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1.
Rev Bras Parasitol Vet ; 29(2): e022819, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32609247

RESUMO

The aim of this study was to evaluate the efficiency of different substrates for larval development of Ctenocephalides felis felis during its biological cycle. Eight hundred eggs of C. felis felis from a flea maintenance colony were used. Different diets were formulated, in which the main substrates were meat flour, powdered milk, sugar, lyophilized bovine blood, tick metabolites and lyophilized egg. The flea eggs were placed in test tubes (10 per tube) and approximately 2 g of the diet to be tested was added to each tube. There were 10 replicates for each substrate. After 28 days, each tube was evaluated individually for the presence of pupae and emerged adults. The following percentages of the larvae completed the cycle to the adult stage: 67% in diets containing tick metabolites; 55%, meat flour; 39%, dehydrated bovine blood; 14%, powdered milk; and less than 1% in diets containing sugar, lyophilized bovine blood, lyophilized egg or wheat bran. It was concluded that among the diets tested, the one constituted by tick metabolites as the substrate was shown to be the most satisfactory for maintaining a laboratory colony of C. felis felis, followed by the one containing meat flour.


Assuntos
Ctenocephalides , Técnicas de Cultura , Animais , Gatos , Ctenocephalides/crescimento & desenvolvimento , Técnicas de Cultura/métodos , Larva/crescimento & desenvolvimento
2.
PLoS One ; 15(4): e0230927, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32243457

RESUMO

INTRODUCTION: Sputum specimen decontamination steps are essential due to the presence of other saprophytic and infectious organisms. However, they negatively affect the mycobacterial recovery. In addition, little is known about the Mycobacterium tuberculosis killing efficacy of the PANTA (polymyxin-B, amphotericin-B, nalidixic acid, trimethoprim, azilocillin) antibiotics. Moreover, M. tuberculosis can be present in more than one metabolic population, but the effect of different growth characteristics on the mycobacterial growth indicator tube (MGIT) based time-to-positive (TTP) is not well studied. METHODS: We performed-(1) experiments using the solid agar and MGIT method to determine the effect of the NALC-NaOH decontamination method, (2) concentration-response studies with each individual antibiotic in the PANTA, and (3) the effect of the M. tuberculosis metabolic population on the TTP. TTP was recorded using the Epicenter software and exponential growth equation was used to calculate the doubling time of the bacteria, whereas, CFU/mL was analyzed using the Inhibitory Sigmoid Emax model for each antibiotic. RESULTS: Decontamination resulted in 4.36+0.13 log10 CFU/mL difference in cultures treated with NALC-NaOH versus no decontamination process and the limit of detection decreased from 1.47 log10 CFU/mL to the 0.42 log10 CFU/mL following NALC-NaOH treatment. PANTA at currently used antibiotic concentrations, did not had negative effect on mycobacterial recovery. Exponential growth model estimated doubling time for the log-phase growth M. tuberculosis as 2.04 days, for the semi-dormant bacilli as 2.80 days, and 6.37 days for the anaerobic cultures. CONCLUSION: Specimen decontamination method negatively affect the laboratory diagnosis of M. tuberculosis, polymyxin-B and nalidixic acid have anti-tuberculosis efficacy at high concentrations, and the doubling time of different metabolic population should be considered when deciding the time-in-protocol for the MGIT system.


Assuntos
Técnicas de Laboratório Clínico , Técnicas de Cultura/métodos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Manejo de Espécimes/métodos , Tuberculose/diagnóstico , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/fisiologia , Tuberculose/tratamento farmacológico
3.
Prostate ; 80(6): 518-526, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32084293

RESUMO

BACKGROUND: Current in vitro modeling systems do not fully reflect the biologic and clinical diversity of prostate cancer (PCa). Organoids are 3D in vitro cell cultures that recapitulate disease heterogeneity, retain prostate gland architecture, and mirror parental tumor characteristics. METHODS: To make better use of organoid models in the PCa research field, we provide a review of cutting-edge prostate organoid methodologies, applications, and limitations. RESULTS: We summarize methodologies for the establishment of benign prostate and PCa organoids and describe some of the model's practical applications and challenges. We highlight the patient-derived xenograft (PDX)-organoid interface model, which may allow for the generation of organoids from primary and rare PCa subtypes. Finally, we discuss potential future utilizations of PCa organoids in the realms of drug development and precision oncology. CONCLUSIONS AND FUTURE DIRECTIONS: Organoids represent a quasi in vivo modeling system that can be easily amenable to genetic modification and functional studies. As such, organoids may serve as an intermediate preclinical model for studying PCa. Future directions may include the refinement of culturing conditions to increase drug response fidelity in PCa organoids. The PDX-organoid interface model may enable the future establishment of primary and rare subtype PCa organoid lines.


