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1.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502240

RESUMO

Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.


Assuntos
Anticoagulantes/farmacologia , Plaquetas/fisiologia , Respiração Celular/fisiologia , Meios de Cultura/farmacologia , Mitocôndrias/fisiologia , Ativação Plaquetária , Plaquetas/efeitos dos fármacos , Citometria de Fluxo , Humanos , Mitocôndrias/efeitos dos fármacos
2.
Molecules ; 26(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34361724

RESUMO

Orchids are rich treasure troves of various important phytomolecules. Among the various medicinal orchids, Ansellia africana stands out prominently in the preparing of various herbal medicines due to its high therapeutic importance. The nodal explants of A. africana were sampled from asymbiotically germinated seedlings on basal Murashige and Skoog (MS) medium and were micropropagated in MS medium supplemented with 3% sucrose and 10 µM meta topolin (mT) + 5 µM naphthalene acetic acid (NAA) +15 µM indole butyric acid (IBA) + 30 µM phloroglucinol (PG). In the present study, the essential oil was extracted by hydrodistillation and the oleoresins by the solvent extraction method from the micropropagated A. africana. The essential oil and the oleoresins were analysed by Gas Chromatography (GC) and GC/MS (Mass spectrometry). A total of 84 compounds were identified. The most predominant components among them were linoleic acid (18.42%), l-ascorbyl 2,6-dipalmitate (11.50%), linolenic acid (10.98%) and p-cresol (9.99%) in the essential oil; and eicosane (26.34%), n-butyl acetate (21.13%), heptadecane (16.48%) and 2-pentanone, 4-hydroxy-4-methyl (11.13%) were detected in the acetone extract; heptadecane (9.40%), heneicosane (9.45%), eicosane (6.40%), n-butyl acetate (14.34%) and styrene (22.20%) were identified and quantified in the ethyl acetate extract. The cytotoxic activity of essential oil and oleoresins of micropropagated A. africana was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) assay on Vero cells compared to the standard drug doxorubicin chloride. The present research contains primary information about the therapeutic utility of the essential oil and oleoresins of A. africana with a promising future research potential of qualitative and quantitative improvement through synchronised use of biotechnological techniques.


Assuntos
Citotoxinas/isolamento & purificação , Óleos Voláteis/isolamento & purificação , Orchidaceae/química , Extratos Vegetais/isolamento & purificação , Plântula/química , Acrilatos/isolamento & purificação , Alcanos/isolamento & purificação , Animais , Ácido Ascórbico/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Cresóis/isolamento & purificação , Meios de Cultura/química , Meios de Cultura/farmacologia , Citotoxinas/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Hidroponia/métodos , Ácido Linoleico/isolamento & purificação , Extração Líquido-Líquido/métodos , Óleos Voláteis/farmacologia , Orchidaceae/metabolismo , Palmitatos/isolamento & purificação , Pentanóis/isolamento & purificação , Pentanonas/isolamento & purificação , Extratos Vegetais/farmacologia , Plantas Medicinais , Plântula/metabolismo , África do Sul , Estireno/isolamento & purificação , Células Vero , Ácido alfa-Linoleico/isolamento & purificação
3.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34204008

RESUMO

Assisted reproductive technologies impact transcriptome and epigenome of embryos and can result in long-term phenotypic consequences. Whole-genome DNA methylation profiles from individual bovine blastocysts in vivo- and in vitro-derived (using three sources of protein: reproductive fluids, blood serum and bovine serum albumin) were generated. The impact of in vitro culture on DNA methylation was analyzed, and sex-specific methylation differences at blastocyst stage were uncovered. In vivo embryos showed the highest levels of methylation (29.5%), close to those produced in vitro with serum, whilst embryos produced in vitro with reproductive fluids or albumin showed less global methylation (25-25.4%). During repetitive element analysis, the serum group was the most affected. DNA methylation differences between in vivo and in vitro groups were more frequent in the first intron than in CpGi in promoters. Moreover, hierarchical cluster analysis showed that sex produced a stronger bias in the results than embryo origin. For each group, distance between male and female embryos varied, with in vivo blastocyst showing a lesser distance. Between the sexually dimorphic methylated tiles, which were biased to X-chromosome, critical factors for reproduction, developmental process, cell proliferation and DNA methylation machinery were included. These results support the idea that blastocysts show sexually-dimorphic DNA methylation patterns, and the known picture about the blastocyst methylome should be reconsidered.


