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1.
Cochrane Database Syst Rev ; 9: CD007421, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32876946

RESUMO

BACKGROUND: This is an update of a Cochrane Review first published in the Cochrane Library (2010, Issue 7). To increase the success rate of assisted reproductive technologies (ARTs), adherence compounds such as hyaluronic acid (HA) have been introduced into subfertility management. Adherence compounds are added to the embryo transfer medium to increase the likelihood of embryo implantation, with the potential for higher clinical pregnancy and live birth rates. OBJECTIVES: To determine whether adding adherence compounds to embryo transfer media could improve pregnancy outcomes, including improving live birth and decreasing miscarriage, in women undergoing assisted reproduction. SEARCH METHODS: We searched the Cochrane Gynaecology and Fertility Group Trials Register, CENTRAL, MEDLINE, Embase, and PsycINFO electronic databases on 7 January 2020 for randomised controlled trials that examined the effects of adherence compounds in embryo transfer media on pregnancy outcomes. Furthermore, we communicated with experts in the field, searched trials registries, checked reference lists of relevant studies, and conference abstracts were handsearched. SELECTION CRITERIA: Only truly randomised controlled trials comparing embryo transfer media containing functional concentrations of adherence compounds to media with no or low adherence compound concentrations were included. DATA COLLECTION AND ANALYSIS: Two review authors selected trials for inclusion according to the above criteria, after which the same two review authors independently extracted data for subsequent analysis. Statistical analysis was performed according to the guidelines developed by Cochrane. We combined data to calculate pooled risk ratios (RRs) and 95% confidence intervals (CIs). We assessed statistical heterogeneity using the I² statistic. We used GRADE methods to assess the overall quality of evidence for the main comparisons. MAIN RESULTS: We analysed 26 studies with a total of 6704 participants. Overall, the certainty of evidence was low to moderate: the main limitations were imprecision and/or heterogeneity. Compared to embryos transferred in media containing no or low (0.125 mg/mL) HA, the addition of functional (0.5 mg/mL) HA concentrations to the transfer media probably increases the live birth rate (RR 1.21, 95% CI 1.1 to 1.31; 10 RCTs, N = 4066; I² = 33%; moderate-quality evidence). This suggests that if the chance of live birth following no HA addition in media is assumed to be 33%, the chance following HA addition would be between 37% and 44%. The addition of HA may slightly decrease miscarriage rates (RR 0.82, 95% CI 0.67 to 1.00; 7 RCTs, N = 3091; I² = 66%; low-quality evidence). Nevertheless, when only studies with low risk of bias were included in the analysis, there was no conclusive evidence of a difference in miscarriage rates (RR 0.96, 95% CI 0.75 to 1.23; N = 2219; I² = 36%). Adding HA to transfer media probably results in an increase in both clinical pregnancy (RR 1.16, 95% CI 1.09 to 1.23; 17 studies, N = 5247; I² = 40%; moderate-quality evidence) and multiple pregnancy rates (RR 1.45, 95% CI 1.24 to 1.70; 7 studies, N = 3337; I² = 36%; moderate-quality evidence). We are uncertain of the effect of HA added to transfer media on the rate of total adverse events (RR 0.86, 95% CI 0.40 to 1.84; 3 studies, N = 1487; I² = 0%; low-quality evidence). AUTHORS' CONCLUSIONS: Moderate-quality evidence shows improved clinical pregnancy and live birth rates with the addition of HA as an adherence compound in embryo transfer media in ART. Low-quality evidence suggests that adding HA may slightly decrease miscarriage rates, but when only studies at low risk of bias were included in the analysis, the results were inconclusive. HA had no clear effect on the rate of total adverse events. The increase in multiple pregnancy rates may be due to combining an adherence compound and transferring more than one embryo. Further studies of adherence compounds with single embryo transfer need to be undertaken.


