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1.
PLoS One ; 15(8): e0236822, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764772

RESUMO

Various marine fungi have been shown to produce interesting, bioactive compounds, but scaling up the production of these compounds can be challenging, particularly because little is generally known about how the producing organisms grow. Here we assessed the suitability of using 100-well BioScreen plates or 96-well plates incubated in a robot hotel to cultivate eight filamentous marine fungi, six sporulating and two non-sporulating, to obtain data on growth and substrate (glucose, xylose, galactose or glycerol) utilisation in a high throughput manner. All eight fungi grew in both cultivation systems, but growth was more variable and with more noise in the data in the Cytomat plate hotel than in the BioScreen. Specific growth rates between 0.01 (no added substrate) and 0.07 h-1 were measured for strains growing in the BioScreen and between 0.01 and 0.27 h-1 for strains in the plate hotel. Three strains, Dendryphiella salina LF304, Penicillium chrysogenum KF657 and Penicillium pinophilum LF458, consistently had higher specific growth rates on glucose and xylose in the plate hotel than in the BioScreen, but otherwise results were similar in the two systems. However, because of the noise in data from the plate hotel, the data obtained from it could only be used to distinguish between substrates which did or did not support growth, whereas data from BioScreen also provided information on substrate preference. Glucose was the preferred substrate for all strains, followed by xylose and galactose. Five strains also grew on glycerol. Therefore it was important to minimise the amount of glycerol introduced with the inoculum to avoid misinterpreting the results for growth on poor substrates. We concluded that both systems could provide physiological data with filamentous fungi, provided sufficient replicates are included in the measurements.


Assuntos
Ascomicetos/crescimento & desenvolvimento , Penicillium/crescimento & desenvolvimento , Água do Mar/microbiologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/isolamento & purificação , Meios de Cultura/química , Meios de Cultura/farmacologia , Glucose/farmacologia , Glicerol/farmacologia , Penicillium/efeitos dos fármacos , Penicillium/isolamento & purificação , Xilose/farmacologia
2.
PLoS One ; 15(6): e0234180, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32511278

RESUMO

The autophagy-endolysosomal pathway is an evolutionally conserved degradation system that is tightly linked to a wide variety of physiological processes. Dysfunction of this system is associated with many pathological conditions such as cancer, inflammation and neurodegenerative diseases. Therefore, monitoring the cellular autophagy-endolysosomal activity is crucial for studies on the pathogenesis as well as therapeutics of such disorders. To this end, we here sought to create a novel means exploiting Keima, an acid-stable fluorescent protein possessing pH-dependent fluorescence excitation spectra, for precisely monitoring the autophagy-endolysosomal system. First, we generated three lines of transgenic (tg) mouse expressing monomeric Keima-fused MAP1LC3B (mKeima-LC3B). Then, these tg mice were subjected to starvation by food-restriction, and also challenged to neurodegeneration by genetically crossing with a mouse model of amyotrophic lateral sclerosis; i.e., SOD1H46R transgenic mouse. Unexpectedly, despite that a lipidated-form of endogenous LC3 (LC3-II) was significantly increased, those of mKeima-LC3B (mKeima-LC3B-II) were not changed under both stressed conditions. It was also noted that mKeima-LC3B-positive aggregates were progressively accumulated in the spinal cord of SOD1H46R;mKeima-LC3B double-tg mice, suggestive of acid-resistance and aggregate-prone natures of long-term overexpressed mKeima-LC3B in vivo. Next, we characterized mouse embryonic fibroblasts (MEFs) derived from mKeima-LC3B-tg mice. In contrast with in vivo, levels of mKeima-LC3B-I were decreased under starved conditions. Furthermore, when starved MEFs were treated with chloroquine (CQ), the abundance of mKeima-LC3B-II was significantly increased. Remarkably, when cultured medium was repeatedly changed between DMEM (nutrient-rich) and EBSS (starvation), acidic/neutral signal ratios of mKeima-LC3B-positive compartments were rapidly and reversibly shifted, which were suppressed by the CQ treatment, indicating that intraluminal pH of mKeima-LC3B-positive vesicles was changeable upon nutritional conditions of culture media. Taken together, although mKeima-LC3B-tg mice may not be an appropriate tool to monitor the autophagy-endolysosomal system in vivo, mKeima-LC3B must be one of the most sensitive reporter molecules for monitoring this system under in vitro cultured conditions.


