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1.
Emerg Microbes Infect ; 9(1): 1415-1417, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-32496967

RESUMEN

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.


Asunto(s)
Aerosoles/química , Betacoronavirus/crecimiento & desarrollo , Infecciones por Coronavirus/virología , Medios de Cultivo/química , Neumonía Viral/virología , Saliva Artificial/química , Salvia/virología , Microbiología del Aire , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/efectos de la radiación , Infecciones por Coronavirus/transmisión , Oscuridad , Humanos , Humedad , Cinética , Pandemias , Neumonía Viral/transmisión
2.
Viruses ; 12(6)2020 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-32517266

RESUMEN

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.


Asunto(s)
Betacoronavirus/fisiología , Infecciones por Coronavirus/virología , Neumonía Viral/virología , Ensayo de Placa Viral/métodos , Inactivación de Virus , Animales , Betacoronavirus/efectos de los fármacos , Betacoronavirus/crecimiento & desarrollo , Betacoronavirus/patogenicidad , Celulosa , Chlorocebus aethiops , Medios de Cultivo/química , Formaldehído , Guanidinas/farmacología , Células HEK293 , Humanos , Pandemias , Fenoles/farmacología , Propiolactona/farmacología , Sefarosa , Células Vero
4.
Ecotoxicol Environ Saf ; 193: 110350, 2020 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-32114242

RESUMEN

Epidemiological studies have shown associations between exposure to environmental extremely low frequency magnetic fields (ELF-MF) and health effects, but the mechanisms of ELF-MF induced biological effects remain unclear. We hypothesized that ELF-MF may regulate functions of tissues or cells via its effects on surrounding environment, e.g., culture medium. To test this hypothesis, we investigated the effects of 50 Hz MF on the relative permittivity of zebrafish embryos culture medium as well as of MF-exposed medium on zebrafish embryos development. The responses of medium to 50 Hz MF exposure were evaluated by a phase-sensitive surface plasmon resonance (SPR) system. The results demonstrated that MF treatment decreased relative permittivity of zebrafish embryos medium in a dose and time-dependent way. Interestingly, the decreased permittivity induced by MF exposure gradually recovered and approached to the base level when the exposure was removed off. However, MF-exposed medium did not trigger adverse consequences of embryos during zebrafish embryonic development, including mortality, malformation, hatching and heart rate when the MF pre-exposed medium was subjected to one cell-stage embryos. Moreover, the MF-exposed medium did not induce apoptosis of zebrafish embryos at 48 and 72 h post fertilization. Our data demonstrated that the relative permittivity of zebrafish embryos medium was decreased by MF exposure, whereas this decrease failed to result in abnormal development of zebrafish embryos.


Asunto(s)
Desarrollo Embrionario , Campos Magnéticos/efectos adversos , Animales , Apoptosis , Medios de Cultivo , Pez Cebra/embriología
5.
PLoS One ; 15(3): e0229556, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32196504

RESUMEN

The heterotrophic microalgae Crypthecodinium cohnii was usually cultivated in complex medium containing glucose, yeast extract and sea salt. For the preparation of DHA with highest purity, a new defined medium without the yeast extract was developed. Different inoculated densities, C/N ratios, temperatures, culture volumes and glucose additions were investigated to optimize the algal growth rate and DHA production. The growth period in C. cohnii was shortened from 12-14 days to 7-8 days, the OD600 was enhanced from 2.0 to 3.0, the glucose consumption was accelerated and used up on day 3-4, and the DHA content in culture were increased from 10 to 45 nmoles/300 µl batch. It was found that C. cohnii had optimal growth and DHA accumulation in 25 °C, 0.2 inoculated density, 5-10 C/N ratio, 5:1 air/culture volume ratio. This is the first time DHA production using C.cohnii has been optimized in synthetic medium. This allows preparation of uniformly radiolabeled 13C- and 14C-DHA.


