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1.
Molecules ; 26(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34500599

RESUMO

Agaricus bisporus (J.E. Lange) Imbach is one the most popular species of edible mushrooms in the world because of its taste and nutritional properties. In the research, repeatability of accumulation of bioelements and biomass yield in experimentally chosen in vitro culture medium, was confirmed. The in vitro cultures were conducted on the modified Oddoux medium enriched with bioelements (Mg, Zn, Cu, Fe). The aim of the study was to create an effective method of sampling, which enabled non-invasive monitoring of metals concentrations changes in the medium, during increase of biomass in in vitro cultures. The first, indirect method of sampling was applied. The non-invasive probe (a dipper) for in vitro culture was used; hence, the highest biomass increase and metals accumulation were gained. The method also guaranteed culture sterility. The second method, a direct one, interfered the in vitro culture conditions and growth of mycelium, and as a consequence the lower biomass increase and metals' accumulation were observed. Few cases of contaminations of mycelium in in vitro cultures were observed. The proposed method of non-invasive sampling of the medium can be used to monitor changes in the concentrations of metals in the medium and their accumulation in the mycelium in natural environment. Changes in concentrations of the selected metals over time, determined by the method of atomic absorption spectrometry, made it possible to correlate the obtained results with the specific stages of A. bisporus mycelium development and to attempt to explain the mechanism of sampling metals from the liquid substrate.


Assuntos
Agaricus/metabolismo , Biotecnologia/métodos , Meios de Cultura/metabolismo , Biomassa , Metais/metabolismo , Micélio/metabolismo , Espectrofotometria Atômica/métodos
2.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445120

RESUMO

Serum albumin physically interacts with fatty acids, small molecules, metal ions, and several other proteins. Binding with a plethora of bioactive substances makes it a critical transport molecule. Albumin also scavenges the reactive oxygen species that are harmful to cell survival. These properties make albumin an excellent choice to promote cell growth and maintain a variety of eukaryotic cells under in vitro culture environment. Furthermore, purified recombinant human serum albumin is mostly free from impurities and modifications, providing a perfect choice as an additive in cell and tissue culture media while avoiding any regulatory constraints. This review discusses key features of human serum albumin implicated in cell growth and survival under in vitro conditions.


Assuntos
Células Eucarióticas/metabolismo , Albumina Sérica Humana/metabolismo , Animais , Linhagem Celular , Meios de Cultura/metabolismo , Humanos
4.
Microbes Environ ; 36(3)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34433738

RESUMO

Current challenges in the anaerobic bioremediation of benzene are the lack of capable cultures and limited knowledge on the biodegradation pathway. Under methanogenic conditions, benzene may be mineralized by syntrophic interactions between microorganisms, which are poorly understood. The present study developed an optimized formula for anoxic medium to successfully promote the growth of the putative benzene degrader Deltaproteobacterium Hasda-A and enhance the benzene degradation activity of methanogenic enrichment cultures. Within 70| |d of incubation, the benzene degradation activity and relative abundance of Hasda-A in cultures in the new defined medium increased from 0.5 to >3| |mg L-1 d-1 and from 2.5% to >17%, respectively. Together with Hasda-A, we found a strong positive relationship between the abundances of superphylum OD1 bacteria, three methanogens (Methanoregula, Methanolinea, and Methanosaeta) and benzene degradation activity. The syntrophic relationship between these microbial taxa and Hasda-A was then demonstrated in a correlation analysis of longitudinal data. The involvement of methanogenesis in anaerobic benzene mineralization was confirmed by inhibition experiments. The high benzene degradation activity and growth of Hasda-A were quickly recovered in successive dilutions of enrichment cultures, proving the feasibility of using the medium developed in the present study to produce highly capable cultures. The present results will facilitate practical applications in bioremediation and research on the molecular mechanisms underlying benzene activation and syntrophic interactions in benzene mineralization.


