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1.
BMC Pulm Med ; 23(1): 323, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-37658311

RESUMO

BACKGROUND: Cystic fibrosis (CF) is a life-limiting disorder that is characterised by respiratory tract inflammation that is mediated by a range of microbial pathogens. Small colony variants (SCVs) of common respiratory pathogens are being increasingly recognised in CF. The aim of this systematic review is to investigate the prevalence of SCVs, clinical characteristics and health outcomes for patients with CF, and laboratory diagnostic features of SCVs compared to non-small colony variants (NCVs) for a range of Gram-positive and Gram-negative respiratory pathogens. METHODS: A literature search was conducted (PubMed, Web of Science, Embase and Scopus) in April 2020 to identify articles of interest. Data pertaining to demographic characteristics of participants, diagnostic criteria of SCVs, SCV prevalence and impact on lung function were extracted from included studies for analysis. RESULTS: Twenty-five of 673 studies were included in the systematic review. Individuals infected with SCVs of Staphylococcus aureus (S. aureus) were more likely to have had prior use of the broad-spectrum antibiotic trimethoprim sulfamethoxazole (p < 0.001), and the prevalence of SCVs in patients infected with S. aureus was estimated to be 19.3% (95% CI: 13.5% to 25.9%). Additionally, patients infected with SCVs of Gram-negative and Gram-positive pathogens were identified to have a lower forced expiratory volume in one second percentage predicted (-16.8, 95% CI: -23.2 to -10.4) than those infected by NCVs. Gram-positive SCVs were commonly described as small and non-haemolytic, grown on Mannitol salt or blood agar for 24 h at 35°C and confirmed using tube coagulase testing. CONCLUSION: The findings of this systematic review demonstrate that SCVs of S. aureus have a high prevalence in the CF community, and that the occurrence of SCVs in Gram-positive and Gram-negative pathogens is linked to poorer respiratory function. Further investigation is necessary to determine the effect of infection by SCVs on the CF population.


Assuntos
Fibrose Cística , Humanos , Staphylococcus aureus , Pacientes , Antibacterianos/uso terapêutico , Meios de Cultura
2.
Molecules ; 28(17)2023 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-37687160

RESUMO

Numerous compounds obtained from the medicinal mushroom Ganoderma lucidum have evidenced renowned bioactive characteristics. Controlled fermentation to generate fungal mycelia confers several advantages, specifically when the valorization of agro-industrial streams as fermentation feedstocks is included. Submerged fermentation of a newly isolated Greek strain of G. lucidum was performed using conventional synthetic media and, also, grape pomace extract (GPE) and cheese whey permeate (CWP) under static and shaking conditions. Under shaking conditions, maximum biomass with GPE and supplementation with organic nitrogen reached 17.8 g/L. The addition of an elicitor in CWP resulted in a significant improvement in biomass production that exceeded synthetic media. Overall, agitation demonstrated a positive impact on biomass productivity and, therefore, on process optimization. Crude intracellular and extracellular polysaccharides were extracted and evaluated regarding antioxidant activity and polysaccharide and protein content. FTIR analysis confirmed the preliminary chemical characterization of the crude extracts. This study introduces the design of a bioprocessing scenario to utilize food industry by-products as onset feedstocks for fungal bioconversions to obtain potential bioactive molecules within the concept of bioeconomy.


Assuntos
Queijo , Reishi , Vitis , Soro do Leite , Proteínas do Soro do Leite , Meios de Cultura
3.
Arch Microbiol ; 205(10): 332, 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37707605

RESUMO

Mesophilic and thermophilic methanogens belonging to the hydrogenotrophic, methylotrophic, and acetotrophic groups were isolated from Indian hot spring environments using BY and BCYT growth media. Following initial Hinf I-based PCR-RFLP screening, 70 methanogens were sequenced to ascertain their identity. These methanogens were phylogenetically and physiologically diverse and represented different taxa distributed across three physiological groups, i.e., hydrogenotrophs (53), methylotrophs (14) and acetotrophs (3). Overall, methanogens representing three families, five genera, and ten species, including two putative novel species, were recognized. The highest number and diversity of methanogens was observed at 40 â„ƒ, dominated by Methanobacterium (10; 3 species), Methanosarcina (9; 3 species), Methanothermobacter (7; 2 species), Methanomethylovorans (5; 1 species) and Methanoculleus (3; 1 species). Both putative novel methanogen species were isolated at 40 â„ƒ and belonged to the genera Methanosarcina and Methanobacterium. At 55 â„ƒ, limited diversity was observed, and resulted in the isolation of only two genera of methanogens, i.e., Methanothermobacter (28; 2 species) and Methanosarcina (4; 1 species). At 70 â„ƒ, only members of the genus Methanothermobacter (5; 2 species) were isolated, whereas no methanogen could be cultured at 85 â„ƒ. Ours is the first study that documents the extensive range of cultivable methanogenic archaea inhabiting hot springs across various geothermal provinces of India.