Assuntos
Organoides/patologia , Neoplasias da Próstata/patologia , Animais , Técnicas de Cultura/métodos , Xenoenxertos , Humanos , Masculino , Próstata/citologia , Próstata/patologia
4.
BMC Res Notes ; 12(1): 818, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31856898

RESUMO

OBJECTIVE: Parafilm M® is a moisture-resistant thermoplastic commonly used to seal Nematode Growth Media (NGM) agar plates on which the nematode Caenorhabditis elegans is cultured. This practice reduces media dehydration and microbial contamination. However, the effects on C. elegans individuals of placing this barrier between the external environment and the interior of the NGM plate are currently unknown. Our research aims to determine if this common practice engenders developmental changes, such as growth, that could subsequently and unintentionally alter experimental data. We compared the larval growth over 48 h of animals cultured on Parafilm-wrapped and unwrapped control NGM plates. RESULTS: Wrapping culture plates with Parafilm significantly accelerated and increased larval growth, with a 0.87 µm/h increase in growth rate (~ 6%) and a 37.90 µm increase in the change in growth (Δgrowth; ~ 5%) over 48 h. Therefore, C. elegans investigators should be aware that wrapping their experimental cultures with Parafilm may result in statistically detectable changes in worm growth and possibly other developmental processes.


Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Técnicas de Cultura/métodos , Ágar , Animais , Meios de Cultura , Larva/crescimento & desenvolvimento , Parafina
5.
PLoS One ; 14(12): e0226616, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31887170

RESUMO

This paper reports the diversity of fungi associated with substrates collected at a shallow hydrothermal vent field at Kueishan Island, Taiwan, using both culture-based and metabarcoding methods. Culture of fungi from yellow sediment (with visible sulfur granules), black sediment (no visible sulfur granules), the vent crab Xenograpsus testudinatus, seawater and, animal egg samples resulted in a total of 94 isolates. Species identification based on the internal transcribed spacer regions of the rDNA revealed that the yellow sediment samples had the highest species richness with 25 species, followed by the black sediment (23) and the crab (13). The Ascomycota was dominant over the Basidiomycota; the dominant orders were Agaricales, Capnodiales, Eurotiales, Hypocreales, Pleosporales, Polyporales and Xylariales. Hortaea werneckii was the only common fungus isolated from the crab, seawater, yellow and black sediment samples. The metabarcoding analysis amplifying a small fragment of the rDNA (from 18S to 5.8S) recovered 7-27 species from the black sediment and 12-27 species from the yellow sediment samples and all species belonged to the Ascomycota and the Basidiomycota. In the yellow sediments, the dominant order was Pleosporales and this order was also dominant in the black sediment together with Sporidiobolales. Based on the results from both methods, 54 and 49 species were found in the black and yellow sediments, respectively. Overall, a higher proportion of Ascomycota (~70%) over Basidiomycota was recovered in the yellow sediment and the two phyla were equally abundant in the black sediment. The top five dominant fungal orders in descending order based on species richness were Pleosporales>Eurotiales>Polyporales>Hypocreales>Capnodiales in the black sediment samples, and Polyporales>Pleosporales>Eurotiales>Capnodiales>Hypocreales in the yellow sediment samples. This study is the first to observe a high diversity of fungi associated with various substrates at a marine shallow water hydrothermal vent ecosystem. While some fungi found in this study were terrestrial species and their airborne spores might have been deposited into the marine sediment, several pathogenic fungi of animals, including Acremonium spp., Aspergillus spp., Fusarium spp., Malassezia spp., Hortaea werneckii, Parengyodontium album, and Westerdykella dispersa, were recovered suggesting that these fungi may be able to cause diseases of marine animals.