Assuntos
Benzeno/metabolismo , Meios de Cultura/química , Deltaproteobacteria/metabolismo , Metano/metabolismo , Methanosarcinales/metabolismo , Anaerobiose , Biodegradação Ambiental , Crescimento Quimioautotrófico , Técnicas de Cocultura , Meios de Cultura/metabolismo , Deltaproteobacteria/crescimento & desenvolvimento , Methanosarcinales/crescimento & desenvolvimento
5.
Biomolecules ; 11(8)2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34439843

RESUMO

Cancer cell culture is routinely performed under superphysiologic O2 levels and in media such as Dulbecco's Modified Eagle Medium (DMEM) with nutrient composition dissimilar to mammalian extracellular fluid. Recently developed cell culture media (e.g., Plasmax, Human Plasma-Like Medium (HPLM)), which are modeled on the metabolite composition of human blood plasma, have been shown to shift key cellular activities in several cancer cell lines. Similar effects have been reported with respect to O2 levels in cell culture. Given these observations, we investigated how media composition and O2 levels affect cellular energy metabolism and mitochondria network structure in MCF7, SaOS2, LNCaP, and Huh7 cells. Cells were cultured in physiologic (5%) or standard (18%) O2 levels, and in physiologic (Plasmax) or standard cell culture media (DMEM). We show that both O2 levels and media composition significantly affect mitochondrial abundance and network structure, concomitantly with changes in cellular bioenergetics. Extracellular acidification rate (ECAR), a proxy for glycolytic activity, was generally higher in cells cultured in DMEM while oxygen consumption rates (OCR) were lower. This effect of media on energy metabolism is an important consideration for the study of cancer drugs that target aspects of energy metabolism, including lactate dehydrogenase activity.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/metabolismo , Micronutrientes/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Oxigênio/metabolismo , Linhagem Celular Tumoral , Metabolismo Energético , Humanos
6.
Molecules ; 26(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299537

RESUMO

Microbial natural products are an invaluable resource for the biotechnological industry. Genome mining studies have highlighted the huge biosynthetic potential of fungi, which is underexploited by standard fermentation conditions. Epigenetic effectors and/or cultivation-based approaches have successfully been applied to activate cryptic biosynthetic pathways in order to produce the chemical diversity suggested in available fungal genomes. The addition of Suberoylanilide Hydroxamic Acid to fermentation processes was evaluated to assess its effect on the metabolomic diversity of a taxonomically diverse fungal population. Here, metabolomic methodologies were implemented to identify changes in secondary metabolite profiles to determine the best fermentation conditions. The results confirmed previously described effects of the epigenetic modifier on the metabolism of a population of 232 wide diverse South Africa fungal strains cultured in different fermentation media where the induction of differential metabolites was observed. Furthermore, one solid-state fermentation (BRFT medium), two classic successful liquid fermentation media (LSFM and YES) and two new liquid media formulations (MCKX and SMK-II) were compared to identify the most productive conditions for the different populations of taxonomic subgroups.


Assuntos
Epigênese Genética/genética , Fungos/genética , Folhas de Planta/microbiologia , Produtos Biológicos/metabolismo , Vias Biossintéticas/genética , Biotecnologia/métodos , Meios de Cultura/metabolismo , Fermentação/genética , Genoma Fúngico/genética , Metabolômica/métodos , África do Sul
7.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209215

RESUMO

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has been linked to several human malignancies and shown to promote tumorigenesis. The purpose of this study was to explore the relative abundance of pro-brain-derived neurotrophic factor (proBDNF) and mature BDNF (mBDNF) in A549 (p53 wild-type) and H1299 (p53-null) lung cancer cell media. Higher levels of proBDNF were detected in the media of A549 cells than in H1299 cell media. Using inhibitors, we found that the levels of proBDNF and mBDNF in the media are likely regulated by PI3K, AKT, and NFκB. However, the largest change in these levels resulted from MMP2/9 inhibition. Blocking p53 function in A549 cells resulted in increased mBDNF and decreased proBDNF, suggesting a role for p53 in regulating these levels. The ratio of proBDNF/mBDNF was not affected by MMP2 knockdown but increased in the media of both cell lines upon knockdown of MMP9. Downregulation of either MMP2 or MMP9 by siRNA showed that MMP9 siRNA treatment of either A549 or H1299 cells resulted in decreased cell viability and increased apoptosis, an effect diminished upon the same treatment with proBDNF immunodepleted media, suggesting that MMP9 regulates the cytotoxic effects induced by proBDNF in lung cancer cells.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Meios de Cultura/metabolismo , Neoplasias Pulmonares/metabolismo , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Células A549 , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Mutação , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
8.
Methods Mol Biol ; 2289: 23-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270061