Assuntos
Meios de Cultura/química , Implantação do Embrião/efeitos dos fármacos , Adesivo Tecidual de Fibrina/farmacologia , Ácido Hialurônico/farmacologia , Técnicas de Reprodução Assistida , Adesivos Teciduais/farmacologia , Aborto Espontâneo/epidemiologia , Adulto , Implantação do Embrião/fisiologia , Feminino , Humanos , Nascimento Vivo/epidemiologia , Gravidez , Gravidez Múltipla/estatística & dados numéricos , Ensaios Clínicos Controlados Aleatórios como Assunto
2.
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
3.
Methods Mol Biol ; 2203: 241-261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833217

RESUMO

Coronavirus entry encompasses the initial steps of infection, from virion attachment to genome release. Advances in fluorescent labeling of viral and cellular components and confocal imaging enable broad spectrum studies on this process. Here, we describe methods for visualization of coronavirus entry into immortalized cell lines and 3D tissue culture models.


Assuntos
Coronavirus/patogenicidade , Interações Hospedeiro-Patógeno/fisiologia , Microscopia Confocal/métodos , Linhagem Celular , Coronavirus/isolamento & purificação , Meios de Cultura/química , Endocitose , Humanos , Proteínas do Nucleocapsídeo/metabolismo , Ácidos Tri-Iodobenzoicos/química , Internalização do Vírus
4.
PLoS One ; 15(8): e0236700, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32750088

RESUMO

Mycobacterial culture remains the gold standard for the diagnosis of tuberculosis. However, an appropriate digestion and decontamination method (DDM) is essential for the effective recovery of tubercle bacilli in culture. Therefore, the current study was designed to compare the performance of papain-cetylpyridinium chloride [papain-CPC] and pepsin-cetylpyridinium chloride [pepsin-CPC] DDMs against N-acetyl L-Cysteine-sodium hydroxide (NALC-NaOH) DDM for recovery of mycobacteria from clinically suspected pulmonary tuberculosis cases. To evaluate papain-CPC, pepsin-CPC and NALC-NaOH DDMs, sputum samples (N = 1381) were cultured on Löwenstein-Jensen medium and the results were compared. The papain-CPC DDM showed sensitivity, specificity, positive predictive value, and negative predictive value of 100%, 93.27%, 71.7%, and 100%, respectively as compared to NALC-NaOH DDM. Similarly, pepsin-CPC DDM demonstrated sensitivity, specificity, positive predictive value and negative predictive value of 98.94%, 94.7%, 76.11%, and 99.81%, respectively. In summary, both papain-CPC and pepsin-CPC DDMs are highly sensitive and specific techniques for recovery of mycobacteria as compared to NALC-NaOH DDM. However, when the overall performances of all DDMs compared, papain-CPC DDM isolated increased number of mycobacterial isolates with comparatively higher numbers of colonies on LJ media than both pepsin-CPC and NALC-NaOH DDMs, indicating its potential to replace the NALC-NaOH DDM for recovery of mycobacteria from sputum samples.


Assuntos
Técnicas Bacteriológicas/métodos , Meios de Cultura/química , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Pulmonar/microbiologia , Acetilcisteína/química , Cetilpiridínio/química , Humanos , Papaína/química , Pepsina A/química , Sensibilidade e Especificidade , Manejo de Espécimes
5.
Ecotoxicol Environ Saf ; 204: 111042, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32738626