Assuntos
Biodiversidade , Técnicas de Cultura/métodos , Código de Barras de DNA Taxonômico/métodos , Fungos/isolamento & purificação , Fontes Hidrotermais/microbiologia , Ascomicetos/isolamento & purificação , Basidiomycota/isolamento & purificação , Fungos/genética , Fungos/patogenicidade , Sedimentos Geológicos/microbiologia , Taiwan
6.
PLoS One ; 14(11): e0222831, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31703058

RESUMO

Metagenomic sequencing of fecal DNA can usefully characterise an individual's intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.


Assuntos
Antibacterianos/farmacologia , Técnicas de Cultura/métodos , Farmacorresistência Bacteriana/genética , Enterobacteriaceae/genética , Fezes/microbiologia , Análise de Sequência de DNA/métodos , Cefalosporinas/farmacologia , Biologia Computacional , DNA Bacteriano , Enterobacteriaceae/efeitos dos fármacos , Infecções por Enterobacteriaceae/tratamento farmacológico , Infecções por Enterobacteriaceae/genética , Humanos
7.
An Acad Bras Cienc ; 91(3): e20180439, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31531531

RESUMO

The orchid seed banks of Atlantic Forest may be considered a key strategy for the conservation of species threatened with extinction by indiscriminate collection or habitat destruction. The aim of this study was to evaluate the seed viability, to choose the best culture medium for the asymbiotic germination and evaluate germination, after storage for different periods and temperatures for the Brazilian native orchids: Gomesa praetexta (Rchb.f.) M.W.Chase & N.H.Williams, Gomesa forbesii (Hook.) M.W.Chase & N.H.Williams, Gomesa recurva R.Br. and Grandiphyllum divaricatum (Lindl.) Docha Neto. Knudson C (KC), Murashige & Skoog (MS), half-strength MS (1/2 MS macro- and micro-nutrients) and Woody Plant Medium (WPM) culture media were tested for germination. The WPM culture medium was the best for asymbiotic germination of all species evaluated, with high germination percentages and improved seedling development. Seeds of G. divaricatum, G. praetexta, G. recurva and G. forbesii indicated orthodox behavior, with high viability rates after 12 months of storage, being recommended the storage temperature of -80°C for the first three species and -20°C for G. forbesii. The protocol developed in the present study was efficient for seed bank storage, in vitro germination and seedling production of G. divaricatum and G. praetexta, contributing to conservation strategies of these species.


Assuntos
Técnicas de Cultura/métodos , Germinação/fisiologia , Orchidaceae/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Aclimatação , Brasil , Meios de Cultura , Espécies em Perigo de Extinção , Florestas , Orchidaceae/classificação , Banco de Sementes
8.
PLoS One ; 14(9): e0221930, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31490970

RESUMO

Since carotenoids are important as natural colorants, antioxidants, neutraceutics and pharmaceutics, the aim of the present study was to find a new good source of these pigments. We hereby report a green microalga Asterarcys quadricellulare PUMCC 5.1.1 as a new and good producer of carotenoids. The organism produced 35±1.75 µg carotenoids mg-1 dry biomass during stationary phase in control cultures. The growth and carotenoids production by the test microalga were optimized by varying nutrient growth media, pH, nitrogen and phosphate source, salinity, light quality, intensity and duration. The optimized conditions for carotenoid production were: Bold basal (BB) medium with pH 8.5, containing with10 mM nitrate, 3.5 mM phosphate and 0.17 mM salinity and illuminated with blue light with 60 µmol m-2 s-1 photon flux light intensity. Cultivation of cultures in the above mentioned optimized conditions resulted in nearly 3.0 fold increase in carotenoid production compared to the control cultures grown in unmodified BB medium. Using HPTLC, four carotenoids have been identified as ß-carotene, lutein, astaxanthin and canthaxanthin. Further, carotenoids were also separated and purified by flash chromatography and the amounts of purified carotenoids were determined by HPLC. The organism produced 47.0, 28.7, 15.5 and 14.0 µg ß-carotene, lutein, astaxanthin and canthaxanthin mg-1 dry biomass, respectively, under optimized conditions. The amount of total carotenoids (118 µg mg-1 dry biomass) produced by Asterarcys quadricellulare PUMCC 5.1.1 under optimized culture conditions was significantly higher than control cultures. Thus, this microalgal strain is a promising candidate for carotenoid production at commercial level.