RESUMO

Anther and microspore culture for producing haploid plants are very complex systems and include general effects where the specific effects must be identified and optimized to develop culture systems capable of producing the large numbers of haploids required by breeding programs. These general effects include genotype, physiological state of the source plant, age of the anthers and microspores, preculture treatments, culture conditions, and culture media. Design of experiments (DoE) is an experimental approach specifically designed to identify and optimize the multiple factors that make up complex systems, and is ideally suited for developing in vitro systems to produce haploids. The basic DoE strategy starts by screening multiple factors thought to affect the responses being measured. Screening identifies factors with large and small effects. Factors with large effects are used to manipulate the system, and are moved to the DoE optimization phase such as response surface methodology. Factors with small or trivial effects are eliminated from further consideration, and this simplifies the system. The basic concepts of fractional factorial designs and how to use them are explained. Fractional factorials are the most important DoE screening tool and are the first experiments run before DoE optimization experiments. To illustrate the unique properties of fractional factorials, a detailed example is provided that includes all of the calculations so that no statistical software is required.


Assuntos
Melhoramento Vegetal/métodos , Plantas/genética , Meios de Cultura/metabolismo , Genótipo , Haploidia , Software
9.
Methods Mol Biol ; 2289: 71-85, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270063

RESUMO

As in any other economically important crop, the possibility of producing fully homozygous, doubled haploid lines in cucumber allows for faster and cheaper breeding. At present, the fastest way to doubled haploidy is the production of cucumber haploid plants and duplication of their chromosomes to make them doubled haploid. In this chapter, we describe a complete protocol to successfully produce cucumber doubled haploid plants, including the evaluation of their ploidy level by flow cytometry. Briefly, this protocol involves a first step of anther culture to induce microspores to divide and proliferate forming calli. The calli produced are isolated from anthers and transferred first to a liquid medium and then to a solid medium to induce organogenesis. Organogenic shoots will eventually give rise to entire DH plants.


Assuntos
Cucumis sativus/genética , Melhoramento Vegetal/métodos , Técnicas de Cultura de Tecidos/métodos , Meios de Cultura/metabolismo , Flores/genética , Haploidia
10.
Methods Mol Biol ; 2289: 249-261, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270075

RESUMO

African violet (Saintpaulia ionantha) is an herbaceous perennial of the Gesneriaceae family. Because almost all the cultivars are heterozygous, pure lines are useful for both classical and new breeding approaches. A shortcut to obtain purebred lines involves the production of doubled haploid strains produced from anther-derived haploids. In this chapter, a protocol for culturing African violet anthers is described in detail.


Assuntos
Técnicas de Cultura de Células/métodos , Flores/genética , Magnoliopsida/genética , Melhoramento Vegetal/métodos , Regeneração/genética , Meios de Cultura/metabolismo , Haploidia
11.
Methods Mol Biol ; 2289: 289-299, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270078

RESUMO

Doubled haploid technology allows for producing completely homozygous plants in one generation, which is a very efficient and fast method compared to the production of near-homozygous lines by selfing through conventional breeding methods. However, grain legumes are known to be recalcitrant for most of the in vitro approaches to doubled haploidy. In the last years, significant advances have been made with several legume species through in vitro methods. Chickpea is one of the most important legume species. Several reports have documented the successful generation of haploid plants through anther culture. These reports also showed that successful production of chickpea haploids was achieved when time- and labor-consuming physical stresses such as centrifugation and electroporation were applied to anthers as a pretreatment. In this chapter, we present an efficient and simple anther culture protocol for production of chickpea haploid plants using high concentrations of 2,4-D and silver nitrate in the culture medium, but without applying any physical stresses to anthers.


Assuntos
Técnicas de Cultura de Células/métodos , Cicer/genética , Flores/genética , Melhoramento Vegetal/métodos , Meios de Cultura/metabolismo , Fabaceae/genética , Haploidia , Estresse Fisiológico/genética
12.
Food Microbiol ; 99: 103839, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119090

RESUMO

The use of non-Saccharomyces yeast together with S. cerevisiae in winemaking is a current trend. Apart from the organoleptic modulation of the wine, the composition of the resulting yeast lees is different and may thus impact malolactic fermentation (MLF). Yeasts of Saccharomyces cerevisiae, Torulaspora delbrueckii and Metschnikowia pulcherrima were inactivated and added to a synthetic wine. Three different strains of Oenococcus oeni were inoculated and MLF was monitored. Non-Saccharomyces lees, especially from some strains of T. delbrueckii, showed higher compatibility with some O. oeni strains, with a shorter MLF and a maintained bacterial cell viability. The supplementation of lees increased nitrogen compounds available by O. oeni. A lower mannoprotein consumption was related with longer MLF. Amino acid assimilation by O. oeni was strain specific. There may be many other compounds regulating these yeast lees-O. oeni interactions apart from the well-known mannoproteins and amino acids. This is the first study of MLF with different O. oeni strains in the presence of S. cerevisiae and non-Saccharomyces yeast lees to report a strain-specific interaction between them.