Asunto(s)
Medios de Cultivo/química , Dinoflagelados/crecimiento & desarrollo , Ácidos Docosahexaenoicos/biosíntesis , Biomasa , Dinoflagelados/metabolismo , Fermentación/fisiología , Microalgas/crecimiento & desarrollo
6.
World J Microbiol Biotechnol ; 36(3): 46, 2020 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-32140791

RESUMEN

Azotobacter vinelandii is a microorganism with biotechnological potential because its ability to produce alginate and polyhydroxybutyrate. Large-scale biotechnological processes are oriented to sustainable production by using biomass hydrolysates that are mainly composed by glucose and xylose. In the present study, it was observed that A. vinelandii was unable to consume xylose as the sole carbon source and that glucose assimilation in the presence of xylose was negatively affected. Adaptive Laboratory Evolution (ALE) was used as a metabolic engineering tool in A. vinelandii, to improve both carbohydrate assimilation. As a result of ALE process, the CT387 strain was obtained. The evolved strain (CT387) grown in shaken flask cultivations with xylose (8 g L-1) and glucose (2 g L-1), showed an increase of its specific growth rate (µ), as well as of its glucose and xylose uptake rates of 2, 6.45 and 3.57-fold, respectively, as compared with the parental strain. At bioreactor level, the µ, the glucose consumption rate and the relative expression of gluP that codes for the glucose permease in the evolved strain were also higher than in the native strain (1.53, 1.29 and 18-fold, respectively). Therefore, in the present study, we demonstrated the potential of ALE as a metabolic engineering tool for improving glucose and xylose consumption in A. vinelandii.


Asunto(s)
Azotobacter vinelandii/metabolismo , Glucosa/metabolismo , Ingeniería Metabólica/métodos , Xilosa/metabolismo , Azotobacter vinelandii/genética , Azotobacter vinelandii/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomasa , Reactores Biológicos , Medios de Cultivo/química , Fermentación , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Bacteriano/aislamiento & purificación
7.
PLoS Negl Trop Dis ; 14(3): e0008104, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32119669

RESUMEN

Approximately one-third of the global population is at risk of Plasmodium vivax infection, and an estimated 7.51 million cases were reported in 2017. Although, P. vivax research is currently limited by the lack of a robust continuous in vitro culture system for this parasite, recent work optimizing short-term ex vivo culture of P. vivax from cryopreserved isolates has facilitated quantitative assays on synchronous parasites. Pairing this improved culture system with low-input Smart-seq2 RNAseq library preparation, we sought to determine whether transcriptional profiling of P. vivax would provide insight into the differential survival of parasites in different culture media. To this end we probed the transcriptional signature of three different ex vivo P. vivax samples in four different culture media using only 1000 cells for each time point taken during the course of the intraerythrocytic development cycle (IDC). Using this strategy, we achieved similar quality transcriptional data to previously reported P. vivax transcriptomes. We found little effect with varying culture media on parasite transcriptional signatures, identified many novel gametocyte-specific genes from transcriptomes of FACS-isolated gametocytes, and determined invasion ligand expression in schizonts in biological isolates and across the IDC. In total, these data demonstrate the feasibility and utility of P. vivax RNAseq-based transcriptomic studies using minimal biomass input to maximize experimental capacity.


Asunto(s)
Eritrocitos/parasitología , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Malaria Vivax/parasitología , Plasmodium vivax/crecimiento & desarrollo , Adolescente , Niño , Preescolar , Medios de Cultivo/química , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Parasitología/métodos , Plasmodium vivax/genética , Análisis de Secuencia de ARN
8.
Microbiol Res ; 236: 126454, 2020 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-32200250