Assuntos
Autofagia/fisiologia , Endossomos/metabolismo , Proteínas Luminescentes/genética , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Endossomos/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Lisossomos/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inanição , Superóxido Dismutase-1/genética , Imagem com Lapso de Tempo
3.
Arch Microbiol ; 202(8): 2181-2188, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32519021

RESUMO

Bacterial quorum sensing (QS) system regulates the production of most costly but sharable extracellular products (public goods) in a growth-phase-dependent manner, and the development of this energy-intensive process is susceptible to environmental changes. However, the role of nutrient factors in dominating the QS-mediated cooperative interaction and intracellular metabolism still remains less understood. Here we studied the performance of QS system by growing Pseudomonas aeruginosa under different nutrient and culture conditions. The results of comparative-transcriptomic analyses revealed that carbon source-limitation was the main factor suppressing the activation of QS system, and a substantial number of public-good-encoding genes were induced when phosphorus is limiting in short-term culture. By contrast, although the QS regulation of P. aeruginosa in all the cultures was generally decreased along with the enrichment of QS-deficient individuals during evolution, limitation of different nutrient factors had discrepant effects in directing the formation of population structure by coordinating the production of public goods and primary metabolism, especially the starch and sucrose metabolism. These findings demonstrate the pleiotropy of QS regulation in balancing the development of cooperative behavior and metabolism, and provide a reference for further understanding the role of QS system in causing persistent infections.


Assuntos
Pseudomonas aeruginosa/fisiologia , Percepção de Quorum/fisiologia , Meios de Cultura/farmacologia , Nutrientes/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos
4.
Arch Microbiol ; 202(8): 2233-2243, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32533206

RESUMO

Selenium nanoparticles (SeNPs) are attractive nanomaterials for application in medical diagnosis, because their toxicities are lower than the elemental selenium which is a functional element and essential for human. In the current study, SeNPs synthesis capability of a novel soil originated indigenous Bacillus isolate was investigated. In this context, effects of processing conditions (SeO2 concentration, pH, temperature, and time), and yeast extract supplementation on the synthesis of SeNPs have been tested. In addition, nanoparticles were characterized and antioxidant capacity was determined. The cell-free supernatant of the bacterium, which was obtained after the cultivation of the isolate in nutrient broth at 33 °C for 24 h, was used for the synthesis. During the synthesis color change from light yellow to red-orange was an indication of the formation of SeNPs. Effect of SeO2 concentration was tested on the formation of nanoparticles and at concentrations higher than 10 mM, there was no formation of nanoparticles. The best production was achieved at 6.4 mM concentration, at pH 9 and 33 °C in 72 h. Field emission scanning electron microscopy (FESEM) images revealed that SeNPs were spherical in shape having the diameters between 31 and 335 nm, and the average diameter was determined to be 126 nm. Energy dispersive X-ray spectroscopy analysis confirmed the presence of elemental selenium. SeNPs possessed significant antioxidant activity that the scavenging capacity was up to 56.5 ± 5% (IC50 322.8 µg/mL).


Assuntos
Bacillus/efeitos dos fármacos , Bacillus/metabolismo , Meios de Cultura/farmacologia , Nanopartículas/química , Selênio/química , Antioxidantes/análise , Microscopia Eletrônica de Varredura , Nanopartículas/ultraestrutura
5.
J Fr Ophtalmol ; 43(6): 477-483, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32444133

RESUMO

BACKGROUND AND PURPOSE: The purpose of this study is to compare two alternative methods of collecting and transporting media for the diagnosis of corneal ulcers, as not all clinical settings have conventional culture materials and transport media available. METHODS: In this open-label, prospective, comparative, and randomized study, patients with clinical suspicion of infectious keratitis with high risk of loss of vision had corneal specimens collected using two methods and transport media: Eswab scraping with Amies transport medium and 23-gauge needle scraping in BACTEC Peds broth. The order of each collection method was randomized. The samples were processed by standard methods, comparing the positivity frequencies for both by parametric and nonparametric tests, according to normality criteria. RESULTS: Corneal infiltrates from 40 eyes of 40 patients were analyzed. Culture positivity rate was 50% for Eswab and 35% for 23-gauge needle (P=0.258). The overall growth rate of the two methods combined was not higher than with the swab alone. The results obtained with a swab were not influenced by the collection sequence (P=0.112); however, the positivity rate was significantly higher when the sample taken with the needle was performed first (P=0.046). CONCLUSIONS: The single sample Eswab method of collection and transportation for the diagnosis of high risk corneal ulcers is a valid alternative and can be used in cases in which, for various reasons, there is no access to the full set of traditional culture materials.


Assuntos
Córnea , Úlcera da Córnea/patologia , Infecções Oculares Bacterianas/patologia , Ceratite/patologia , Manejo de Espécimes/métodos , Coleta de Tecidos e Órgãos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Córnea/patologia , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/microbiologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/microbiologia , Feminino , Humanos , Ceratite/diagnóstico , Ceratite/microbiologia , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Tecidos/métodos , Transportes , Adulto Jovem
6.
Nat Commun ; 11(1): 1528, 2020 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-32251294

RESUMO

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations.