Assuntos
Euryarchaeota , Fontes Termais , Humanos , Archaea/genética , Meios de Cultura , Índia
4.
Nat Commun ; 14(1): 5757, 2023 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-37717013

RESUMO

Elucidating genome-scale regulatory networks requires a comprehensive collection of gene expression profiles, yet measuring gene expression responses for every transcription factor (TF)-gene pair in living prokaryotic cells remains challenging. Here, we develop pooled promoter responses to TF perturbation sequencing (PPTP-seq) via CRISPR interference to address this challenge. Using PPTP-seq, we systematically measure the activity of 1372 Escherichia coli promoters under single knockdown of 183 TF genes, illustrating more than 200,000 possible TF-gene responses in one experiment. We perform PPTP-seq for E. coli growing in three different media. The PPTP-seq data reveal robust steady-state promoter activities under most single TF knockdown conditions. PPTP-seq also enables identifications of, to the best of our knowledge, previously unknown TF autoregulatory responses and complex transcriptional control on one-carbon metabolism. We further find context-dependent promoter regulation by multiple TFs whose relative binding strengths determined promoter activities. Additionally, PPTP-seq reveals different promoter responses in different growth media, suggesting condition-specific gene regulation. Overall, PPTP-seq provides a powerful method to examine genome-wide transcriptional regulatory networks and can be potentially expanded to reveal gene expression responses to other genetic elements.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Escherichia coli , Escherichia coli/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Meios de Cultura , Redes Reguladoras de Genes , Homeostase
5.
Sci Rep ; 13(1): 15190, 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37709845

RESUMO

In this study, the potential of Chlorella sorokiniana JD1-1 for biodiesel production was evaluated using domestic wastewater (DWW) as a diluent for locally-generated livestock wastewater (LWW). This strategy aimed to provide sustainable wastewater treatment, reduce environmental impacts, enhance cost-effectiveness, and promote biodiesel production. LWW was diluted with tap water and DWW at ratios of 75%, 50%, and 25% (v/v), and the effects on microalgal growth, nutrient removal efficiency, and lipid yield were evaluated. Although the maximum biomass concentration was observed in the artificial growth medium (BG-11) (1170 mg L-1), 75% dilution using tap water (610 mg L-1) and DWW (780 mg L-1) yielded results comparable to the exclusive use of DWW (820 mg L-1), suggesting a potential for substitution. Total nitrogen (TN) removal rates were consistently high under all conditions, particularly in samples with higher concentrations of LWW. Conversely, total phosphorus (TP) concentrations decreased under most conditions, although some displayed large increases. Further studies are necessary to optimize the nutrient balance while maintaining economic feasibility and maximizing biodiesel production.


Assuntos
Chlorella , Microalgas , Animais , Biocombustíveis , Gado , Águas Residuárias , Meios de Cultura , Água
6.
Cell Mol Biol (Noisy-le-grand) ; 69(8): 246-249, 2023 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-37715373

RESUMO

To elucidate the role of microRNA-1284 (miR-1284) in the onset of thyroid cancer (TC) and its underlying mechanism. Differential expressions of miR-1284 in TC and thyroid tissues were detected. Regulatory effects of miR-1284 on proliferative, migratory, apoptotic potentials and cell cycle progression were assessed. In addition, miR-1284 levels in TC tissues and peripheral blood of TC patients were determined as well. Through collecting culture medium and exosomes of PTC cells, changes in miR-1284 levels were examined. MiR-1284 was downregulated in TC than normal thyroid tissues. Overexpression of miR-1284 attenuated proliferative and migratory potentials, but induced apoptosis in TPC-1 and FTC-133 cells. Moreover, overexpression of miR-1284 upregulated E-cadherin and downregulated N-cadherin in papillary TC (PTC) cells. MiR-1284 was downregulated in TC tissues, while its level in the peripheral blood of TC patients was upregulated. Besides, miR-1284 was upregulated in the culture medium and exosomes of PTC cells, which was reversed by Brefeldin A treatment. Overexpression of miR-1284 suppresses proliferative and migratory potentials and induces apoptosis in TC. Upregulated miR-1284 in the peripheral blood of TC patients may be derived from exosomes secreted by PTC cells.