RESUMEN

Freeze-drying technology has been widely considered for decades as a suitable technique to preserve microorganisms. However, protective agents must be added prior to freeze drying to improve the survival and storage stability of the bacteria. The objective of our study was to evaluate the effect of a new protectant medium containing sucrose (10 %), trehalose (10 %), skimmed milk (10 %) and antioxidants on the viability of gut bacteria under different storage conditions. Two strains were tested, Escherichia coli and Akkermansia muciniphila, as examples of facultative aerobic and anaerobic bacteria, respectively. We studied the cell viability and bacterial morphology in 5 fecal samples in the presence and absence of this protectant medium using plating technique, flow cytometry and scanning electron microscopy. The results of bacterial viability assessed by plating method showed that the protectant medium yielded higher survival rates for both strains whatever the storage conditions (85-93 %) compared to normal saline solution (0.36-37.50 %). It also showed its effectiveness on fecal samples, where bacterial viability after freeze-drying was 89.47 ± 7.63 % and 84.01 ± 7.44 %, as evidenced by flow cytometry analysis and plating method. However unprotected samples showed the lowest cell viability at 19.01 ± 12.88 % and 13.23 ± 9.56 %, as measured by flow cytometry and plating method. In addition, bacterial size and shape were conserved in the protectant medium. In contrast, storage without protectant medium severely damaged bacterial morphology. In conclusion, our study is the first to use morphological features as well as culture-dependant and culture-independent tests to evaluate the effectiveness of a new protectant medium.


Asunto(s)
Bacterias , Liofilización , Viabilidad Microbiana , Preservación Biológica , Animales , Bacterias/citología , Bacterias/crecimiento & desarrollo , Medios de Cultivo/química , Leche , Preservación Biológica/métodos , Sacarosa , Trehalosa
9.
PLoS One ; 15(3): e0226467, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32203515

RESUMEN

The aim of this study was to compare the Candida bromcresol green (BCG) medium with the chromogenic (CHROM) Brilliance Candida agar and Sabouraud dextrose agar (SDA) media in regard to their capability of detecting Candida isolates from mono- or dual-species cultures. We prepared Candida isolates' suspensions to obtain mono-species (n = 18) or dual-species (n = 153) culture plates per each medium, and three readers independently observed 513 plates at 24-h, 48-h and 72-h incubation time. We scored reading results as correct, over or under detection compared to the expected species number(s). BCG showed significantly higher correct-detection and lower under-detection rates for all Candida species when observed by at least one reader. At 24-h reading, 12 mono-species cultures had correct (or over) detections in all media, whereas 106, 60 and 78 dual-species cultures had correct (or over) detections in BCG, CHROM or SDA, respectively. BCG provides the basis for an accurate laboratory diagnosis of Candida infections.


Asunto(s)
Agar/química , Candida/aislamiento & purificación , Candidiasis/diagnóstico , Medios de Cultivo/química , Indicadores y Reactivos/química , Candidiasis/microbiología , Humanos , Técnicas Microbiológicas/métodos
10.
Can J Microbiol ; 66(4): 303-312, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32118486

RESUMEN

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.


Asunto(s)
Brochothrix/crecimiento & desarrollo , Pollos/microbiología , Animales , Brochothrix/clasificación , Brochothrix/aislamiento & purificación , Brochothrix/metabolismo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Contaminación de Alimentos/análisis , Carne/microbiología , Temperatura
11.
PLoS One ; 15(3): e0229043, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32182244

RESUMEN

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.


Asunto(s)
Medios de Cultivo/farmacología , Células del Cúmulo/citología , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/crecimiento & desarrollo , Folículo Ovárico/citología , Anfirregulina/farmacología , Animales , Biomarcadores , Células Cultivadas , Medios de Cultivo/química , Células del Cúmulo/metabolismo , Dinoprostona/farmacología , Femenino , Fertilización In Vitro , Fluoresceínas/metabolismo , Meiosis , Modelos Animales , Péptido Natriurético Tipo-C/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Folículo Ovárico/efectos de los fármacos , Ovinos
12.
PLoS One ; 15(3): e0228229, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32214336

RESUMEN

The culture of differentiated human airway epithelial cells allows the study of pathogen-host interactions and innate immune responses in a physiologically relevant in vitro model. As the use of primary cell culture has gained popularity the availability of the reagents needed to generate these cultures has increased. In this study we assessed two different media, Promocell and PneumaCult, during the differentiation and maintenance of well-differentiated primary nasal epithelial cell cultures (WD-PNECs). We compared and contrasted the consequences of these media on WD-PNEC morphological and physiological characteristics and their responses to respiratory syncytial virus (RSV) infection. We found that cultures generated using PneumaCult resulted in greater total numbers of smaller, tightly packed, pseudostratified cells. However, cultures from both media resulted in similar proportions of ciliated and goblet cells. There were no differences in RSV growth kinetics, although more ciliated cells were infected in the PneumaCult cultures. There was also significantly more IL-29/IFNλ1 secreted from PneumaCult compared to Promocell cultures following infection. In conclusion, the type of medium used for the differentiation of primary human airway epithelial cells may impact experimental results.


