO
The promoter of nrdA gene which is related with DNA synthesis was used to construct a DNA damage sensitive biosensor. A recombinant bioluminescent E. coli strain, BBTNrdA, harboring a plasmid with the nrdA promoter fused to the luxCDABE operon, was successfully constructed. Its response to various chemicals including genotoxic chemicals substantiates it as a DNA damage biosensor. In characterization, three different classes of toxicants were used: DNA damaging chemicals, oxidative stress chemicals, and phenolics. BBTNrdA only responded strongly to DNA damaging chemicals, such as nalidixic acid (NDA), mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), and 4-nitroquinoline N-oxide (4-NQO). In contrast, there were no responses from the oxidative stress chemicals and phenolics, except from hydrogen peroxide (H2O2) which is known to cause DNA damage indirectly. Therefore, the results of the study demonstrate that BBTNrdA can be used as a DNA damage biosensor.
RESUMO
In this study, we developed lysosome-alginate beads for application as an oral drug delivery system (ODDS). The beads harboring lysosomes, which have antimicrobial activity, and various concentrations of alginate were characterized and optimized. For application as an ODDS, pH-dependent lysosome-alginate beads were generated, and the level of lysosome release was investigated by using antimicrobial tests. At low pH, lysosomes were not released from the lysosome-alginate beads; however, at neutral pH, similar to the pH in the intestine, lysosome release was confirmed, as determined by a high antimicrobial activity. This study shows the potential of such an ODDS for the in vivo treatment of infection with pathogens.
Assuntos
Alginatos/administração & dosagem , Antibacterianos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Lisossomos/metabolismo , Microesferas , Administração Oral , Antibacterianos/química , Escherichia coli K12/efeitos dos fármacos , Ácido Glucurônico/administração & dosagem , Ácidos Hexurônicos/administração & dosagem , Concentração de Íons de HidrogênioRESUMO
We recently reported that the tetra(ethylene glycol) derivative of benzothiazole aniline, BTA-EG4, acts as an amyloid-binding small molecule that promotes dendritic spine density and cognitive function in wild-type mice. This raised the possibility that BTA-EG4 may benefit the functional decline seen in Alzheimer's disease (AD). In the present study, we directly tested whether BTA-EG4 improves dendritic spine density and cognitive function in a well-established mouse model of AD carrying mutations in APP, PS1 and tau (APPswe;PS1M146V;tauP301L, 3xTg AD mice). We found that daily injections of BTA-EG4 for 2 weeks improved dendritic spine density and cognitive function of 3xTg AD mice in an age-dependent manner. Specifically, BTA-EG4 promoted both dendritic spine density and morphology alterations in cortical layers II/III and in the hippocampus at 6-10 months of age compared to vehicle-injected mice. However, at 13-16 months of age, only cortical spine density was improved without changes in spine morphology. The changes in dendritic spine density correlated with Ras activity, such that 6-10 month old BTA-EG4 injected 3xTg AD mice had increased Ras activity in the cortex and hippocampus, while 13-16 month old mice only trended toward an increase in Ras activity in the cortex. Finally, BTA-EG4 injected 3xTg AD mice at 6-10 months of age showed improved learning and memory; however, only minimal improvement was observed at 13-16 months of age. This behavioral improvement corresponds to a decrease in soluble Aß 40 levels. Taken together, these findings suggest that BTA-EG4 may be beneficial in ameliorating the synaptic loss seen in early AD.
Assuntos
Doença de Alzheimer/complicações , Compostos de Anilina/uso terapêutico , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Espinhas Dendríticas/efeitos dos fármacos , Hipocampo/patologia , Fatores Etários , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Compostos de Anilina/farmacologia , Animais , Modelos Animais de Doenças , Hipocampo/ultraestrutura , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Mutação/genética , Presenilina-1/genética , Proteínas tau/genéticaRESUMO
In vitro genotoxicity tests detect carcinogens that are thought to act primarily via a mechanism involving direct genetic damage. In this study, we constructed a Geno-Tox cell array chip for genotoxicity testing; eight recombinant bioluminescent bacteria were used to successfully fabricate a Geno-Tox cell array chip. Four well-characterized DNA damage chemicals were selected to determine the capabilities of this Geno-Tox array chip, and each strain of the chip was distinctly responsive, according to the specific mode of genotoxic action. Therefore, this Geno-Tox cell array chip could be implemented to characterize and understand the genotoxic modes of impact; thus, it could be used to provide the genotoxic mechanism of action for new drugs or unknown or newly synthesized chemicals in food and the environment.
Assuntos
Bactérias/citologia , Bactérias/genética , Testes de Mutagenicidade/métodos , Análise Serial de Tecidos/métodos , Bactérias/efeitos dos fármacos , Células Imobilizadas/citologia , Células Imobilizadas/efeitos dos fármacos , Dano ao DNA , DNA Recombinante/genética , Engenharia Genética , Mutagênicos/toxicidade , Regiões Promotoras Genéticas/genéticaRESUMO
In this study, we present the successful detection of food-borne pathogens using randomly selected non-sequenced genomic DNA probes-based DNA microarray chips. Three food-borne pathogens, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), and Bacillus cereus, were subjected for the preparation of the DNA microarray probes. Initially, about 50 DNA probes selected randomly from non-sequenced genomic DNA of each pathogen were prepared by using a set of restriction enzyme pairs. The proto-type of DNA microarray chip for detecting three different pathogens simultaneously was fabricated by using those DNA probes prepared for each pathogen. This proto-type DNA microarray has been tested with three target pathogens and additional seven bacteria, and successfully verified with a few cross-hybridized probes. After this primary verification of the DNA microarray hybridization, this proto-type DNA microarray chip was redesigned and successfully optimized by eliminating a few cross-hybridized probes. The specificity of this redesigned DNA microarray chip to each pathogen was confirmed without any serious cross-hybridizations, and its multiplexing capability in its pathogen detection was found to be possible. This randomly selected non-sequenced genomic DNA probes-based DNA microarray was successfully proved to be the high-throughput simultaneous detection chip for the detection of food-borne pathogens, without knowing the exact sequence information of the target bacteria. This could be the first fabrication of DNA microarray chip for the simultaneous detection of different kinds of food-borne pathogens.