Assuntos
Técnicas de Cultura de Células/métodos , Cromossomos Humanos X/genética , DNA Intergênico/genética , Taxa de Mutação , Células-Tronco Pluripotentes/fisiologia , Linhagem Celular , Meios de Cultura/farmacologia , Metilação de DNA , Análise Mutacional de DNA , Epigênese Genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Oxigênio/química , Oxigênio/farmacologia , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
7.
PLoS One ; 15(4): e0220163, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294080

RESUMO

BACKGROUND: Clinical application of mesenchymal stromal cells (MSCs) usually requires an in vitro expansion step to reach clinically relevant numbers. In vitro cell expansion necessitates supplementation of basal mammalian cell culture medium with growth factors. To avoid using supplements containing animal substances, human platelet lysates (hPL) produced from expired and pathogen inactivated platelet concentrates can be used in place of fetal bovine serum. However, globally, most transfusion units are currently not pathogen inactivated. As blood banks are the sole source of platelet concentrates for hPL production, it is important to ensure product safety and standardized production methods. In this proof-of-concept study we assessed the feasibility of producing hPL from expired platelet concentrates with pathogen inactivation applied after platelet lysis by evaluating the retention of growth factors, cytokines, and the ability to support MSC proliferation and tri-lineage differentiation. METHODOLOGY/PRINCIPAL FINDINGS: Bone marrow-derived MSCs (BM-MSCs) were expanded and differentiated using hPL derived from pathogen inactivated platelet lysates (hPL-PIPL), with pathogen inactivation by amotosalen/ultraviolet A treatment applied after lysis of expired platelets. Results were compared to those using hPL produced from conventional expired pathogen inactivated platelet concentrates (hPL-PIPC), with pathogen inactivation applied after blood donation. hPL-PIPL treatment had lower concentrations of soluble growth factors and cytokines than hPL-PIPC treatment. When used as supplementation in cell culture, BM-MSCs proliferated at a reduced rate, but more consistently, in hPL-PIPL than in hPL-PIPC. The ability to support tri-lineage differentiation was comparable between lysates. CONCLUSION/SIGNIFICANCE: These results suggest that functional hPL can be produced from expired and untreated platelet lysates by applying pathogen inactivation after platelet lysis. When carried out post-expiration, pathogen inactivation may provide a valuable solution for further standardizing global hPL production methods, increasing the pool of starting material, and meeting future demand for animal-free supplements in human cell culturing.


Assuntos
Plaquetas/química , Extratos Celulares/farmacologia , Meios de Cultura/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , Segurança do Sangue/métodos , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Extratos Celulares/química , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/química , Furocumarinas/farmacologia , Humanos , Células-Tronco Mesenquimais/citologia
8.
PLoS One ; 15(3): e0229043, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32182244

RESUMO

Oocyte in vitro maturation can be improved by mimicking the intra-follicular environment. Oocyte, cumulus cells, granulosa cells, and circulating factors act as meiotic regulators in follicles and maintain oocyte in the meiotic phase until oocyte becomes competent and ready to be ovulated. In a randomized experimental design, an ovine model was used to optimize the standard in vitro maturation media by Granulosa secreted factors. At first, the development capacity of oocyte derived from medium (>4 to 6 mm) and small (2 to ≤4 mm) size follicles was determined. Differential gene expression of granulosa secreted factors and their receptors were compared between the cumulus cells of the two groups. Then, the best time and concentration for arresting oocytes at the germinal vesicle stage by natriuretic peptide type C (CNP) were determined by nuclear staining in both groups. Oocyte quality was further confirmed by calcein uptake and gene expression. The developmental competence of cumulus oocyte complexes derived from small size follicles that were cultured in the presence of CNP in combination with amphiregulin (AREG) and prostaglandin E2 (PGE2) for 24 h was determined. Finally, embryo quality was specified by assessing expressions of NANOG, SOX2, CDX2, OCT4, and TET1. The cumulus oocyte complexes derived from small size follicles had a lower capacity to form blastocyst in comparison with cumulus oocyte complexes derived from medium size follicles. Prostaglandin E receptor 2 and prostaglandin-endoperoxide synthase 2 had significantly lower expression in cumulus cells derived from small size follicles in comparison with cumulus cells derived from medium size follicles. Natriuretic peptide type C increased the percentage of cumulus oocyte complexes arresting at the germinal vesicle stage in both oocytes derived from medium and small follicles. Gap junction communication was also improved in the presence of natriuretic peptide type C. In oocytes derived from small size follicles; best blastocyst rates were achieved by sequential exposure of cumulus oocyte complexes in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)] and [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. Increased SOX2 expression was observed in [TCM+CNP (6 h), then cultured in TCM+AREG+PGE2 (18h)], while decreased OCT4 expression was observed in [TCM+CNP (6 h), then cultured in conventional IVM supplements+AREG+PGE2 (18h)]. It seems that the natriuretic peptide type C modulates meiotic progression, and oocyte development is probably mediated by amphiregulin and prostaglandin E2. These results may provide an alternative IVM method to optimize in vitro embryo production in sheep and subsequently for humans.