Assuntos
Carotenoides/metabolismo , Clorofíceas/crescimento & desenvolvimento , Clorofíceas/metabolismo , Técnicas de Cultura/métodos , Microalgas/crescimento & desenvolvimento , Microalgas/metabolismo , Carotenoides/isolamento & purificação
9.
Folia Microbiol (Praha) ; 64(5): 615-625, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31363995

RESUMO

We have worked out a rapid 1-day test based on photosynthesis measurements to estimate suitable growth temperature of microalgae cultures. To verify the proposed procedure, several microalgae-Chlorella, Nostoc, Synechocystis, Scenedesmus, and Cylindrospermum-were cultured under controlled laboratory conditions (irradiance, temperature, mixing, CO2, and nutrient supply) to find the optima of photosynthetic activity using the range between 15 and 35 °C. These activities were recorded at each temperature step after 2 h of acclimation which should be sufficient as oxygen production and the PQ cycle are regulated by fast processes. Photosynthetic activity was measured using three techniques-oxygen production/respiration, saturating pulse analysis of fluorescence quenching, and fast fluorescence induction kinetics-to estimate the temperature optima which should correspond to high growth rate. We measured all variables that might have been directly related to growth-photosynthetic oxygen evolution, maximum photochemical yield of PSII, Fv/Fm, relative electron transport rate rETRmax, and the transients Vj and Vi determined by fast fluorescence induction curves. When the temperature optima for photosynthetic activity were verified in growth tests, we found good correlation. For most of tested microalgae strains, temperature around 30 °C was found to be the most suitable at this setting. We concluded that the developed test can be used as a rapid 1-day pre-screening to estimate a suitable growth temperature of microalgae strains before they are cultured in a pilot scale.


Assuntos
Técnicas de Cultura/métodos , Microalgas/crescimento & desenvolvimento , Chlorella/crescimento & desenvolvimento , Chlorella/metabolismo , Chlorella/efeitos da radiação , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Cinética , Luz , Microalgas/metabolismo , Microalgas/efeitos da radiação , Oxigênio/metabolismo , Fotossíntese , Scenedesmus/crescimento & desenvolvimento , Scenedesmus/metabolismo , Scenedesmus/efeitos da radiação , Temperatura
10.
J Cancer Res Clin Oncol ; 145(8): 1949-1976, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31292714

RESUMO

PURPOSE: Efflux transporters of the adenosine triphosphate-binding cassette (ABC)-superfamily play an important role in the development of multidrug resistance (multidrug resistant; MDR) in cancer. The overexpression of these transporters can directly contribute to the failure of chemotherapeutic drugs. Several in vitro and in vivo models exist to screen for the efficacy of chemotherapeutic drugs against MDR cancer, specifically facilitated by efflux transporters. RESULTS: This article reviews a range of efflux transporter-based MDR models used to test the efficacy of compounds to overcome MDR in cancer. These models are classified as either in vitro or in vivo and are further categorised as the most basic, conventional models or more complex and advanced systems. Each model's origin, advantages and limitations, as well as specific efflux transporter-based MDR applications are discussed. Accordingly, future modifications to existing models or new research approaches are suggested to develop prototypes that closely resemble the true nature of multidrug resistant cancer in the human body. CONCLUSIONS: It is evident from this review that a combination of both in vitro and in vivo preclinical models can provide a better understanding of cancer itself, than using a single model only. However, there is still a clear lack of progression of these models from basic research to high-throughput clinical practice.


Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Antineoplásicos/isolamento & purificação , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Modelos Biológicos , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Técnicas de Cultura/métodos , Técnicas de Apoio para a Decisão , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Especificidade de Órgãos , Seleção de Pacientes
11.
Biotechnol J ; 14(11): e1800573, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31329373

RESUMO

The monoclonal antibody (mAb) industry is witnessing unprecedented growth, with an increasing range of new molecules and biosimilars as well as disease targets approved than ever before. Competition necessitates pharmaceutical companies to reduce development/production costs and time-to-market. To this aim, mathematical modeling can aid traditional experiment-only-based process development by reducing the design space, integrating scales, and assisting in identifying optimal operating conditions in less time and with lower expense. Mathematical models have been employed by other industries for control and optimization purposes and are important decisional tools for testing scenarios, process configurations, operating conditions, etc. Herein, a predictive, experimentally validated mathematical model that captures cellular metabolism and growth with cell cycle, cell death (apoptosis), and mAb production in GS-NS0 cells is presented. The model utilizes cellular, metabolic, and gene expression data, highlighting how multiple data sources can be integrated in one tool with the aim of optimizing mammalian cell bioprocessing.