Assuntos
Blastocisto/metabolismo , Reprogramação Celular/genética , Meios de Cultura/farmacologia , Epigênese Genética/efeitos dos fármacos , Caracteres Sexuais , Animais , Blastocisto/efeitos dos fármacos , Bovinos , Cromossomos de Mamíferos/genética , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Feminino , Fertilização In Vitro , Ontologia Genética , Modelos Logísticos , Masculino , Anotação de Sequência Molecular , Análise de Componente Principal
4.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209789

RESUMO

Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.


Assuntos
Materiais Biocompatíveis/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Meios de Cultura/farmacologia , Adulto , Materiais Biocompatíveis/química , Plaquetas/citologia , Meios de Cultura/química , Endotélio/citologia , Feminino , Humanos , Masculino , Teste de Materiais , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Polímeros/farmacologia , Tecidos Suporte/química
5.
Methods Mol Biol ; 2314: 595-609, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235672

RESUMO

Antimicrobial susceptibility testing is the mainstay of tuberculosis drug development programs. In this chapter, we describe methods for determination of the minimum inhibitory concentration of compounds against Mycobacterium tuberculosis growing in liquid media as a function of carbon source, detergent, and environmental stress imposed by acidic pH as well as reactive nitrogen intermediates. Methods for determining the effect of bovine serum albumin in the growth medium on antimicrobial susceptibility are also described. Finally, we provide a method for antimicrobial susceptibility testing on agar medium.


Assuntos
Ágar/química , Antituberculosos/farmacologia , Meios de Cultura/farmacologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Soroalbumina Bovina/metabolismo , Tuberculose/tratamento farmacológico , Carbono/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Estresse Fisiológico , Tuberculose/microbiologia , Tuberculose/patologia
6.
Life Sci ; 282: 119794, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34237312

RESUMO

AIMS: Engineered conduction tissues (ECTs) fabricated from cardiac progenitor cells (CPCs) and collagen sponges were precisely targeted for the treatment of atrioventricular conduction block in our previous studies. However, obvious shrinkage and deformation of ECTs was observed during in vitro culture. According to the literature, it can be speculated that basic fibroblast growth factor (bFGF) may downregulate alpha-smooth muscle actin (α-SMA) produced by CPCs to prevent the shrinkage of CPC-engineered conduction tissues. MAIN METHODS: In this study, culture media with or without bFGF were used for both cell culture and 3D tissue construction. The expression of α-SMA and the size change of engineered tissue were analyzed to evaluate the feasibility of adding bFGF to regulate α-SMA expression and shrinkage of constructs. In addition, cardiac-specific examinations were performed to evaluate the effect of bFGF on cardiac tissue formation. KEY FINDINGS: Supplementation with bFGF efficiently relieved shrinkage of engineered tissue by downregulating the expression of α-SMA at both the cellular and 3D tissue levels. Moreover, bFGF had a positive influence on cardiac tissue formation in terms of cell viability, tissue organization and electrical conduction velocity. SIGNIFICANCE: This study provides a guide for both shape control and quality improvement of CPC-engineered cardiac tissues.


Assuntos
Actinas/genética , Meios de Cultura/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Miocárdio/citologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Animais , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Feminino , Ratos Sprague-Dawley , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Tecidos Suporte/química
7.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205905

RESUMO

Human oral mucosa stem cells (hOMSCs) arise from the neural crest, they can self-renew, proliferate, and differentiate to several cell lines and could represent a good source for application in tissue engineering. Because of their anatomical location, hOMSCs are easy to isolate, have multilineage differentiation capacity and express embryonic stem cells markers such as-Sox2, Oct3/4 and Nanog. We have used SHEM (supplemented hormonal epithelial medium) media and cultured hOMSCs over human amniotic membrane and determined the cell's capacity to differentiate to an epithelial-like phenotype and to express corneal specific epithelial markers-CK3, CK12, CK19, Pan-cadherin and E-cadherin. Our results showed that hOMSCs possess the capacity to attach to the amniotic membrane and express CK3, CK19, Pan-Cadherin and E-Cadherin without induction with SHEM media and expressed CK12 or changed the expression pattern of E-Cadherin to a punctual-like feature when treated with SHEM media. The results observed in this study show that hOMSCs possess the potential to differentiate toward epithelial cells. In conclusion, our results revealed that hOMSCs readily express markers for corneal determination and could provide the ophthalmology field with a therapeutic alternative for tissue engineering to achieve corneal replacement when compared with other techniques. Nevertheless, further studies are needed to develop a predictable therapeutic alternative for cornea replacement.