Asunto(s)
Diferenciación Celular , Medios de Cultivo/química , Células Epiteliales/citología , Células Epiteliales/virología , Nariz/citología , Cultivo Primario de Células/métodos , Virus Sincitiales Respiratorios/fisiología , Línea Celular , Niño , Células Caliciformes/citología , Humanos
13.
Artículo en Inglés | MEDLINE | ID: mdl-32107954

RESUMEN

Microorganisms with efficient organic matter degradation ability are essential for organic waste treatment. In this study, a thermophilic bacterium, Bacillus thermoliquefaciens, was identified to have excellent cellulase, amylase, and protease activity, and provided efficient degradation of food waste. This is the first report on the organic matter degradation potential of B. thermoliquefaciens. Using a "one-variable-at-a-time" approach and response surface methodology, the optimal culture conditions for B. thermoliquefaciens were determined to be a 5% inoculation level, 50 °C culture temperature, 25 mL filling volumes in 250 mL flasks, and 180 rpm shaking for 24 h. The optimized medium was formulated as 1 g Na2HPO4, 1 g KH2PO4, 0.05 g MgSO4, 3 g NaCl, 0.05 g CaCl2, 11.44 g wheat bran powder, 4.92 g soybean meal, and 1 L distilled water at pH 7.12. The maximum biomass attained was 1.57 ± 0.153 × 109 CFU/mL. The cost of this medium was 4.18 times less than that before optimization. This promising result lays a foundation for future industrial application of this bacterium to the degradation of organic waste.


Asunto(s)
Bacillus/crecimiento & desarrollo , Medios de Cultivo/química , Alimentos , Eliminación de Residuos/métodos , Residuos Sólidos , Bacillus/metabolismo , Biodegradación Ambiental , Biomasa , Análisis Costo-Beneficio , Concentración de Iones de Hidrógeno , Temperatura
14.
Ultrasonics ; 103: 106086, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-32070827

RESUMEN

Ultrasonic bioreactors have been used for in vitro experimentation to study cellular responses to low-intensity pulsed ultrasound. The presence of an air interface in these bioreactors contributes to variability in the acoustic pressure field, reducing experimental reproducibility. A multiphysics finite element model was developed to simulate the acoustic field in an in-dish ultrasonic bioreactor, where the transducer is immersed in culture medium above the dish surface, and the effects of replacing air below the dish in the bioreactor with a water layer bounded by an acoustic absorbent layer were evaluated. Frequency domain simulations showed that the spatially-averaged pressure at the dish surface alternated between a minimum and maximum level as the distance between the dish and transducer increased. The ratio of the maximum to minimum level was 6.5-fold when the air interface was present, and this ratio dropped to 1.8-fold with replacement of the air interface. However, radial pressure variability was present with or without the air interface in the bioreactor model. Time-dependent simulations showed that the increase in acoustic pressure to a maximum level after US signal activation and the pressure drop after signal cessation were faster when the water-coupled non-reflective layer was used to replace the air layer below the dish, generating a pressure pattern that more closely followed the applied pulsed ultrasound signal due to reduced wave reflection and interference. Overall, this work showed that having water rather than air in contact with the lower dish surface when paired with an acoustic absorbent layer resulted in a less variable pressure field, providing an improved bioreactor design for in vitro experiments.