RESUMO

Paralytic shellfish poisoning (PSP) toxins have received considerable attention in recent years because of their adverse effects on marine breeding industries and human health. In this study, a reliable method for the analysis of extracellular PSP toxins in the culture medium of marine toxic dinoflagellates was developed for the first time using graphitized carbon black-solid-phase extraction and hydrophilic interaction liquid chromatography-high-resolution mass spectrometry. The limit of quantification of typical PSP toxins in algal culture medium ranged from 0.072 µg/L to 0.151 µg/L under optimal conditions. Satisfactory absolute recoveries (87.5%-102.4%), precision (relative standard deviation ≤ 7.6%), and linearity (R2 ≥ 0.9998) were also achieved. In addition, the proposed method was applied to screen and determine the extracellular PSP toxins of two typical toxigenic dinoflagellates, Alexandrium minutum and Alexandrium tamarense. The total concentrations of the extracellular PSP toxins in A. minutum and A. tamarense over the whole growth period were within 2.0-735.5 and 2.0-19.2 µg/L, respectively. The concentrations of extracellular PSP toxins varied remarkably in the different growth stages of A. minutum and A. tamarense, and the contents of some extracellular PSP toxins were substantially higher than those of intracellular PSP toxins. Therefore, the extracellular PSP toxins released by toxigenic red tide algae cannot be ignored, and their environmental fate, bioavailability, and potential harm to aquatic environment need to be investigated in future studies.


Assuntos
Cromatografia Líquida/métodos , Meios de Cultura/química , Dinoflagelados/metabolismo , Toxinas Marinhas/análise , Espectrometria de Massas/métodos , Extração em Fase Sólida/métodos , Interações Hidrofóbicas e Hidrofílicas , Intoxicação por Frutos do Mar , Fuligem/química
6.
Cochrane Database Syst Rev ; 7: CD013497, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32672358

RESUMO

BACKGROUND: GM-CSF (granulocyte macrophage colony-stimulating factor) is a growth factor that is used to supplement culture media in an effort to improve clinical outcomes for those undergoing assisted reproduction. It is worth noting that the use of GM-CSF-supplemented culture media often adds a further cost to the price of an in vitro fertilisation (IVF) cycle. The purpose of this review was to assess the available evidence from randomised controlled trials (RCTs) on the effectiveness and safety of GM-CSF-supplemented culture media. OBJECTIVES: To assess the effectiveness and safety of GM-CSF-supplemented human embryo culture media versus culture media not supplemented with GM-CSF, in women or couples undergoing assisted reproduction. SEARCH METHODS: We used standard methodology recommended by Cochrane. We searched the Cochrane Gynaecology and Fertility Group Trials Register, CENTRAL, MEDLINE, Embase, CINAHL, LILACS, DARE, OpenGrey, PubMed, Google Scholar, and two trials registers on 15 October 2019, checked references of relevant papers and communicated with experts in the field. SELECTION CRITERIA: We included RCTs comparing GM-CSF (including G-CSF (granulocyte colony-stimulating factor))-supplemented embryo culture media versus any other non-GM-CSF-supplemented embryo culture media (control) in women undergoing assisted reproduction. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures recommended by Cochrane. The primary review outcomes were live birth and miscarriage rate. The secondary outcomes were clinical pregnancy, multiple gestation, preterm birth, birth defects, aneuploidy, and stillbirth rates. We assessed the quality of the evidence using GRADE methodology. We undertook one comparison, GM-CSF-supplemented culture media versus culture media not supplemented with GM-CSF, for those undergoing assisted reproduction. MAIN RESULTS: We included five studies, the data for three of which (1532 participants) were meta-analysed. We are uncertain whether GM-CSF-supplemented culture media makes any difference to the live-birth rate when compared to using conventional culture media not supplemented with GM-CSF (odds ratio (OR) 1.19, 95% confidence interval (CI) 0.93 to 1.52, 2 RCTs, N = 1432, I2 = 69%, low-quality evidence). The evidence suggests that if the rate of live birth associated with conventional culture media not supplemented with GM-CSF was 22%, the rate with the use of GM-CSF-supplemented culture media would be between 21% and 30%. We are uncertain whether GM-CSF-supplemented culture media makes any difference to the miscarriage rate when compared to using conventional culture media not supplemented with GM-CSF (OR 0.75, 95% CI 0.41 to 1.36, 2 RCTs, N = 1432, I2 = 0%, low-quality evidence). This evidence suggests that if the miscarriage rate associated with conventional culture media not supplemented with GM-CSF was 4%, the rate with the use of GM-CSF-supplemented culture media would be between 2% and 5%. Furthermore, we are uncertain whether GM-CSF-supplemented culture media makes any difference to the following outcomes: clinical pregnancy (OR 1.16, 95% CI 0.93 to 1.45, 3 RCTs, N = 1532 women, I2 = 67%, low-quality evidence); multiple gestation (OR 1.24, 95% CI 0.73 to 2.10, 2 RCTs, N = 1432, I2 = 35%, very low-quality evidence); preterm birth (OR 1.20, 95% CI 0.70 to 2.04, 2 RCTs, N = 1432, I2 = 76%, very low-quality evidence); birth defects (OR 1.33, 95% CI 0.59 to 3.01, I2 = 0%, 2 RCTs, N = 1432, low-quality evidence); and aneuploidy (OR 0.34, 95% CI 0.03 to 3.26, I2 = 0%, 2 RCTs, N = 1432, low-quality evidence). We were unable to undertake analysis of stillbirth, as there were no events in either arm of the two studies that assessed this outcome. AUTHORS' CONCLUSIONS: Due to the very low to low quality of the evidence, we cannot be certain whether GM-CSF is any more or less effective than culture media not supplemented with GM-CSF for clinical outcomes that reflect effectiveness and safety. It is important that independent information on the available evidence is made accessible to those considering using GM-CSF-supplemented culture media. The claims from marketing information that GM-CSF has a positive effect on pregnancy rates are not supported by the available evidence presented here; further well-designed, properly powered RCTs are needed to lend certainty to the evidence.