Assuntos
Meios de Cultura/farmacologia , Células do Cúmulo/citologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/citologia , Anfirregulina/farmacologia , Animais , Biomarcadores , Células Cultivadas , Meios de Cultura/química , Células do Cúmulo/metabolismo , Dinoprostona/farmacologia , Feminino , Fertilização In Vitro , Fluoresceínas/metabolismo , Meiose , Modelos Animais , Peptídeo Natriurético Tipo C/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano/efeitos dos fármacos , Ovinos
9.
Artigo em Inglês | MEDLINE | ID: mdl-32208929

RESUMO

The human bronchial epithelium is an important barrier tissue that is damaged or pathologically altered in various acute and chronic respiratory conditions. To represent the epithelial component of respiratory disease, it is essential to use a physiologically relevant model of this tissue. The human bronchial epithelium is a highly organized tissue consisting of a number of specialized cell types. Primary human bronchial epithelial cells (HBEC) can be differentiated into a mucociliated tissue in air-liquid interface (ALI) cultures using appropriately supplemented media under optimized growth conditions. We compared the histology, ciliary length, and function, diffusion, and barrier properties of HBEC from donors with no respiratory disease grown in two different media, PneumaCult-ALI or Bronchial Epithelial Differentiation Medium (BEDM). In the former group, HBEC have a more physiological pseudostratified morphology and mucociliary differentiation, including increased epithelial thickness, intracellular expression of airway-specific mucin protein MUC5AC, and total expression of cilia basal-body protein compared with cells from the same donor grown in the other medium. Baseline expression levels of inflammatory mediators, thymic stromal lymphopoietin (TSLP), soluble ST2, and eotaxin-3 were lower in PneumaCult-ALI. Additionally, the physiological cilia beat frequency and electrical barrier properties with transepithelial electrical resistance were significantly different between the two groups. Our study has shown that these primary cell cultures from the same donor grown in the two media possess variable structural and functional characteristics. Therefore, it is important to objectively validate primary epithelial cell cultures before experimentation to ensure they are appropriate to answer a specific scientific question.


Assuntos
Meios de Cultura/farmacologia , Células Epiteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Ar , Brônquios/citologia , Brônquios/metabolismo , Diferenciação Celular/efeitos dos fármacos , Quimiocina CCL26/genética , Quimiocina CCL26/metabolismo , Cílios/efeitos dos fármacos , Cílios/metabolismo , Meios de Cultura/química , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Voluntários Saudáveis , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Modelos Biológicos , Mucina-5AC/genética , Mucina-5AC/metabolismo , Cultura Primária de Células , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo
10.
Nat Commun ; 11(1): 764, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034154

RESUMO

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.


Assuntos
Autorrenovação Celular/fisiologia , Células-Tronco Embrionárias Humanas/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ativinas/metabolismo , Animais , Blastocisto/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Endoderma/citologia , Endoderma/metabolismo , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Receptor IGF Tipo 1/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Transcriptoma , Inativação do Cromossomo X/fisiologia
11.
J Appl Microbiol ; 129(2): 311-318, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32052540

RESUMO

AIMS: The aim of this study was to develop a novel selective agar for the specific isolation and detection of Bacillus anthracis. METHODS AND RESULTS: Based on published data on antibiotic resistance and susceptibility of B. anthracis and other closely related species of the Bacillus cereus sensu lato group, a new selective agar formulation termed CEFOMA (Bacillus CEreus sensu lato group-specific antibiotics, FOsfomycin, MAcrolides) was developed and evaluated. All tested strains of B. anthracis were able to grow on CEFOMA with the same colony number as on non-selective media, whereas CEFOMA inhibited the growth of the other species within the B. cereus sensu lato group. In comparison to other selective agars, CEFOMA had a superior performance and considerably reduced the total amount of accompanying flora in soil. Furthermore, B. anthracis was successfully isolated from deliberately spiked soil samples. CONCLUSIONS: CEFOMA is a highly promising selective agar for the efficient isolation of B. anthracis from environmental samples with a large bacterial background flora. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolation of B. anthracis from environmental samples is severely impaired by the lack of adequate selective agars which suppress the growth of other bacteria. CEFOMA agar represents an important improvement and suitable alternative to currently used selective agars.