Assuntos
Anticorpos Monoclonais/biossíntese , Apoptose , Ciclo Celular , Modelos Teóricos , Mieloma Múltiplo/metabolismo , Proteínas do Mieloma/metabolismo , Animais , Anticorpos Monoclonais/genética , Medicamentos Biossimilares , Linhagem Celular Tumoral , Técnicas de Cultura/métodos , Regulação Neoplásica da Expressão Gênica , Camundongos , Mieloma Múltiplo/genética
12.
J Vis Exp ; (147)2019 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-31157778

RESUMO

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures - temperature and osmolarity - across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.


Assuntos
Técnicas de Cultura/métodos , Saccharomyces cerevisiae/citologia , Software , Automação , Ciclo Celular , Proliferação de Células , Fenótipo , Saccharomyces cerevisiae/genética
13.
Methods Mol Biol ; 1993: 47-59, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148077

RESUMO

Mouse models have been used to study the physiology and pathogenesis of the skin. However, propagation of mouse primary epidermal keratinocytes remains challenging. In this chapter, we introduce a newly developed protocol that enables long-term expansion of p63+ mouse epidermal keratinocytes in low-Ca2+ media without the need of progenitor cell purification steps or support by a feeder cell layer. Pharmacological inhibition of TGF-ß signaling in crude preparations of mouse epidermis robustly increases proliferative capacity of p63+ epidermal progenitor cells while preserving their ability to differentiate. Suppression of TGF-ß signaling also permits p63+ epidermal keratinocytes to form macroscopically large clones in 3T3-J2 feeder cell co-culture. This simple and efficient approach will facilitate the use of mouse models by providing p63+ primary epidermal keratinocytes in quantity.


Assuntos
Técnicas de Cultura/métodos , Queratinócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Técnicas de Cocultura , Células Epidérmicas/efeitos dos fármacos , Células Epidérmicas/metabolismo , Células Alimentadoras , Queratinócitos/efeitos dos fármacos , Camundongos , Fator de Crescimento Transformador beta/metabolismo
14.
Carbohydr Polym ; 219: 63-76, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31151547

RESUMO

Bacterial cellulose (BC) is an organic compound produced by certain types of bacteria. In natural habitats, the majority of bacteria synthesize extracellular polysaccharides, such as cellulose, which form protective envelopes around the cells. Many methods are currently being investigated to enhance cellulose growth. The various celluloses produced by different bacteria possess different morphologies, structures, properties, and applications. However, the literature lacks a comprehensive review of the different methods of BC production, which are critical to BC properties and their final applications. The aims of this review are to provide an overview of the production of BC from different culture methods, to analyze the characteristics of particular BC productions, to indicate existing problems associated with different methods, and to choose suitable culture approaches for BC applications in different fields. The main goals for future studies have also been discussed here.


Assuntos
Bactérias/metabolismo , Materiais Biocompatíveis , Celulose , Técnicas de Cultura/métodos , Fermentação , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , Celulose/química , Celulose/metabolismo
15.
Intensive Care Med ; 45(8): 1082-1092, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31209523

RESUMO

PURPOSE: To compare bacteria recovered by standard cultures and metataxonomics, particularly with regard to ventilator-associated pneumonia (VAP) pathogens, and to determine if the presence of particular bacteria or microbiota in tracheal and oropharyngeal secretions during the course of intubation was associated with the development of VAP. METHODS: In this case-control study, oropharyngeal secretions and endotracheal aspirate were collected daily in mechanically ventilated patients. Culture and metataxonomics (16S rRNA gene-based taxonomic profiling of bacterial communities) were performed on serial upper respiratory samples from patients with late-onset definite VAP and their respective controls. RESULTS: Metataxonomic analyses showed that a low relative abundance of Bacilli at the time of intubation in the oropharyngeal secretions was strongly associated with the subsequent development of VAP. On the day of VAP, the quantity of human and bacterial DNA in both tracheal and oropharyngeal secretions was significantly higher in patients with VAP than in matched controls with similar ventilation times. Molecular techniques identified the pathogen(s) of VAP found by culture, but also many more bacteria, classically difficult to culture, such as Mycoplasma spp. and anaerobes. CONCLUSIONS: Molecular analyses of respiratory specimens identified markers associated with the development of VAP, as well as important differences in the taxa abundance between VAP and controls. Further prospective trials are needed to test the predictive value of these markers, as well as the relevance of uncultured bacteria in the pathogenesis of VAP.