Assuntos
Diferenciação Celular/genética , Epitélio Corneano/crescimento & desenvolvimento , Células-Tronco Mesenquimais/citologia , Mucosa Bucal/crescimento & desenvolvimento , Âmnio/crescimento & desenvolvimento , Células Cultivadas , Córnea/citologia , Córnea/crescimento & desenvolvimento , Córnea/metabolismo , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio Corneano/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Mucosa Bucal/citologia , Engenharia Tecidual/tendências
8.
FEBS Lett ; 595(14): 1886-1901, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34096057

RESUMO

Eukaryotes harbour a conserved signalling pathway, called General Amino Acid Control (GAAC) in Saccharomyces cerevisiae, for overcoming amino acid starvation. Upon starvation, the protein kinase Gcn2, which phosphorylates the eukaryotic translation initiation factor eIF2α, becomes stimulated to trigger the GAAC response. Genetic studies suggest that Yih1, which is the yeast homolog of mammalian IMPACT and which binds monomeric actin, inhibits Gcn2 when released from actin. Here, we found that D56A substitution in actin (the act1-9 allele) leads to reduced eIF2α phosphorylation, suggesting that the Asp56 residue is required for full Gcn2 activation. In the act1-9 mutant, Yih1 overexpression further enhanced the sensitivity to amino acid starvation-inducing drugs and further impaired eIF2α phosphorylation, suggesting that Gcn2 inhibition was mediated via Yih1. The D56A substitution may impair the actin-Yih1 interaction, directly or indirectly, thereby increasing the amount of Yih1 available to inhibit Gcn2.


Assuntos
Actinas/genética , Substituição de Aminoácidos , Ácido Aspártico/química , Fator de Iniciação 2 em Eucariotos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Actinas/química , Actinas/metabolismo , Alanina/química , Alanina/metabolismo , Alelos , Ácido Aspártico/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Compostos de Sulfonilureia/farmacologia
9.
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
10.
Am J Physiol Cell Physiol ; 321(1): C72-C81, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34010067

RESUMO

Estradiol (E2) and selective estrogen receptor modulators (SERMs) have broad-ranging cellular effects that include mitochondrial respiration and reactive oxygen species (ROS) metabolism. Many of these effects have been studied using cell culture models. Recent advances have revealed the extent to which cellular metabolism is affected by the culture environment. Cell culture media with metabolite composition similar to blood plasma [e.g., Plasmax, Human Plasma-Like Medium (HPLM)] alter cellular behaviors including responses to drugs. Similar effects have been observed with respect to O2 levels in cell culture. Given these observations, we investigated whether the effects of E2 and SERMs are also influenced by media composition and O2 level during cell culture experiments. We analyzed mitochondrial network characteristics, cellular oxidative metabolism, and H2O2 production in C2C12 myoblasts growing in physiological (5%) or standard cell culture (18%)O2 and in physiological (Plasmax) or standard cell culture [Dulbecco's modified Eagle's medium (DMEM)] media. Although E2 significantly lowered H2O2 production from cells growing in 18% O2/DMEM (standard cell culture), it had no effect on cells growing in Plasmax. Moreover, culture conditions significantly altered the effects of E2 and SERMs on mitochondrial abundance and network characteristics. These results indicate that the effects of E2 and SERMs on various aspects of cell physiology strongly depends on growth conditions, which in turn emphasizes the need to consider this carefully in cell culture experiments.


Assuntos
Meios de Cultura/farmacologia , Estradiol/farmacologia , Mitocôndrias/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Oxigênio/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose , Técnicas de Cultura de Células , Linhagem Celular , Meios de Cultura/química , Fluorescência , Genes Reporter , Peróxido de Hidrogênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo
11.
J Drugs Dermatol ; 20(5): 538-545, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33938706