Assuntos
Ágar/química , Bacillus anthracis/isolamento & purificação , Meios de Cultura/química , Ágar/farmacologia , Antibacterianos/análise , Antibacterianos/farmacologia , Bacillus anthracis/crescimento & desenvolvimento , Bacillus cereus/efeitos dos fármacos , Contagem de Colônia Microbiana , Meios de Cultura/farmacologia , Microbiologia do Solo , Especificidade da Espécie
12.
Arch Microbiol ; 202(4): 875-885, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31894393

RESUMO

The objective of this study was to assess the effects of some culture conditions [temperature (20, 30, 37 °C), incubation time (48, 72, 120 h), pH (5.0, 6.0, 7.0), NaCl concentration (0, 3, 6%), carbon (glucose, fructose, lactose), nitrogen (sodium nitrate, ammonium sulfate, bacto-peptone), and mineral sources (calcium carbonate, ferric chloride)] on the exopolysaccharide (EPS) production by lactic acid bacteria (LAB) strains (belonging to Lactobacillus (L.) plantarum, L. namurensis, and Pediococcus (P.) ethanolidurans species) isolated from naturally fermented pickles. The maximum EPS production was determined at 30 °C and pH 6.0. The highest amount of EPS was obtained after 120 h of incubation, with glucose as carbon source, bacto-peptone as nitrogen source and calcium carbonate as mineral source for most of the tested strains. The EPS formation was not stimulated by NaCl, indicating that EPS formation of the tested strains was not a stress response. L. plantarum MF460 produced the highest amount of EPS at 30 °C after 48 h of incubation, which was 515.48 mg/L. One of the most pronounced results of this study was that the EPS production of L. plantarum MF556 strain was increased up to 512.81 mg/L with the addition of calcium carbonate to MRS medium. The effect of different culture conditions, particularly of incubation time, carbon, nitrogen, and mineral sources, on the EPS production often vary depending on the strain. Therefore, these apparent strain specific results demonstrated that the optimum culture conditions for the enhanced EPS production should be specifically determined for each LAB strain.


Assuntos
Meios de Cultura/farmacologia , Alimentos e Bebidas Fermentados/microbiologia , Microbiologia de Alimentos , Lactobacillales/efeitos dos fármacos , Lactobacillales/metabolismo , Polissacarídeos Bacterianos/biossíntese , Fermentação , Lactobacillales/isolamento & purificação , Temperatura
13.
Gene ; 734: 144381, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-31978510

RESUMO

Down-regulation of stemness genes expression is important in differentiation therapy against cancer stem cells (CSCs). The aim of this study was to evaluate the Oct4 , Sox2, Nanog, and C-myc expression in rat breast cancer stem cells (LA7) which treated with human ovarian follicular fluid (FF), replicative senescent fibroblast culture supernatant (P14), and 16 h serum starved fibroblast supernatant (16 h-SFS). The cells were exposed to these biological fluids for 24 h, 72 h, and 7 days. Stem-loop RT-qPCR assay was used to quantify the expression of above mentioned genes. Results showed that FF had the least cytotoxic effect on the LA7 cells. Except for Nanog gene, exposure of LA7 cell line to 16 h-SFS and P14 decreased significantly expression of the three other genes after 24 h (P < 0.05). Nanog and Sox2 genes expression was also decreased in LA7 cells which have been already treated with FF for 24 h. Moreover, compared to the control solution, the expression of Oct4 increased significantly after 7 days exposure to FF (P < 0.05). Annexin V-PE /7-AAD-, acridine orange/ethidium bromide staining and doubling time assays revealed apoptosis and necrosis induction by these biological fluids in LA7 cells. Moreover, in an in vitro model of metastasis assay, i.e., scratch test, these fluids exhibited anti-LA7 migration activity which culminated in 16 h-SFS treated cells. Generally, this study showed that FF, 16 h-SFS, and P14 have positive effects on down-regulation of Nanog, Oct4, Sox2 and C-myc expression, and consequently can increase the differentiation of breast cancer stem cells. For the first time, this study provided some evidence indicating that some biological fluids have potential to differentiate the CSCs, show anti- survival, growth-, and cell migration activity.