Assuntos
Malatos/metabolismo , Oenococcus/metabolismo , Vinho/microbiologia , Leveduras/metabolismo , Meios de Cultura/metabolismo , Fermentação , Filogenia , Leveduras/classificação , Leveduras/genética
13.
Food Microbiol ; 99: 103813, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119100

RESUMO

Tyramine is one of the most toxic biogenic amines and it is produced commonly by lactic acid bacteria in fermented food products. In present study, we investigated the influence of selected nisin-producing Lactococcus lactis subsp. lactis strains and their cell-free supernatants (CFSs) on tyramine production by four Lactobacillus and two Lactiplantibacillus strains isolated from cheese and beer. Firstly, we examined the antimicrobial effect of the CFSs from twelve Lactococcus strains against tested tyramine producers by agar-well diffusion assay. Six Lactococcus strains whose CFSs showed the highest antimicrobial effect on tyramine producers were further studied. Secondly, we investigated the influence of the selected six Lactococcus strains and their respective CFSs on tyramine production by tested Lactobacillus and Lactiplantibacillus strains in MRS broth supplemented with 2 g.L-1 of l-tyrosine. Tyramine production was monitored by HPLC-UV. The tyramine formation of all tested Lactobacillus and Lactiplantibacillus strains was not detected in the presence of Lc. lactis subsp. lactis CCDM 71 and CCDM 702, and their CFSs. Moreover, the remainder of the investigated Lactococcus strains (CCDM 670, CCDM 686, CCDM 689 and CCDM 731) and their CFSs decreased tyramine production significantly (P < 0.05) - even suppressing it completely in some cases - in four of the six tested tyramine producing strains.


Assuntos
Antibacterianos/farmacologia , Cerveja/microbiologia , Queijo/microbiologia , Meios de Cultura/farmacologia , Lactobacillaceae/efeitos dos fármacos , Lactobacillus/efeitos dos fármacos , Lactococcus lactis/química , Tiramina/farmacologia , Antibacterianos/análise , Antibacterianos/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Meios de Cultura/metabolismo , Lactobacillaceae/crescimento & desenvolvimento , Lactobacillaceae/isolamento & purificação , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/isolamento & purificação , Lactococcus lactis/metabolismo , Tiramina/análise , Tiramina/metabolismo
14.
Food Microbiol ; 99: 103818, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119103

RESUMO

Conventional Salmonella detection is time consuming, often employing a 24-h pre-enrichment step in buffered peptone water (BPW), followed by a 24-h selective enrichment in either Rappaport Vassiliadis (RV) or tetrathionate (TT) broths before streaking onto selective indicator agar. To reduce this time, we sought to optimize pre-enrichment for Salmonella recovery by evaluating the addition of selective chemicals to BPW. Duplicate samples each representative of 500 carcasses were collected by catching processing water drip under moving carcass shackle lines immediately after feather removal in each of nine commercial processing plants. Carcass drip samples were cultured under selective pre-enrichment conditions in parallel with BPW pre-enrichment followed by RV and TT selective enrichment. Addition of bile salts (1 g/L) and novobiocin (0.015 g/L) resulted in Salmonella recovery from 89% samples when plated directly after pre-enrichment compared to 67% recovery in non-selective BPW alone. Salmonella serovar identities were determined using CRISPR-SeroSeq. Overall, serovars matched between selective pre-enrichment and traditional enrichment methods. These data suggest that increasing the selectivity of Salmonella pre-enrichment step may lessen the need for a separate selective enrichment step thereby reducing time required for Salmonella isolation by 24 h.