Asunto(s)
Acústica , Reactores Biológicos , Análisis de Elementos Finitos , Fenómenos Biofísicos , Medios de Cultivo , Diseño de Equipo , Presión , Programas Informáticos , Propiedades de Superficie , Transductores , Ultrasonido , Agua
15.
J Agric Food Chem ; 68(8): 2485-2492, 2020 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-32049524

RESUMEN

Employing isotope incubation studies, the biosynthetic pathway leading to a series of benzylic derivatives was elucidated in the fermentation broth of the edible mushroom Ischnoderma resinosum (P. Karst). Twenty-six hydroxy- and methoxy- benzylic derivatives were screened by gas chromatography-mass spectrometry (GC-MS) of which 13 were detected in the culture media. Results from the isotope incubation studies showed the transformation of both benzyl alcohol and benzoic acid into benzaldehyde. Benzaldehyde was then converted into 4-methoxybenzaldehyde via hydroxylation and subsequent methylation of the 4-C position. The resulting 4-methoxybenzaldehyde was then hydroxylated in the 3-C position followed by methylation into 3,4-dimethoxybenzaldehyde. Based on these findings, a novel metabolic scheme for the biosynthesis of benzylic derivatives in I. resinosum was proposed. The knowledge of the biosynthetic pathway was utilized to produce 4-hydroxy-3-methoxybenzaldehyde (vanillin) from 4-hydroxy-3-methoxybenzoic acid (vanillic acid). This is the first report to elucidate the biosynthetic pathway of benzyl derivatives and production of vanillin from I. resinosum.


Asunto(s)
Benzaldehídos/metabolismo , Polyporales/metabolismo , Benzaldehídos/análisis , Ácido Benzoico/análisis , Ácido Benzoico/metabolismo , Alcohol Bencilo/análisis , Alcohol Bencilo/metabolismo , Biotransformación , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Fermentación , Cromatografía de Gases y Espectrometría de Masas , Polyporales/química , Polyporales/genética , Ácido Vanílico/análisis , Ácido Vanílico/metabolismo
16.
Syst Appl Microbiol ; 43(2): 126066, 2020 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-32019686

RESUMEN

On-going studies of phytoplankton-bacterioplankton interactions at the long-term ecological research site Helgoland Roads have indicated that many of the heterotrophic bacterial taxa have not yet been cultivated. A high-throughput approach combining whole cell matrix-assisted laser desorption ionization - time of flight mass spectroscopy with 16S rRNA gene sequencing was applied to the spring bloom of 2016. Aiming at an assessment of cultivability during a spring bloom, cultivation on solid marine media had to be used since dilution to extinction would not have been feasible for a high-throughput approach, as performed in this study. A total of 5023 isolates were obtained from nine weekly samples on eight different solid media between the early-bloom and post-bloom periods. Most of the 4136 strains identified affiliated with Bacteroidetes (13.3%), Gammaproteobacteria (26.9%), Alphaproteobacteria (40.6%) and Actinobacteria (6.7%). Of the 271 operational phylogenetic units (OPUs) identified, 13 are likely to represent novel genera and 143 novel species. A comparison with 16S rRNA gene tag data indicated that most of the isolates were rather rare in surface waters, with the exception of five OPUs affiliating with Rhodobacteraceae, Polaribacter, Psychromonas and Pseudoalteromonas. The effort yielded many novel isolates, yet most of the abundant heterotrophic bacteria still remained elusive. The large strain collection obtained will not only provide insights into the succession of the cultivable fraction of the bacterioplankton, but also enable fine-tuned taxonomic and physiological follow-up studies for improving our knowledge on heterotrophic bacteria in North Sea waters.


Asunto(s)
Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Fitoplancton/crecimiento & desarrollo , Fitoplancton/metabolismo , Agua de Mar/microbiología , Bacterias/clasificación , Bacterias/genética , Medios de Cultivo , ADN Bacteriano/genética , Eutrofización , Procesos Heterotróficos , Mar del Norte , Filogenia , Fitoplancton/clasificación , Fitoplancton/genética , ARN Ribosómico 16S/genética , Estaciones del Año , Agua de Mar/química , Análisis de Secuencia de ADN
17.
Microbes Environ ; 35(1)2020.
Artículo en Inglés | MEDLINE | ID: mdl-32009018

RESUMEN

We previously demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving phosphate and agar separately (termed the "PS protocol"), improved the culturability of aerobic microorganisms by reducing the generation of reactive oxygen species. We herein investigated the effects of the PS protocol on the cultivation of anaerobic microorganisms using sludge from a wastewater treatment system as a microbial source. The application of the PS protocol increased colony numbers and the frequency of phylogenetically novel isolates under aerobic, nitrate reduction, and fermentation conditions. The PS protocol is useful for isolating both aerobic and anaerobic microorganisms.