Assuntos
Líquidos Corporais/fisiologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/genética , Células-Tronco Neoplásicas , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Meios de Cultura/farmacologia , Regulação para Baixo , Feminino , Líquido Folicular/fisiologia , Genes myc , Humanos , Proteína Homeobox Nanog/genética , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genética
14.
PLoS One ; 15(1): e0220020, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31971939

RESUMO

BACKGROUND: In a previous study we found a significant correlation between dystocia and hyponatraemia that developed during labour. The present study examined a possible causal relationship. In vitro studies often use area under the curve (AUC) determined by frequency and force of contractions as a measure of myometrial contractility. However, a phase portrait plot of isometric contraction, obtained by plotting the first derivate of contraction against force of contraction, could indicate that bi-or multiphasic contractions might be less effective compared to the smooth contractions. MATERIAL AND METHODS: Myometrial biopsies were obtained from 17 women undergoing elective caesarean section at term. Each biopsy was divided into 8 strips and mounted isometrically in a force transducer. Seven biopsies were used in the first part of the study when half of the strips were immersed in the hyponatraemic study solution S containing Na+ 120 mmol/L and observed for 1 hour, followed by 1 hour in normonatraemic control solution C containing Na+ 136 mmol/L, then again in S for 1 hour, and finally 1 hour in C. The other half of the strips were studied in reverse order, C-S-C-S. The remaining ten biopsies were included in the second part of the study. Response to increasing doses of oxytocin (OT) in solutions S and C was studied. In the first part of the study we calculated AUC, and created phase portrait plots of two different contractions from the same strip, one smooth and one biphasic. In both parts of the study we registered frequency and force of contractions, and described appearance of the contractions. RESULTS: First part of the study: Mean (median) contractions per hour in C: 8.7 (7.6), in S 14,3 (13). Mean (SD) difference between groups 5.6 (4.2), p = 0.018. Force of contractions in C: 11.8 (10.2) mN, in S: 10.8 (9.2) mN, p = 0.09, AUC increased in S; p = 0.018. Bi-/multiphasic contractions increased from 8% in C to 18% in S, p = 0.001. All changes were reversible in C. Second part of the study: Frequency after OT 1.65 x 10-9 M in C:3.4 (2.9), in S: 3.8 (3.2), difference between groups: p = 0.48. After OT 1.65 x 10-7 M in C: 7.8 (8.9), increase from previous OT administration: p = 0.09, in S: 8.7 (9.0), p = 0.04, difference between groups, p = 0.32. Only at the highest dose of OT dose was there an increase in force of contraction in S, p = 0.05, difference between groups, p = 0.33. Initial response to OT was more frequently bi/multiphasic in S, reaching significance at the highest dose of OT(1.65 x 10-7 M), p = 0.015. when almost all contractions were bi/multiphasic. CONCLUSION: Hyponatraemia reversibly increased frequency of contractions and appearance of bi-or multiphasic contractions, that could reduce myometrial contractility. This could explain the correlation of hyponatraemia and instrumental delivery previously observed. Contractions in the hyponatraemic solution more frequently showed initial multiphasic contractions when OT was added in increasing doses. Longer lasting labours carry the risk both of hyponatraemia and OT administration, and their negative interaction could be significant. Further studies should address this possibility.


Assuntos
Meios de Cultura/farmacologia , Miométrio/efeitos dos fármacos , Ocitocina/farmacologia , Sódio/farmacologia , Contração Uterina/efeitos dos fármacos , Adulto , Área Sob a Curva , Biópsia , Cesárea , Meios de Cultura/química , Distocia/metabolismo , Distocia/fisiopatologia , Feminino , Humanos , Hiponatremia/metabolismo , Hiponatremia/fisiopatologia , Contração Isométrica/efeitos dos fármacos , Modelos Biológicos , Miométrio/metabolismo , Projetos Piloto , Gravidez , Técnicas de Cultura de Tecidos
15.
Int J Mol Sci ; 21(2)2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952279

RESUMO

To investigate environmental factors that contribute to ultraviolet A (UVA)-induced oxidative stress, which accelerates the senescence and toxicity of skin cells, we irradiated human fibroblasts cultured in commonly used essential media with UVA and evaluated their viability and production of reactive oxygen species. The viability of fibroblasts exposed to a single dose of 3.6 J/cm2 UVA was not reduced when cultured in Hanks balanced salt solution, but it was significantly decreased when cultured in Dulbecco's modified Eagle's medium (DMEM), which contains various amino acids and vitamins. Furthermore, cell viability was not reduced when fibroblasts were cultured in DMEM and treated with a hydrogen peroxide (H2O2) scavenger such as glutathione or catalase added after UVA irradiation. In addition, we confirmed that the production of H2O2 was dramatically increased by UVA photosensitization when riboflavin (R) coexisted with amino acids such as tryptophan (T), and found that R with folic acid (F) produced high levels of H2O2 after UVA irradiation. Furthermore, we noticed that R and F or R and T have different photosensitization mechanisms since NaN3, which is a singlet oxygen quencher, suppressed only R and T photosensitization. Lastly, we examined the effects of antioxidants (L-ascorbic acid, trolox, L-cysteine, and L-histidine), which are singlet oxygen or superoxide or H2O2 scavengers, on R and F or on R and T photosensitization, and found that 1 mM ascorbic acid, Trolox, and L-histidine were strongly photosensitized with R, and produced significant levels of H2O2 during UVA exposure. However, 1 mM L-cysteine dramatically suppressed H2O2 production by UVA photosensitization. These data suggest that a low concentration of R-derived photosensitization is elicited by different mechanisms depending on the coexisting vitamins and amino acids, and regulates cellular oxidative stress by producing H2O2 during UVA exposure.