Assuntos
Técnicas Bacteriológicas/métodos , Contaminação de Alimentos/análise , Microbiologia de Alimentos/métodos , Aves Domésticas/microbiologia , Salmonella/crescimento & desenvolvimento , Animais , Meios de Cultura/química , Meios de Cultura/metabolismo , Manipulação de Alimentos , Salmonella/isolamento & purificação , Salmonella/metabolismo
15.
Food Microbiol ; 99: 103826, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34119111

RESUMO

The aim of the present study was to evaluate the effect of oxygen availability (aerobic, hypoxic and anoxic conditions) and sub-optimal pH (6.2 and 5.5) in a structured medium (10% w/V gelatin) on the growth of two immobilized L. monocytogenes strains (C5, 6179) at 10 °C and their subsequent acid resistance (pH 2.0, e.g., gastric acidity). Anaerobic conditions resulted in lower bacterial population (P < 0.05) (7.8-8.2 log CFU/mL) at the end of storage than aerobic and hypoxic environment (8.5-9.0 log CFU/mL), a phenomenon that was intensified at lower pH (5.5), where no significant growth was observed for anaerobically grown cultures. Prolonged habituation of L. monocytogenes (15 days) at both pH increased its acid tolerance resulting in max. 10 times higher t4D (appx. 60 min). The combined effect though of oxygen availability and suboptimal pH on L. monocytogenes acid resistance was found to vary with the strain. Anoxically grown cultures at pH 5.5 exhibited the lowest tolerance towards lethal acid stress, with countable survivors occurring only until 20 min of exposure at pH 2.0. Elucidating the role of oxygen limiting conditions, often encountered in structured foods, on acid resistance of L. monocytogenes, would assist in assessing the capacity of L. monocytogenes originated from different food-related niches to withstand gastric acidity and possibly initiate infection.


Assuntos
Ácidos/metabolismo , Meios de Cultura/metabolismo , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/metabolismo , Oxigênio/metabolismo , Anaerobiose , Células Imobilizadas/química , Células Imobilizadas/metabolismo , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Listeria monocytogenes/química
16.
Nat Microbiol ; 6(6): 783-791, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34017106

RESUMO

As bacteria transition from exponential to stationary phase, they change substantially in size, morphology, growth and expression profiles. These responses also vary between individual cells, but it has proved difficult to track cell lineages along the growth curve to determine the progression of events or correlations between how individual cells enter and exit dormancy. Here, we developed a platform for tracking more than 105 parallel cell lineages in dense and changing cultures, independently validating that the imaged cells closely track batch populations. Initial applications show that for both Escherichia coli and Bacillus subtilis, growth changes from an 'adder' mode in exponential phase to mixed 'adder-timers' entering stationary phase, and then a near-perfect 'sizer' upon exit-creating broadly distributed cell sizes in stationary phase but rapidly returning to narrowly distributed sizes upon exit. Furthermore, cells that undergo more divisions when entering stationary phase suffer reduced survival after long periods of dormancy but are the only cells observed that persist following antibiotic treatment.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Escherichia coli/crescimento & desenvolvimento , Antibacterianos/farmacologia , Bacillus subtilis/citologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo
17.
BMC Cancer ; 21(1): 629, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34044797

RESUMO

BACKGROUND: Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. METHODS: Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. RESULTS: Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. CONCLUSION: In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Metformina/farmacologia , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Células 3T3 BALB , Carcinógenos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/induzido quimicamente , Meios de Cultura/metabolismo , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Interações Medicamentosas , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Glucose/metabolismo , Humanos , Metformina/uso terapêutico , Metilcolantreno/toxicidade , Camundongos , Mitomicina/farmacologia , Mitomicina/uso terapêutico
18.
Medicine (Baltimore) ; 100(21): e25808, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34032694

RESUMO

ABSTRACT: MicroRNAs play important roles in gestational diabetes mellitus (GDM), and this study aimed to elucidate the clinical significance of miR-96-5p in diagnosing GDM.There are 123 pregnant women diagnosed with GDM and 123 healthy pregnant women were enrolled as control participants. Placenta and plasma samples from the patients and control participants were collected, and quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to determine miR-96-5p expression levels. Moreover, a receiver operating characteristic (ROC) curve was established to evaluate the significance of miR-96-5p in diagnosing GDM. HRT-8/SVneo trophoblasts were cultured under high glucose conditions and treated with miR-96-5p mimics, and cell viability was examined.miR-96-5p levels were significantly decreased in both the placenta and plasma samples of patients with GDM. The ROC curve indicated that miR-96-5p can serve as a diagnostic biomarker for GDM with high sensitivity and specificity. Moreover, miR-96-5p levels were markedly low under high glucose conditions, and the overexpression of miR-96-5p increased the viability, respectively, of trophoblasts in vitro.miR-96-5p may participate in the pathogenesis of GDM by exerting effects on the viability of trophoblasts.