Assuntos
MicroRNAs , Neoplasias da Glândula Tireoide , Humanos , Neoplasias da Glândula Tireoide/genética , Câncer Papilífero da Tireoide , Divisão Celular , Meios de Cultura , MicroRNAs/genética
7.
Microb Cell Fact ; 22(1): 178, 2023 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-37689693

RESUMO

BACKGROUND: γ-aminobutyric acid (GABA) is a bioactive compound produced by lactic acid bacteria (LAB). The diversity of GABA production in the Lactococcus genus is poorly understood. Genotypic and phenotypic approaches were therefore combined in this study to shed light on this diversity. A comparative genomic study was performed on the GAD-system genes (gadR, gadC and gadB) involved in GABA production in 36 lactococci including L. lactis and L. cremoris species. In addition, 132 Lactococcus strains were screened for GABA production in culture medium supplemented with 34 mM L-glutamic acid with or without NaCl (0.3 M). RESULTS: Comparative analysis of the nucleotide sequence alignments revealed the same genetic organization of the GAD system in all strains except one, which has an insertion sequence element (IS981) into the PgadCB promoter. This analysis also highlighted several deletions including a 3-bp deletion specific to the cremoris species located in the PgadR promoter, and a second 39-bp deletion specific to L. cremoris strains with a cremoris phenotype. Phenotypic analysis revealed that GABA production varied widely, but it was higher in L. lactis species than in L. cremoris, with an exceptional GABA production of up to 14 and 24 mM in two L. lactis strains. Moreover, adding chloride increased GABA production in some L. cremoris and L. lactis strains by a factor of up to 16 and GAD activity correlated well with GABA production. CONCLUSIONS: This genomic analysis unambiguously characterized the cremoris phenotype of L. cremoris species and modified GadB and GadR proteins explain why the corresponding strains do not produce GABA. Finally, we found that glutamate decarboxylase activity revealing GadB protein amount, varied widely between the strains and correlated well with GABA production both with and without chloride. As this protein level is associated to gene expression, the regulation of GAD gene expression was identified as a major contributor to this diversity.


Assuntos
Cloretos , Lactococcus , Fenótipo , Meios de Cultura , Ácido gama-Aminobutírico
8.
Biomedica ; 43(Sp. 1): 216-228, 2023 08 31.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-37721906

RESUMO

INTRODUCTION: For over a century, Sporothrix schenckii was considered the sole species responsible for sporotrichosis. In 2007, scientific community confirmed the disease could be caused by various Sporothrix species. These species differed in their virulence factors and their antifungal sensitivity. OBJECTIVE: This study aims to characterize 42 Colombian clinical isolates of Sporothrix spp. phenotypically and genotypically. MATERIAL AND METHODS: Forty-two clinical isolates were characterized using phenotypic methods. It involved various culture media to determine their growth range at different temperatures and to assess the type and distribution of pigment and colony texture. Microscopic morphology was evaluated through microcultures, as well as the conidia diameter, type of sporulation, and morphology. Additionally, the assimilation of carbohydrates was selected as a physiological trait for species identification. Genotyping of 40 isolates was performed through partial amplification of the calmodulin gene, followed by sequence analysis. RESULTS: Molecular studies enabled the identification of 32 isolates of S. schenckii and 8 isolates of S. globosa. The combination of phenotypic and genotypic methods eased these species characterizations and the recognition keys development based on parameters such as growth diameter at 25 and 30 ºC, colony texture (membranous or velvety) on potato dextrose agar, and microscopic morphology with predominance of pigmented triangular, elongated oval globose, or subglobose conidia. CONCLUSIONS: Confirmation of the phenotypic characteristics and molecular analysis is crucial for identifying Sporothrix species and determining adequate treatment. This study represents the first phenotypical and genotypical characterization of clinical isolates of Sporothrix spp. reported in Colombia.