Assuntos
Aminoácidos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Peróxido de Hidrogênio/metabolismo , Riboflavina/farmacologia , Raios Ultravioleta , Vitaminas/farmacologia , Aminoácidos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Humanos , Recém-Nascido , Masculino , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/efeitos da radiação , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/metabolismo , Vitaminas/metabolismo
16.
Int J Oncol ; 56(2): 606-617, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894296

RESUMO

Abnormal metabolism serves a critical role in the development and progression of different types of malignancies including glioblastoma (GBM), and may therefore serve as a promising target for treatment of cancer. Preclinical studies have indicated that a ketogenic diet (KD) may exhibit beneficial effects in patients with GBM; however, the underlying mechanisms remain incompletely understood. The aim of the present study was to evaluate the effects of a KD on glioma stem­like cells (GSCs), by culturing patient­derived primary GSCs as well as a GSC cell line in glucose­restricted, ß­hydroxybutyrate­containing medium (BHB­Glow) which was used to mimic clinical KD treatment. GSCs cultured in BHB­Glow medium exhibited reduced proliferation and increased apoptosis compared with cells grown in the control medium. Furthermore, decreased expression of stem cell markers, diminished self­renewal in vitro, and reduced tumorigenic capacity in vivo, providing evidence that the stemness of GSCs was compromised. Mechanistically, culturing in BHB­Glow medium reduced glucose uptake and inhibited glycolysis in GSCs. Furthermore, culturing in the BHB­Glow medium resulted in morphological and functional disturbances to the mitochondria of GSCs. These metabolic changes may have reduced ATP production, promoted lactic acid accumulation, and thus, increased the production of reactive oxygen species (ROS) in GSCs. The expression levels and activation of mammalian target of rapamycin, hypoxia­inducible factor 1 and B­cell lymphoma 2 were decreased, consistent with the reduced proliferation of GSCs in BHB­Glow medium. ROS scavenging reversed the inhibitory effects of a KD on GSCs. Taken together, the results demonstrate that treatment with KD inhibited proliferation of GSCs, increased apoptosis and attenuated the stemness in GSCs by increasing ROS production.


Assuntos
Ácido 3-Hidroxibutírico/farmacologia , Neoplasias Encefálicas/dietoterapia , Dieta Cetogênica , Glioblastoma/dietoterapia , Células-Tronco Neoplásicas/patologia , Adolescente , Adulto , Idoso , Animais , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Glioblastoma/cirurgia , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais Cultivadas , Adulto Jovem
17.
Ann Biol Clin (Paris) ; 78(1): 47-53, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31974073

RESUMO

Rapid and accurate identification of pathogens involved in urinary tract infections helps to guide antimicrobial therapy. Chromogenic agars provide presumptive identification directly from primary isolation media. They have been intended to make the bacterial isolation and identification process easier and faster. Our study aimed to compare the performance and the cost of the CPS ID3® and the Uriselect4® chromogenic agars with the conventional method for the isolation and identification of urinary tract infections bacteria. We included 301 urinary samples in a prospective study conducted in May 2018 in the clinical laboratory of the National institute of nutrition and food technology of Tunis. Isolates from routine media were identified using API® system while isolates from chromogenic media were directly identified by colony color with reference to the manufacturer's recommendations. Chromogenic media yielded more pure positive cultures and allowed better isolation of Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, Morganella morganii and Streptococcus agalactiae. Sensitivity and specificity of the presumptive identification of most commonly isolated uropathogens were higher with the Uriselect4® medium than with the CPS ID3® medium. Chromogenic media yielded the identification of pathogenic organisms 24 hours sooner than the conventional method in approximately 63 % of cases with the CPS ID3® medium and in 77.7% of cases with the Uriselect4® medium. Chromogenic media allowed a much better isolation of bacteria commonly involved in urinary tract infections with a quick, easy and accurate presumptive identification especially with the Uriselect4® medium.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/microbiologia , Técnicas Bacteriológicas/métodos , Compostos Cromogênicos/farmacologia , Meios de Cultura/farmacologia , Infecções Urinárias/microbiologia , Ágar/química , Antibacterianos/farmacologia , Bactérias/classificação , Infecções Bacterianas/diagnóstico , Técnicas Bacteriológicas/normas , Compostos Cromogênicos/química , Meios de Cultura/química , Testes Diagnósticos de Rotina/métodos , Testes Diagnósticos de Rotina/normas , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Sensibilidade e Especificidade , Urinálise/métodos , Urinálise/normas , Infecções Urinárias/diagnóstico
18.
Arch Microbiol ; 202(1): 209-212, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31482327