Assuntos
Biomarcadores/análise , Microbiota , Pneumonia Associada à Ventilação Mecânica/microbiologia , APACHE , Adulto , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Técnicas de Cultura/instrumentação , Técnicas de Cultura/métodos , Feminino , Humanos , Unidades de Terapia Intensiva/organização & administração , Masculino , Pessoa de Meia-Idade , Orofaringe/microbiologia , Pneumonia Associada à Ventilação Mecânica/mortalidade , Estudos Prospectivos , RNA Ribossômico 16S/análise , Respiração Artificial/efeitos adversos , Suíça , Traqueia/microbiologia
16.
Acta Odontol Latinoam ; 32(1): 36-43, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31206573

RESUMO

Several studies have tried to associate the presence of different pathogens with the onset and progression ofperiodontitis, reporting a wide variety of results from different populations and environments. The aim of this study was to determine the main periodontal pathogens present in the subgingival biofilm of Dominican patients with periodontitis, by using specific microbiological culturing techniques. Periodontitis patients were selected after a full-mouth periodontal evaluation, and assigned to different periodontitis groups based on percentage of affected locations. Subgingival samples were collected and analyzed by means of specific culture techniques. Anaerobic counts, frequency of detection and proportions of target pathogens were calculated. Variables were analyzed by means of Student's T-test or chi-square test. Twenty-nine subjects were recruited, of whom 17 were diagnosed with generalized periodontitis (GenP) and 12 with localized periodontitis (LocP). The most prevalent bacterial species in both groups was Prevotella intermedia (94.1% in GenP and 91.7% in LocP), followed by Porphyromonas gingivalis (88.2% in GenP and 83.3% in LocP). Total microbiota in subgingival samples was 1.3 x107 colony-forming units (CFU)/mL (standard deviation, SD=1.5 x107) and 9.6x10s CFU/mL (SD=1.1 x107) in GenP and LocP subjects, respectively, though differences were not statistically significant (p=0.222). The highest counts were observed for P gingivalis in both groups, with mean concentration 2.5x10s CFU/mL (6.1x10s) in GenP and 2.9x10s CFU/mL (5x10s) in LocP, with no statistically significant difference (p=0.879). These results suggest that relevant periodontal pathogens are found with diversity and abundance in the subgingival microbiota of adult Dominican patients with periodontitis.


Assuntos
Infecções Bacterianas/microbiologia , Técnicas de Cultura/métodos , Bactérias Gram-Negativas/isolamento & purificação , Periodontite/microbiologia , Adulto , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Infecções Bacterianas/epidemiologia , Biofilmes , Estudos Transversais , República Dominicana/epidemiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Periodontite/classificação , Periodontite/epidemiologia , Porphyromonas gingivalis/isolamento & purificação , Prevalência , Prevotella intermedia/isolamento & purificação
17.
Microb Pathog ; 134: 103574, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31170450

RESUMO

The present study was aimed to assess the prevalence and efficiency of techniques for the diagnosis of bovine tuberculosis (bTB) including enzyme-linked immunosorbent assay (ELISA), Gamma interferon assay (IFN-γ) and polymerase chain reaction (PCR) in comparison to skin tuberculin test and culture technique. A total of 2600 cross-breed dairy cattle in Menoufia and Daqahlia governorates were tested by the single intradermal tuberculin test where the disease prevalence was 1.8%. Serum and whole blood samples were collected from positive tuberculin reactors for ELISA and IFN-γ assay, respectively. After slaughtering of positive tuberculin reactors, the post-mortem examination was carried out and tissue samples were collected for the bacteriological examination and PCR. The percentage of visible lesions of tuberculin reactors was 78.7%, while non-visible lesions were 21.27%. Culture technique revealed that the percentage of bTB was 63.8%. The ELISA and IFN-γ assay using short-term culture filtrate (ST-CF) prepared antigen revealed higher sensitivity (72.3% and 82.9%) than the bovine purified protein derivative (PPD-B) antigen. Although prepared ST-CF antigen has great efficiency and eligibility for the diagnosis of bTB, PCR appeared to have a higher sensitivity (85.1%) than other diagnostic methods when dealing with post-mortem samples. Gamma interferon assay using ST-CF antigen is recommended for antemortem diagnosis of bTB in cattle.