RESUMO

BACKGROUND: Applied topically, growth factors, cytokines, and other components in bovine colostrum are known to affect collagen biosynthesis, thus offering promise as a therapeutic modality in wound healing, delay in skin aging, and skin rejuvenation. OBJECTIVE: To demonstrate the protective effect that liposomal bovine colostrum exerts on skin aging using telomere length as an aging biomarker. METHODS: Human fibroblasts were cultured for 8 weeks with colostrum at three concentrations (0.125%, 0.25%, 0.50%). Cells were cultured and assayed both under standard conditions, as well as with H2O2 added as an agent of oxidative stress. Alterations in proliferation rates, telomere lengths, and telomere shortening rates (TSRs) were determined in each treatment group and compared. RESULTS: Colostrum increased the proliferation rate of the fibroblast control cells and the addition of H2O2(without colostrum) decreased the proliferation rates of the fibroblast control cells. Under standard culture conditions, telomeres shortened progressively over 8 weeks and the addition of colostrum reduced the rate of telomere shortening. Under oxidative stress conditions (H2O2 – induced) the TSR increased; however, treatment with colostrum appeared to attenuate this increase. CONCLUSIONS: Under normal culture conditions and after both 4 weeks and 8 weeks of treatment, liposomal bovine colostrum appears to exert a protective effect on telomere length erosion. Under culture conditions of oxidative stress and after 8 weeks of treatment, colostrum appears to exert a protective effect on telomere length erosion. These results suggest that topical treatment of the liposomal bovine colostrum formulation would enhance skin health as the skin ages. J Drugs Dermatol. 20(5):538-545. doi:10.36849/JDD.5851.


Assuntos
Colostro/química , Rejuvenescimento , Envelhecimento da Pele/efeitos dos fármacos , Animais , Bovinos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/farmacologia , Feminino , Fibroblastos , Peróxido de Hidrogênio/metabolismo , Lipossomos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Gravidez , Cultura Primária de Células , Pele/citologia , Envelhecimento da Pele/genética , Telômero/metabolismo , Encurtamento do Telômero/efeitos dos fármacos
12.
Microb Cell Fact ; 20(1): 102, 2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34001083

RESUMO

As treatment of Staphylococcus aureus (S. aureus) osteomyelitis is often hindered by the development of antibiotic tolerance, novel antibacterial therapeutics are required. Here we found that the cell-free supernatant of Bacillus subtilis (B. subtilis CFS) killed planktonic and biofilm S. aureus, and increased S. aureus susceptibility to penicillin and gentamicin as well. Further study showed that B. subtilis CFS suppressed the expression of the genes involved in adhesive molecules (Cna and ClfA), virulence factor Hla, quorum sensing (argA, argB and RNAIII) and biofilm formation (Ica and sarA) in S. aureus. Additionally, our data showed that B. subtilis CFS changed the membrane components and increased membrane permeabilization of S. aureus. Finally, we demonstrated that B. subtilis CFS increased considerably the susceptibility of S. aureus to penicillin and effectively reduced S. aureus burdens in a mouse model of implant-associated osteomyelitis. These findings support that B. subtilis CFS may be a potential resistance-modifying agent for ß-lactam antibiotics against S. aureus.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/crescimento & desenvolvimento , Meios de Cultura/farmacologia , Osteomielite/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Antibacterianos/química , Bacillus subtilis/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Biofilmes/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Meios de Cultura/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Osteomielite/tratamento farmacológico , Percepção de Quorum , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética
13.
J Med Microbiol ; 70(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34048334

RESUMO

Introduction. Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE) are the most common pathogens from the genus Staphylococcus causing biofilm-associated infections. Generally, biofilm-associated infections represent a clinical challenge. Bacteria in biofilms are difficult to eradicate due to their resistance and serve as a reservoir for recurring persistent infections.Gap Statement. A variety of protocols for in vitro drug activity testing against staphylococcal biofilms have been introduced. However, there are often fundamental differences. All these differences in methodical approaches can then be reflected in the form of discrepancies between results.Aim. In this study, we aimed to develop optimal conditions for staphylococcal biofilm formation on pegs. The impact of peg surface modification was also studied.Methodology. The impact of tryptic soy broth alone or supplemented with foetal bovine serum (FBS) or human plasma (HP), together with the impact of the inoculum density of bacterial suspensions and the shaking versus the static mode of cultivation, on total biofilm biomass production in SA and SE reference strains was studied. The surface of pegs was modified with FBS, HP, or poly-l-lysine (PLL). The impact on total biofilm biomass was evaluated using the crystal violet staining method and statistical data analysis.Results. Tryptic soy broth supplemented with HP together with the shaking mode led to crucial potentiation of biofilm formation on pegs in SA strains. The SE strain did not produce biofilm biomass under the same conditions on pegs. Preconditioning of peg surfaces with FBS and HP led to a statistically significant increase in biofilm biomass formation in the SE strain.Conclusion. Optimal cultivation conditions for robust staphylococcal biofilm formation in vitro might differ among different bacterial strains and methodical approaches. The shaking mode and supplementation of cultivation medium with HP was beneficial for biofilm formation on pegs for SA (ATCC 29213) and methicillin-resistant SA (ATCC 43300). Peg conditioning with HP and PLL had no impact on biofilm formation in either of these strains. Peg coating with FBS showed an adverse effect on the biofilm formation of these strains. By contrast, there was a statistically significant increase in biofilm biomass production on pegs coated with FBS and HP for SE (ATCC 35983).