RESUMO

Different methods to analyze Streptococcus agalactiae biofilm formation have been investigated, but standardized protocols have not been developed. We compared S. agalactiae biofilm production among different atmospheres and growth media. Biofilm formation was studied in 32 isolates from bovine mastitis cases grown in Tryptone Soy Broth (TSB), Todd Hewitt Broth (THB), Luria Bertani Broth (LB) and Brain Heart Infusion (BHI), under two atmospheres, aerobic and 5% CO2. Regardless of the culture medium, growth under 5% CO2 resulted in a greater proportion of biofilm formation (65.63%), as compared with aerobic conditions (39.84%). Regardless of the atmosphere, the chances of biofilm formation were greater for isolates grown in TSB, as compared with THB [Odds ratio (OR) = 3.02], BHI (OR = 4.57), or LB (OR = 10.20). Thus, we suggest the use of 5% CO2 atmosphere and TSB in biofilm formation assays by Group-B streptococci (GBS) isolated from intramammary infections.


Assuntos
Biofilmes/crescimento & desenvolvimento , Mastite Bovina/microbiologia , Leite/microbiologia , Streptococcus agalactiae/fisiologia , Animais , Atmosfera , Bovinos , Meios de Cultura/farmacologia , Feminino , Streptococcus agalactiae/efeitos dos fármacos
19.
Artigo em Inglês | MEDLINE | ID: mdl-31678517

RESUMO

Full thickness models (FTMs) are 3D-cultured human skin models that mimic many aspects of native human skin (NHS). However, their stratum corneum (SC) lipid composition differs from NHS causing a reduced skin barrier. The most pronounced differences in lipid composition are a reduction in lipid chain length and increased monounsaturated lipids. The liver-X-receptor (LXR) activates the monounsaturated lipid synthesis via stearoyl-CoA desaturase-1 (SCD-1). Therefore, the aim was to improve the SC lipid synthesis of FTMs by LXR deactivation. This was achieved by supplementing culture medium with LXR antagonist GSK2033. LXR agonist T0901317 was added for comparison. Subsequently, epidermal morphogenesis, lipid composition, lipid organization and the barrier functionality of these FTMs were assessed. We demonstrate that LXR deactivation resulted in a lipid composition with increased overall chain lengths and reduced levels of monounsaturation, whereas LXR activation increased the amount of monounsaturated lipids and led to a reduction in the overall chain length. However, these changes did not affect the barrier functionality. In conclusion, LXR deactivation led to the development of FTMs with improved lipid properties, which mimic the lipid composition of NHS more closely. These novel findings may contribute to design interventions to normalize SC lipid composition of atopic dermatitis patients.


Assuntos
Meios de Cultura/farmacologia , Receptores X do Fígado/antagonistas & inibidores , Cultura Primária de Células/métodos , Pele/efeitos dos fármacos , Sulfonamidas/farmacologia , Ceramidas/metabolismo , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Avaliação Pré-Clínica de Medicamentos/métodos , Ácidos Graxos não Esterificados , Humanos , Hidrocarbonetos Fluorados/farmacologia , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/metabolismo , Morfogênese/efeitos dos fármacos , Pele/crescimento & desenvolvimento , Pele/metabolismo , Estearoil-CoA Dessaturase/metabolismo
20.
Theriogenology ; 142: 207-215, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31614287

RESUMO

One of the major challenges of artificial reproductive technologies is to develop new methods for producing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo before oocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), a purine nucleoside found in follicular fluid, on the inhibition of oocyte meiotic resumption and the production of blastocysts. The results showed the efficacy of ADO to inhibit oocyte meiotic resumption. The use of ADO (3 mM) during a pre-in vitro maturation (pre-IVM) culture period of 6 h resulted in a significant increase (p < 0.05) of blastocysts compared to control conditions with no pre-IVM culture period. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yield was time- and concentration-dependent with an optimum effect at 3 mM for 6 h. Supplementing the ADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermal growth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptional analyses of ADO-treated cumulus cells revealed that NRP1, RELN, MAN1A1, THRA and GATM were up-regulated. Finally, bioinformatic analysis identified mitochondrial function as the top canonical pathway affected by ADO. This opens up new opportunities for further investigations.


Assuntos
Adenosina/farmacologia , Bovinos , Técnicas de Cultura de Células/métodos , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Bovinos/embriologia , Bovinos/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/métodos , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário/genética , Feminino , Fertilização In Vitro/métodos , Fertilização In Vitro/veterinária , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Análise em Microsséries , Oócitos/citologia , Oócitos/fisiologia
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