Assuntos
Meios de Cultura/química , Fertilização In Vitro/métodos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Aborto Espontâneo/epidemiologia , Aneuploidia , Viés , Intervalos de Confiança , Anormalidades Congênitas/epidemiologia , Feminino , Humanos , Nascimento Vivo , Gravidez , Taxa de Gravidez , Gravidez Múltipla/estatística & dados numéricos , Nascimento Prematuro/epidemiologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Técnicas de Reprodução Assistida
7.
PLoS One ; 15(7): e0236739, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730333

RESUMO

Rhodopseudomonas palustris PS3 is one of the purple phototrophic non-sulfur bacteria (PNSB), which have plant growth-promoting effects on various plants. To expand the scale of PS3 fermentation in a time- and cost-effective fashion, the purpose of this work was to evaluate the use of low-cost materials as culture media and to optimize the culture conditions via response surface methodology. Corn steep liquor (CSL) and molasses were identified as potential materials to replace the nitrogen and carbon sources, respectively, in the conventional growth medium. The optimum culture conditions identified through central composite design were CSL, 39.41 mL/L; molasses, 32.35 g/L; temperature, 37.9°C; pH, 7.0; and DO 30%. Under the optimized conditions, the biomass yield reached 2.18 ± 0.01 g/L at 24 hours, which was 7.8-fold higher than that under the original medium (0.28 ± 0.01 g/L). The correlation between the predicted and experimental values of the model was over 98%, which verified the validity of the response models. Furthermore, we verified the effectiveness of the R. palustris PS3 inoculant grown under the newly developed culture conditions for plant growth promotion. This study provides a potential strategy for improving the fermentation of R. palustris PS3 in low-cost media for large-scale industrial production.


Assuntos
Carbono/metabolismo , Meios de Cultura/química , Meios de Cultura/economia , Nitrogênio/metabolismo , Desenvolvimento Vegetal , Rodopseudomonas/crescimento & desenvolvimento , Meios de Cultura/metabolismo , Fermentação , Microbiologia Industrial , Rodopseudomonas/metabolismo
8.
Emerg Microbes Infect ; 9(1): 1415-1417, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-526750

RESUMO

SARS-CoV-2, the causative agent of the COVID-19 pandemic, may be transmitted via airborne droplets or contact with surfaces onto which droplets have deposited. In this study, the ability of SARS-CoV-2 to survive in the dark, at two different relative humidity values and within artificial saliva, a clinically relevant matrix, was investigated. SARS-CoV-2 was found to be stable, in the dark, in a dynamic small particle aerosol under the four experimental conditions we tested and viable virus could still be detected after 90 minutes. The decay rate and half-life was determined and decay rates ranged from 0.4 to 2.27 % per minute and the half lives ranged from 30 to 177 minutes for the different conditions. This information can be used for advice and modelling and potential mitigation strategies.