Assuntos
Diabetes Gestacional/diagnóstico , MicroRNAs/análise , Adulto , Biomarcadores/análise , Biomarcadores/metabolismo , Glicemia/análise , Estudos de Casos e Controles , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/metabolismo , Diabetes Gestacional/sangue , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patologia , Feminino , Glucose/metabolismo , Teste de Tolerância a Glucose , Humanos , MicroRNAs/agonistas , MicroRNAs/metabolismo , Placenta/patologia , Gravidez , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trofoblastos
19.
Aging (Albany NY) ; 13(8): 11646-11664, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879632

RESUMO

OBJECTIVE: To investigate the heterogeneous responses of in vitro expanded chondrocytes, which were cultured in an interleukin (IL)-1ß -induced inflammatory environment. METHOD: Human articular chondrocytes were expanded, in vitro, for 13 days and treated with IL-1ß for 0, 24, and 48 h. Cells were collected and subjected to single-cell RNA sequencing. Multiple bioinformatics tools were used to determine the signatures that define chondrocyte physiology. RESULTS: Two major cell clusters with distinct expression patterns were identified at the initial phase and were with heterogeneous variation that coincides with inflammation progress. They transformed into two terminal cell clusters one of which exhibited OA-phenotype and proinflammatory characteristics through two paths, "response-to-inflammation" and "atypical response-to-inflammation", respectively. The involved cell clusters exhibited intrinsic relationship with cell types within native cartilage from OA patients. Genes controlling cell transformation to OA-phenotype were relating to the tumor necrosis factor (TNF) signaling pathway via NFKB, up-regulated KRAS signaling and the IL2/STAT5 signaling pathway and pathways relating to apoptosis and reactive oxygen species. CONCLUSION: The in vitro expanded chondrocytes under IL-1ß-induced inflammatory progression behave heterogeneously. One of the initial cell clusters could transform into a proinflammatory subpopulation through a termed response-to-inflammation path, which may serve as the core target to alleviate OA progression.


Assuntos
Condrócitos/patologia , Regulação da Expressão Gênica/imunologia , Osteoartrite/imunologia , Transdução de Sinais/genética , Cartilagem Articular/citologia , Células Cultivadas , Criança , Condrócitos/imunologia , Biologia Computacional , Meios de Cultura/metabolismo , Humanos , Interleucina-1beta/metabolismo , Osteoartrite/genética , Osteoartrite/patologia , Cultura Primária de Células , RNA-Seq , Transdução de Sinais/imunologia , Análise de Célula Única
20.
Biomolecules ; 11(4)2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805227

RESUMO

How cancer cells utilize nutrients to support their growth and proliferation in complex nutritional systems is still an open question. However, it is certainly determined by both genetics and an environmental-specific context. The interactions between them lead to profound metabolic specialization, such as consuming glucose and glutamine and producing lactate at prodigious rates. To investigate whether and how glucose and glutamine availability impact metabolic specialization, we integrated computational modeling on the genome-scale metabolic reconstruction with an experimental study on cell lines. We used the most comprehensive human metabolic network model to date, Recon3D, to build cell line-specific models. RNA-Seq data was used to specify the activity of genes in each cell line and the uptake rates were quantitatively constrained according to nutrient availability. To integrated both constraints we applied a novel method, named Gene Expression and Nutrients Simultaneous Integration (GENSI), that translates the relative importance of gene expression and nutrient availability data into the metabolic fluxes based on an observed experimental feature(s). We applied GENSI to study hepatocellular carcinoma addiction to glucose/glutamine. We were able to identify that proliferation, and lactate production is associated with the presence of glucose but does not necessarily increase with its concentration when the latter exceeds the physiological concentration. There was no such association with glutamine. We show that the integration of gene expression and nutrient availability data into genome-wide models improves the prediction of metabolic phenotypes.


Assuntos
Meios de Cultura/metabolismo , Regulação Neoplásica da Expressão Gênica , Biomassa , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Modelos Biológicos , Fosforilação Oxidativa/efeitos dos fármacos
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