Asunto(s)
Bacterias Anaerobias/crecimiento & desarrollo , Bacterias Anaerobias/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Medios de Cultivo/química , Agar , Bacterias Anaerobias/clasificación , Bacterias Anaerobias/metabolismo , Recuento de Colonia Microbiana , Fermentación , Nitratos/metabolismo , Fosfatos , Filogenia , ARN Ribosómico 16S/genética , Aguas del Alcantarillado/microbiología , Esterilización
18.
Microbes Environ ; 35(1)2020.
Artículo en Inglés | MEDLINE | ID: mdl-32037377

RESUMEN

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.


Asunto(s)
Genoma Bacteriano/genética , Elementos de la Serie de los Lantanoides/metabolismo , Proteobacteria/genética , Proteobacteria/aislamiento & purificación , Oxidorreductasas de Alcohol/genética , Proteínas Bacterianas/genética , Medios de Cultivo/metabolismo , Sedimentos Geológicos/microbiología , Metales de Tierras Raras/metabolismo , Metano/metabolismo , Methylosinus/clasificación , Methylosinus/genética , Methylosinus/aislamiento & purificación , Methylosinus/metabolismo , Estanques/microbiología , Proteobacteria/clasificación , Proteobacteria/fisiología , ARN Ribosómico 16S/genética
19.
Nat Commun ; 11(1): 764, 2020 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-32034154

RESUMEN

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.


Asunto(s)
Autorrenovación de las Células/fisiología , Células Madre Embrionarias Humanas/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Activinas/metabolismo , Animales , Blastocisto/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Medios de Cultivo/farmacología , Endodermo/citología , Endodermo/metabolismo , Membranas Extraembrionarias/citología , Membranas Extraembrionarias/metabolismo , Fibroblastos/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/fisiología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor IGF Tipo 1/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma , Inactivación del Cromosoma X/fisiología
20.
PLoS One ; 15(2): e0229654, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32106262

RESUMEN

Human hepatocytes are essential materials in pharmaceutical researches. Not only primary human hepatocytes (PHH) but also human iPS cell-derived hepatocyte-like cells (human iPS-HLCs) are expected to be applied as materials for pharmaceutical researches. To date, several culture media have been developed for culturing human hepatocytes. However, there have been no reports comparing these media to determine which is most suitable for culturing human hepatocytes. In this study, we compared five commercial media (Hepatocyte Culture Medium (HCM), HepatoZYME-SFM, Cellartis Power Primary HEP Medium, DMEM/F12, and William's E Medium (WEM)) to determine which is most suitable for culturing PHH and human iPS-HLCs. In hepatic differentiation of human iPS cells (day 14-25 of differentiation), albumin (ALB) and urea secretion abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM or WEM. During maintenance of human iPS-HLCs, ALB and urea producing abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM. Importantly, we found that human iPS-HLCs cultured in HCM were maintained for 3 weeks or more without impairment of their hepatic functions. These results suggest that it is necessary to select an optimal medium for hepatic differentiation and maintenance of human iPS-HLCs. In the case of PHH culture, there was little difference in hepatic functions among the five media. However, the CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM and WEM. In conclusion, it is important to select the optimal medium for specific application when carrying out pharmaceutical researches using human hepatocytes.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Medios de Cultivo , Hepatocitos/citología , Hepatocitos/metabolismo , Albúminas/metabolismo , Diferenciación Celular , Línea Celular , Células Cultivadas , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Urea/metabolismo
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