Introducción: Por más de un siglo se creyó que Sporothrix schenckii era la única especie responsable de la esporotricosis. Sin embargo, en el 2007, se consideró que podría ser causada por diferentes especies de Sporothrix, que difieren en sus factores de virulencia y su sensibilidad a los antifúngicos. Objetivo: Caracterizar fenotípica y genotípicamente 42 aislamientos clínicos colombianos de Sporothrix spp. Materiales y métodos: Se caracterizaron 42 aislamientos clínicos mediante métodos fenotípicos. Se usaron varios medios de cultivo para determinar el rango de crecimiento a diferentes temperaturas, el tipo y la distribución del pigmento, y la textura de las colonias. Se evaluó la morfología microscópica por microcultivos mediante la determinación del diámetro, el tipo de esporulación y la morfología de las conidias. La asimilación de carbohidratos se usó como una característica fisiológica para identificar las especies. La genotipificación de los 40 aislamientos se llevó a cabo mediante la amplificación parcial del gen que codifica para la calmodulina y se confirmó por secuenciación. Resultados: Mediante estudios moleculares, se identificaron 32 aislamientos de S. schenckii y ocho de S. globosa. La combinación de métodos fenotípicos y genotípicos permitió caracterizar las especies y construir claves para su reconocimiento, con base en parámetros como el diámetro de crecimiento a 25 y 30 ºC, la textura de las colonias (membranosa, aterciopelada) en agar papa dextrosa y la morfología microscópica con predominio de conidias (triangulares pigmentadas, ovales globosas elongadas, subglobosas). Conclusiones: La caracterización fenotípica y los análisis moleculares son necesarios para identificar las especies de Sporothrix y, de esta forma, elegir el tratamiento indicado. Esta es la primera caracterización fenotípica y genotípica reportada de aislamientos clínicos colombianos de Sporothrix spp.


Assuntos
Sporothrix , Colômbia , Sporothrix/genética , Genótipo , Fenótipo , Antifúngicos , Meios de Cultura
10.
BMJ Open Ophthalmol ; 8(Suppl 2): A4-A5, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37604569

RESUMO

PURPOSE: To analyse the microbiologic control results taken during the processing of hypothermic and cultured corneas for endothelial transplants comparing the two groups from January to September 2022. METHODS: The microbiological controls of hypothermic corneas prepared for DSAEK or DMEK are: Transport Eusol control (pre-manipulation) and new Eusol control (post-manipulation). In cultured corneas the number of controls is increased to 4: first culture medium, evaluation culture medium, transport medium 24 hours post-evaluation and transport medium post-manipulation. RESULTS: A total of 1438 corneas were processed for transplant during the 9 months studied (321 fresh corneas and 1113 cultured corneas). A total of 557 corneas were prepared for DSAEK or DMEK, from which 89 (15,98%) were hypothermic corneas and 468 (84.O2%) were cultured. From hypothermic corneas, 65 were cut for DSAEK and with 24 corneas, pre-stripping for DMEK was done. In the case of cultured corneas, 187 were cut for DSAEK and with 281 pre-stripping for DMEK was done. The number of corneas with positive results in the microbiological controls were 15 (16,85%) in the case of fresh corneas (in 7 corneas that were prepared for DSAEK and in 8 for DMEK) and 4 cases (0,85%) in cultured corneas (in 3 corneas for DSAEK and in 1 corneas for DMEK) resulting in a clear difference between both preservation methods. Bio-surveillance notifications notified during the studied period have been a total of 5, from which 2 were SAE in hypothermic corneas and other 2 were SAE and 1 SAR, in cultured corneas, all for endothelial transplantations. CONCLUSION: The number of positive results for microorganisms was higher in the case of hypothermic corneas and the Bio-surveillance notifications were also a little bit higher in hypothermic corneas (2,25%) comparing to organ cultured corneas (0.64%). The management of an eye bank with both preservation systems is challenging with its advantages and disadvantages. The main disadvantage of hypothermic corneas is the risk of not detecting contaminations because the corneas are released without any definitive results but it is compensated by the fact that they allow us to respond to emergencies, tissue returns, apart from the economic aspect.