Assuntos
Bactérias/genética , Bactérias/isolamento & purificação , Sondas de DNA/genética , Microbiologia de Alimentos/métodos , Genoma Bacteriano/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , Bacillus cereus/genética , Salmonella typhimurium/genética , Staphylococcus aureus/genéticaRESUMO
We report on a novel approach to predict the mode of genotoxic action of chemicals using a series of DNA damage specific bioluminescent bacteria. For this, a group of seven different DNA damage sensing recombinant bioluminescent strains were employed. Each of these strains was tested against model DNA damaging agents, such as mitomycin C (MMC), 1-methyl-1-nitroso-N-methylguanidine (MNNG), nalidixic acid (Nal) and 4-nitroquinoline N-oxide (4-NQO). These biosensors were grouped based on their responses to a specific mode of genotoxic action, such as (a) DNA damage cascade response (biosensor with nrdA-, dinI- and sbmC-lux), (b) SOS response or DNA repair (strains carrying recA-, recN- and sulA-lux), and (c) DNA damage potentially by alkylation (biosensor with alkA-lux). The differential response patterns and its strength of these strains to various model genotoxicants allowed classifying the chemical's potential genotoxic mode. Therefore, it is possible to elucidate and classify the mode of genotoxic impacts of an unknown sample and that together they may be utilized in the pre-screening steps of new drugs, newly synthesized chemicals, food and environmental contaminants.
Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , Dano ao DNA/efeitos dos fármacos , DNA Bacteriano/efeitos dos fármacos , Escherichia coli/genética , Testes de Mutagenicidade/métodos , Mutagênicos/farmacologia , Escherichia coli/efeitos dos fármacos , Mutagênicos/análiseRESUMO
A new oxidative stress-responsive bacterial biosensor was constructed using the promoter of the pgi gene fused to the luxCDABE reporter. This strain (PGRFM) responded in a dose-dependent manner to methyl viologen (MV), a model redox chemical that results in oxidative stress. The responses of strain PGRFM to redox chemicals was strongly dependent on the available carbon source. For example, when the strain was grown under nutrient-limited conditions in the presence of glucose or gluconate it was capable of responding to low MV concentrations (0.6-19.3ppm), whereas the same cells grown in LB (a nutrient rich media) only responded to higher concentrations (4.9-625ppm). This allowed us to select PGRFM's growth conditions and extend the range of concentrations at which a stress-inducing chemical could be detected. Further, strain PGRFM responded to structural analogs of MV (i.e., ethyl and benzyl viologen), demonstrating that this strain is responsive to the presence of superoxide radicals, regardless of the chemical by which they are generated. Strain PGRFM's response patterns to these analogs were distinct from each other, which determined their strength to induce oxidative stress. As well, a significant induction was seen when this strain was exposed to hydrogen peroxide, illustrating that strain PGRFM is responsive in the presence of both the superoxide (O(2)(-)) and hydroxyl (OH) radicals.
Assuntos
Bioensaio/instrumentação , Técnicas Biossensoriais/instrumentação , Proteínas de Escherichia coli/análise , Escherichia coli/fisiologia , Medições Luminescentes/instrumentação , Proteínas Luminescentes/genética , Paraquat/isolamento & purificação , Bioensaio/métodos , Técnicas Biossensoriais/métodos , Contagem de Colônia Microbiana/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Escherichia coli/efeitos dos fármacos , Proteínas Luminescentes/análise , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Paraquat/administração & dosagem , Regiões Promotoras Genéticas/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In this study, we describe a straightforward strategy to develop whole cell-based biosensors using fusions of the bacterial bioluminescence genes and the promoters from chemically responsive genes within Escherichia coli, in which chemical target-responsive genes were screened by using the information of gene expression data obtained from DNA microarray analysis. Paraquat was used as a model chemical to trigger gene expression changes of E. coli and to show the DNA microarray-assisted development of whole cell-based biosensors. Gene expression data from the DNA microarray were obtained by time course analysis (10, 30, and 60 min) after exposure to paraquat. After clustering gene expression data obtained by time course analysis, a group of highly expressed genes over the all time courses could be classified. Within this group, three genes expressed highly for overall time points were selected and promoters of these genes were used as fusion partners with reporter genes, lux CDABE, to construct whole cell-based biosensors. The constructed biosensors recognized the presence of model inducer, paraquat, and structural analogue chemicals of paraquat with a high specificity, and the results were reconfirmed by using DNA microarray experiments for those structural analogues. This strategy to develop whole cell-based biosensors assisted by DNA microarray information should be useful in general for constructing chemical-specific or stress-specific biosensors with a high-throughput manner.