Assuntos
Aerossóis/química , Betacoronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/virologia , Meios de Cultura/química , Pneumonia Viral/virologia , Saliva Artificial/química , Salvia/virologia , Microbiologia do Ar , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/efeitos da radiação , Infecções por Coronavirus/transmissão , Escuridão , Humanos , Umidade , Cinética , Pandemias , Pneumonia Viral/transmissão
9.
PLoS One ; 15(6): e0234192, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479562

RESUMO

Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family.


Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supressão Genética , Ubiquinona/biossíntese , Substituição de Aminoácidos , Asparagina , Meios de Cultura/química , Regulação Fúngica da Expressão Gênica , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquinona/genética
10.
Viruses ; 12(6)2020 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-32517266

RESUMO

In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcrystalline cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Ensaio de Placa Viral/métodos , Inativação de Vírus , Animais , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/patogenicidade , Celulose , Chlorocebus aethiops , Meios de Cultura/química , Formaldeído , Guanidinas/farmacologia , Células HEK293 , Humanos , Pandemias , Fenóis/farmacologia , Propiolactona/farmacologia , Sefarose , Células Vero
11.
J Vis Exp ; (159)2020 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-32510508

RESUMO

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Meios de Cultura/química , Gânglios Parassimpáticos/citologia , Neurônios/citologia , Células-Tronco Pluripotentes/citologia , Células Cultivadas , Humanos , Reprodutibilidade dos Testes
12.
Emerg Microbes Infect ; 9(1): 1415-1417, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32496967

RESUMO

SARS-CoV-2, the causative agent of the COVID-19 pandemic, may be transmitted via airborne droplets or contact with surfaces onto which droplets have deposited. In this study, the ability of SARS-CoV-2 to survive in the dark, at two different relative humidity values and within artificial saliva, a clinically relevant matrix, was investigated. SARS-CoV-2 was found to be stable, in the dark, in a dynamic small particle aerosol under the four experimental conditions we tested and viable virus could still be detected after 90 minutes. The decay rate and half-life was determined and decay rates ranged from 0.4 to 2.27 % per minute and the half lives ranged from 30 to 177 minutes for the different conditions. This information can be used for advice and modelling and potential mitigation strategies.


Assuntos
Aerossóis/química , Betacoronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/virologia , Meios de Cultura/química , Pneumonia Viral/virologia , Saliva Artificial/química , Salvia/virologia , Microbiologia do Ar , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/efeitos da radiação , Infecções por Coronavirus/transmissão , Escuridão , Humanos , Umidade , Cinética , Pandemias , Pneumonia Viral/transmissão
13.
J Oleo Sci ; 69(5): 467-477, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32378550

RESUMO

Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. In the present study, thirty-seven bacterial isolates were isolated from soil contaminated with waste cooking oil, dairy waste etc. from Shimla and Solan district of H.P. Out of 37 isolates, the isolate RL-1, which gave maximum activity, was identified as Bacillus licheniformis MH061919. The optimization of various production parameters resulted in maximum activity at inoculum age of 24 h and inoculum size of 1.5% (v/v). Esterase gave considerable activity in production medium containing sodium chloride (0.5 % w/v), galactose (1%, w/v), coconut oil (2.0%, v/v) and beef extract (0.3%, w/v) at a temperature of 45℃ and pH 8.5.The enzyme production was enhanced by 3-fold after optimization of production parameters. Further, on optimizing reaction conditions, enzyme gave maximum activity at a temperature of 45℃ and pH 8.5. The para-nitrophenyl acetate (p-NPA) was found to be optimum substrate and metal ions and detergents have inhibitory effect on esterase activity.