Assuntos
Córnea , Bancos de Olhos , Córnea/cirurgia , Meios de Cultura , Contaminação de Medicamentos
11.
Int J Food Microbiol ; 404: 110367, 2023 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-37597274

RESUMO

Progress in oenological biotechnology now makes it possible to control alcoholic (AF) and malolactic (MLF) fermentation processes for the production of wines. Key factors in controlling these processes and enhancing wine quality include the use of selected strains of non-Saccharomyces species, Saccharomyces cerevisiae, and Oenococcus oeni, as well as the method of inoculation (co-inoculation or sequential) and the timing of inoculation. In the present work, we investigated the effects of different inoculation strategies of two Torulaspora delbrueckii (Td-V and Td-P) strains followed by S. cerevisiae. Times (two, four, and six days) and types (co-inoculation and sequential) of inoculation were evaluated on the AF of a synthetic grape must. Furthermore, this synthetic medium was optimized by adding linoleic acid and ß-sitosterol to simulate the natural grape must and facilitate reproducible results in potential assays. Subsequently, the wines obtained were inoculated with two strains of Oenococcus oeni to carry out MLF. Parameters after AF were analysed to observe the impact of wine composition on the MLF performance. The results showed that the optimization of the must through the addition of linoleic acid and ß-sitosterol significantly enhanced MLF performance. This suggests that these lipids can positively impact the metabolism of O. oeni, leading to improved MLF efficiency. Furthermore, we observed that a 4-day contact period with T. delbrueckii leads to the most efficient MLF process and contributed to the modification of certain AF metabolites, such as the reduction of ethanol and acetic acid, as well as an increase in available nitrogen. The combination of Td-P with Oo-VP41 for 4 or 6 days during MLF showed that it could be the optimal option in terms of efficiency. By evaluating different T. delbrueckii inoculation strategies, optimizing the synthetic medium and studying the effects on wine composition, we aimed to gain insights into the relationship between AF conditions and subsequent MLF performance. Through this study, we aim to provide valuable insights for winemakers and researchers in the field of wine production and will contribute to a better understanding of the complex interactions between these species in the fermentation process.


Assuntos
Torulaspora , Vitis , Fermentação , Saccharomyces cerevisiae , Ácido Linoleico , Meios de Cultura
12.
Cells ; 12(16)2023 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-37626914

RESUMO

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.


Assuntos
Medula Óssea , Células-Tronco Mesenquimais , Humanos , Meios de Cultura/farmacologia , Suplementos Nutricionais , Ácido Láctico
13.
Int J Mol Sci ; 24(16)2023 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-37628714

RESUMO

The heart is one of the major organs affected by microwave radiation, and these effects have been extensively studied. Previous studies have shown that microwave-radiation-induced heart injury might be related to the abnormal expression and distribution of Cx43. In order to make the research model closer to humans, we used iPSC-CMs as the cell injury model to investigate the biological effect and mechanism of iPSC-CM injury after microwave radiation. To model the damage, iPSC-CMs were separated into four groups and exposed to single or composite S-band (2.856 GHz) and X-band (9.375 GHz) microwave radiation sources with an average power density of 30 mW/cm2. After that, FCM was used to detect cell activity, and ELISA was used to detect the contents of myocardial enzymes and injury markers in the culture medium, and it was discovered that cell activity decreased and the contents increased after radiation. TEM and SEM showed that the ultrastructure of the cell membrane, mitochondria, and ID was damaged. Mitochondrial function was aberrant, and glycolytic capacity decreased after exposure. The electrical conduction function of iPSC-CM was abnormal; the conduction velocity was decreased, and the pulsation amplitude was reduced. Wb, qRT-PCR, and IF detections showed that the expression of Cx43 was decreased and the distribution of Cx43 at the gap junction was disordered. Single or composite exposure to S- and X-band microwave radiation caused damage to the structure and function of iPSC-CMs, primarily affecting the cell membrane, mitochondria, and ID. The composite exposure group was more severely harmed than the single exposure group. These abnormalities in structure and function were related to the decreased expression and disordered distribution of Cx43.


Assuntos
Conexina 43 , Células-Tronco Pluripotentes Induzidas , Humanos , Conexina 43/genética , Micro-Ondas/efeitos adversos , Membrana Celular , Meios de Cultura
14.
Int J Mol Sci ; 24(16)2023 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-37628952

RESUMO

Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-ß-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.


Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , Cavéolas , Osteogênese , Endocitose , Diferenciação Celular , Meios de Cultura
15.
Int J Mol Sci ; 24(16)2023 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-37629041

RESUMO

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor ß (TGFß), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.


Assuntos
Neurônios Receptores Olfatórios , Animais , Camundongos , Camundongos Endogâmicos C57BL , Fibronectinas , Cicatriz , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fator de Crescimento Neural , Axônios , Proteínas da Matriz Extracelular , Colágeno Tipo IV , Meios de Cultura
16.
Int J Mol Sci ; 24(16)2023 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-37629141

RESUMO

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by Prevotella copri (Segatella copri) or Rhodospirillum rubrum inside Escherichia coli cells. Four tRFs imported by E. coli cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of E. coli. Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of E. coli and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria.


Assuntos
Escherichia coli , Espécies Introduzidas , Escherichia coli/genética , Reprodutibilidade dos Testes , RNA de Transferência/genética , Meios de Cultura , RNA
17.
Methods Mol Biol ; 2704: 185-200, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37642845

RESUMO

This chapter describes methods for cultivation and characterization of the growth of Mycolicibacterium spp. mutants in a microbioreactor system in the presence of steroids and/or phytosterols followed by high-throughput mass spectrometry analysis to describe their ability to convert phytosterols into the target steroid androstenedione (AD). We focus on Mycolicibacterium neoaurum NRRL B-3805 ΔkstD which can convert phytosterol into androstenedione (AD) as one of its major steroid products, and mutants thereof with increased tolerance towards this end-product. By using BioLector 48-well plates with optodes at the bottom of each well, bacterial growth can be monitored online despite the turbidity of the growth medium resulting from non-dissolved phytosterol and steroid particles. To cope with the large number of samples that accumulate during growth experiments in microbioreactors and similar formats (e.g., microtiter plates), protocols for extraction and subsequent RapidFire-MS analysis are presented. This reduces the analysis time per sample to 10 s from 10 min required for regular LC-MS analysis.


Assuntos
Androstenodiona , Fitosteróis , Cromatografia Líquida , Meios de Cultura , Esteroides
18.
Diagn Microbiol Infect Dis ; 107(2): 115959, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37536260

RESUMO

The BACT/ALERT® MP Reagent System is a broth culture medium for optimal detection and recovery of mycobacteria from clinical samples. The MP formulation was recently modified to improve detection and recovery times. A multicenter prospective matched pair study design was conducted to validate the performance of improved MP (MP-I) versus current MP (MP-C) bottles utilizing nonsterile and normally sterile samples, except blood, from patients suspected of having mycobacterial infections. A total of 1488 clinical samples were collected to obtain 212 mycobacteria samples by either or both MP culture bottles. MP-I and MP-C sensitivities were 86.6% and 81.4%, respectively, but the difference was not significant (P = 0.163) while specificities were 96.8% and 93.8%, respectively, and that difference was significant (P = 0.002). Overall recovery was 94.34% for MP-I and 88.68% for MP-C (recovery was 100% for both bottles with 52 seeded samples). Overall performance of MP-I was better than MP-C for sensitivity, specificity, and recovery.


Assuntos
Infecções por Mycobacterium , Mycobacterium , Humanos , Estudos Prospectivos , Meios de Cultura , Infecções por Mycobacterium/microbiologia , Kit de Reagentes para Diagnóstico
19.
Sci Rep ; 13(1): 13716, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37607956

RESUMO

The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.


Assuntos
Fibroblastos , Cabras , Animais , Meios de Cultura/farmacologia , Glucose/farmacologia , Técnicas de Cultura de Células
20.
J Microbiol Methods ; 212: 106807, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37573888

RESUMO

A new culture technique that involves a potato slice and enriched Middlebrook 7H9 in a two-part glass tube has been developed to revive dormant or persistent Mycobacterium avium subspecies paratuberculosis and acclimate it to Middlebrook 7H9 liquid media. This method is more efficient than directly introducing the bacteria into the liquid medium.


Assuntos
Mycobacterium avium subsp. paratuberculosis , Paratuberculose , Animais , Meios de Cultura , Paratuberculose/microbiologia
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