Assuntos
Técnicas Bacteriológicas/métodos , Interferon gama/sangue , Mycobacterium bovis/isolamento & purificação , Tuberculose Bovina/diagnóstico , Animais , Bovinos , Técnicas de Cultura/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Mycobacterium bovis/imunologia , Reação em Cadeia da Polimerase/métodos , Prevalência , Sensibilidade e Especificidade , Tuberculina , Teste Tuberculínico/métodos , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/imunologia , Tuberculose Bovina/microbiologia
18.
Yakugaku Zasshi ; 139(5): 663-672, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31061333

RESUMO

Marine environments offer a rich source of natural products with potential therapeutic applications because the ocean covers 70% of the earth's surface and approximately 80% of all living organisms live in the sea. Therefore we have investigated bioactive compounds from marine organisms such as marine sponges, ascidians, and marine-derived microorganisms. This review consists of two topics based on marine natural product chemistry. (1) Protein tyrosine phosphatase (PTP) 1B plays a key role as a negative regulator in the insulin and leptin signaling pathways. Accordingly, the development of PTP1B inhibitors is expected to provide new drugs for type 2 diabetes and obesity. We have been searching for new types of PTP1B inhibitors among marine organisms and identified various PTP1B inhibitors from marine sponges and fungi. This review presents their structural diversities and unique biological properties. (2) In the course of our studies on the induced production of new fungal metabolites, the Palauan marine-derived fungus, Trichoderma cf. brevicompactum TPU199, was found to produce the unusual epipolythiodiketopiperazines, gliovirin and pretrichodermamide A. Long-term static fermentation of the strain induced production of a new dipeptide, dithioaspergillazine A, whereas fermentation of the strain with NaCl, NaBr, and NaI produced the Cl and Br derivatives of pretrichodermamide A and a new iodinated derivative, iododithiobrevamide, respectively. Moreover, DMSO-added seawater medium induced the production of diketopiperazine with the unprecedented trithio-bridge, chlorotrithiobrevamide. This fermentation study on the strain as well as the structures of the metabolites obtained are described in this review.


Assuntos
Organismos Aquáticos/química , Técnicas de Cultura/métodos , Inibidores Enzimáticos/isolamento & purificação , Fungos/metabolismo , Técnicas Microbiológicas/métodos , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Trichoderma/metabolismo , Diabetes Mellitus Tipo 2 , Dicetopiperazinas/metabolismo , Dipeptídeos/metabolismo , Piperazinas/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia
19.
Biogerontology ; 20(4): 457-474, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30989423

RESUMO

It has been repeatedly reported that transposable elements (TE) become active and/or mobile in the genomes of replicatively and stress-induced senescent mammalian cells. However, the biological role of senescence-associated transposon activation and its occurrence and relevance in other eukaryotic cells remain to be elucidated. In the present study, Candida albicans, a prevalent opportunistic fungal pathogen in humans, was used to analyze changes in gene copy number of selected TE, namely Cirt2, Moa and Cmut1 during long-term culture (up to 90 days). The effects of stress stimuli (fluconazole, hydrogen peroxide, hypochlorite) and ploidy state (haploid, diploid, tetraploid cells) were also considered. An increase in copy number of Cirt2 and Moa was the most accented in tetraploid cells after 90 days of culture that was accompanied by changes in karyotype patterns and slightly more limited growth rate compared to haploid and diploid cells. Stress stimuli did not potentiate TE activity. Elevation in chromosomal DNA breaks was also observed during long-term culture of cells of different ploidy, however this was not correlated with increased TE activity. Our results suggest that increased TE activity may promote genomic diversity and plasticity, and cellular heterogeneity during long-term culture of C. albicans cells.


Assuntos
Candida albicans/genética , Senescência Celular/genética , Elementos de DNA Transponíveis/fisiologia , Dosagem de Genes , Variação Genética/genética , Adaptação Fisiológica/genética , Animais , Técnicas de Cultura/métodos , Quebras de DNA , Humanos , Ploidias , Tempo
20.
J Vis Exp ; (144)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30829328

RESUMO

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.


Assuntos
Técnicas de Cultura/métodos , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos
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