Assuntos
Bacillus/enzimologia , Meios de Cultura/química , Técnicas de Cultura/métodos , Esterases/metabolismo , Bacillus/isolamento & purificação , Óleo de Coco , Galactose , Concentração de Íons de Hidrogênio , Nitrofenóis/metabolismo , Carne Vermelha , Cloreto de Sódio , Temperatura , Extratos de Tecidos
14.
J Biosci Bioeng ; 130(2): 195-199, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32370929

RESUMO

Ectoine production using inexpensive and renewable biomass resources has attracted great interest among the researchers due to the low yields of ectoine in current fermentation approaches that complicate the large-scale production of ectoine. In this study, ectoine was produced from corn steep liquor (CSL) and soybean hydrolysate (SH) in replacement to yeast extract as the nitrogen sources for the fermentation process. To enhance the bacterial growth and ectoine production, biotin was added to the Halomonas salina fermentation media. In addition, the effects addition of surfactants such as Tween 80 and saponin on the ectoine production were also investigated. Results showed that both the CSL and SH can be used as the nitrogen source substitutes in the fermentation media. Higher amount of ectoine (1781.9 mg L-1) was produced in shake flask culture with SH-containing media as compared to CSL-containing media. A total of 2537.0 mg L-1 of ectoine was produced at pH 7 when SH-containing media was applied in the 2 L batch fermentation. Moreover, highest amount of ectoine (1802.0 mg L-1) was recorded in the SH-containing shake flask culture with addition of 0.2 µm mL-1 biotin. This study demonstrated the efficacy of industrial waste as the nutrient supplement for the fermentation of ectoine production.


Assuntos
Diamino Aminoácidos/metabolismo , Fermentação , Halomonas/metabolismo , Microbiologia Industrial/métodos , Técnicas de Cultura Celular por Lotes , Biomassa , Biotina/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Resíduos Industriais , Nitrogênio/metabolismo , Soja/química , Zea mays/química
15.
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
16.
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
17.
J Vet Sci ; 21(2): e30, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32233136

RESUMO

Mycoplasma ovipneumoniae (Mo) is difficult to culture, resulting in many difficulties in related research and application. Since nucleotide metabolism is a basic metabolism affects growth, this study conducted a "point-to-point" comparison of the corresponding growth phases between the Mo NM151 strain and the Mycoplasma mycoides subsp. capri (Mmc) PG3 strain. The results showed that the largest difference in nucleotide metabolism was found in the stationary phase. Nucleotide synthesis in PG3 was mostly de novo, while nucleotide synthesis in NM151 was primarily based on salvage synthesis. Compared with PG3, the missing reactions of NM151 referred to the synthesis of deoxythymine monophosphate. We proposed and validated a culture medium with added serine to fill this gap and prolong the stationary phase of NM151. This solved the problem of the fast death of Mo, which is significant for related research and application.


Assuntos
Técnicas Bacteriológicas/veterinária , Meios de Cultura/química , Mycoplasma ovipneumoniae/crescimento & desenvolvimento , Transcriptoma , Técnicas Bacteriológicas/métodos , Mycoplasma ovipneumoniae/metabolismo , Nucleotídeos/metabolismo
18.
Oncol Rep ; 43(6): 1797-1804, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32236615

RESUMO

A tumor contains special types of cells that have characteristics similar to stem cells that aid in tumor initiation, evasion and proliferation and are often resistant to chemotherapy. These cancer stem cells can be differentiated to eradicate their stemness and proliferative capacity by differentiating agents. This study investigated the effect of differentiation on the expression of two immune checkpoint inhibitors, human leukocyte antigen­G (HLA­G) and programmed death ligand­1 (PD­L1). Two cancer cell lines (OVCAR­3­NIH and KATO­III) were treated with adipocyte and neurocyte differentiation media for 14 days. Bone­marrow derived mesenchymal stem cells (BM­MSCs) were used as control healthy stem cells. We found that the cancer cell lines (OVCAR­3­NIH and KATO­III) when subjected to differentiation lost their proliferation ability. BM­MSC proliferation was not halted but was decreased in the adipocyte differentiation media. There was no decrease in the CD90 stem cell marker in the BM­MSCs; however, both cancer cell lines showed decreased CD90 stem cell marker. A significant increase in HLA­G was noted for both the cancer cell lines following adipocyte differentiation. No effect was found for BM­MSCs. Moreover, an increase in PD­L1 in cancer cell lines was found following neurocyte differentiation. Moreover, we found that differentiation resulted in decreased PD­L1 expression in BM­MSCs. Differentiation therapy of cancer stem cells may result in increased immunosuppression ability, hence causing hindrance in the removal of cancer cells. Moreover, the differentiation of healthy stem cells can result in increased immunogenic reactivity owing to a decrease in PD­L1 expression.


Assuntos
Antígeno B7-H1/genética , Antígenos HLA-G/genética , Células-Tronco Mesenquimais/citologia , Neoplasias/genética , Antígeno B7-H1/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura/química , Regulação Neoplásica da Expressão Gênica , Antígenos HLA-G/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Neoplasias/metabolismo , Antígenos Thy-1/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-32233789

RESUMO

Air-liquid interface (ALI) cultures are ex vivo models that are used extensively to study the epithelium of patients with chronic respiratory diseases. However, the in vitro conditions impose a milieu different from that encountered in the patient in vivo, and the degree to which this alters gene expression remains unclear. In this study we employed RNA sequencing to compare the transcriptome of fresh brushings of nasal epithelial cells with that of ALI-cultured epithelial cells from the same patients. We observed a strong correlation between cells cultured at the ALI and cells obtained from the brushed nasal epithelia: 96% of expressed genes showed similar expression profiles, although there was greater similarity between the brushed samples. We observed that while the ALI model provides an excellent representation of the in vivo airway epithelial transcriptome for mechanistic studies, several pathways are affected by the change in milieu.


Assuntos
Mucosa Nasal/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Mucosa Respiratória/metabolismo , Transcriptoma , Idoso , Ar , Fumar Cigarros/efeitos adversos , Meios de Cultura/química , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Anotação de Sequência Molecular , Mucosa Nasal/patologia , Cultura Primária de Células , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Análise de Sequência de RNA , Conchas Nasais/metabolismo , Conchas Nasais/patologia
20.
Can J Microbiol ; 66(4): 303-312, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32118486

RESUMO

Herein we describe a highly structured, filamentous growth phenotype displayed by an isolate of the food spoilage microorganism Brochothrix thermosphacta. The growth morphology of this B. thermosphacta strain (strain BII) was dependent on environmental factors such as the growth media, incubation temperatures, and the inoculum concentration. Inoculation of cultures in highly dilute suspensions resulted in the formation of isolated, tight aggregates resembling fungal growth in liquid media. This same strain also formed stable, mesh-like structures in 6-well tissue culture plates under specific growth conditions. The complex growth phenotype does not appear to be unique to strain BII but was common among B. thermosphacta strains isolated from chicken. Light and electron micrographs showed that the filaments of multiple BII cells can organize into complex, tertiary structures resembling multistranded cables. Time-lapse microscopy was employed to monitor the development of such aggregates over 18 h and revealed growth originating from short filaments into compact ball-like clusters that appeared fuzzy due to protruding filaments or cables. This report is the first to document this complex filamentous growth phenotype in a wild-type bacterial isolate of B. thermosphacta.


Assuntos
Brochothrix/crescimento & desenvolvimento , Galinhas/microbiologia , Animais , Brochothrix/classificação , Brochothrix/isolamento & purificação , Brochothrix/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Contaminação de Alimentos/análise , Carne/microbiologia , Temperatura
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