Assuntos
Técnicas Bacteriológicas/instrumentação , Biofilmes/crescimento & desenvolvimento , Staphylococcus/fisiologia , Animais , Técnicas Bacteriológicas/métodos , Biofilmes/classificação , Biofilmes/efeitos dos fármacos , Biomassa , Meios de Cultura/química , Meios de Cultura/farmacologia , Matriz Extracelular de Substâncias Poliméricas/classificação , Matriz Extracelular de Substâncias Poliméricas/efeitos dos fármacos , Humanos , Especificidade da Espécie , Staphylococcus/classificação , Staphylococcus/efeitos dos fármacos
14.
Cell Prolif ; 54(6): e13049, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33960560

RESUMO

OBJECTIVES: Mouse embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos and cultured in different culture media with varied pluripotency. Sporadically, a small population of ESCs exhibit 2-cell stage embryonic features in serum containing medium. However, whether ESCs can transit into 2-cell embryo-like (2C-like) cells in the chemically defined media remains largely unknown. MATERIALS AND METHODS: We established a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase the subpopulation of 2C-like cells. Further test the pluripotency and 2C features of ESCs cultured in RA. 2C reporter-positive cells were selected by FACS; the level of protein was detected via immunofluorescence staining and western blot; the level gene expressions were measured by RNA-seq. RESULTS: Retinoic acid drives a NELFA (negative elongation factor A)-mediated 2C-like state in mouse ESCs, characterized with 2C-specific transcriptional networks and the ability to contribute trophectoderm (TE) when injected into developing embryos. In addition, RA treatment triggers DNA hypomethylation, active histone modification, suppressed glycolysis metabolism and reduced protein synthesis activity of ESCs. CONCLUSIONS: We showed that RA has a broader role in 2C-like cells state, not only is one of the upstream regulators of the 2C-like state in chemically defined media but also illuminates genetic and epigenetic regulations that govern ESCs to 2C-like transition.


Assuntos
Meios de Cultura/farmacologia , Epigênese Genética/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos ICR , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo
15.
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
16.
Viruses ; 13(4)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923408

RESUMO

Aspergillus and Pseudomonas compete in nature, and are the commonest bacterial and fungal pathogens in some clinical settings, such as the cystic fibrosis lung. Virus infections of fungi occur naturally. Effects on fungal physiology need delineation. A common reference Aspergillus fumigatus strain, long studied in two (of many) laboratories, was found infected with the AfuPmV-1 virus. One isolate was cured of virus, producing a virus-free strain. Virus from the infected strain was purified and used to re-infect three subcultures of the virus-free fungus, producing six fungal strains, otherwise isogenic. They were studied in intermicrobial competition with Pseudomonasaeruginosa. Pseudomonas culture filtrates inhibited forming or preformed Aspergillus biofilm from infected strains to a greater extent, also seen when Pseudomonas volatiles were assayed on Aspergillus. Purified iron-chelating Pseudomonas molecules, known inhibitors of Aspergillus biofilm, reproduced these differences. Iron, a stimulus of Aspergillus, enhanced the virus-free fungus, compared to infected. All infected fungal strains behaved similarly in assays. We show an important consequence of virus infection, a weakening in intermicrobial competition. Viral infection may affect the outcome of bacterial-fungal competition in nature and patients. We suggest that this occurs via alteration in fungal stress responses, the mechanism best delineated here is a result of virus-induced altered Aspergillus iron metabolism.


Assuntos
Aspergillus fumigatus/fisiologia , Aspergillus fumigatus/virologia , Micovírus/patogenicidade , Interações entre Hospedeiro e Microrganismos/fisiologia , Interações Microbianas , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Meios de Cultura/química , Meios de Cultura/farmacologia , Ferro/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/fisiologia
17.
Am J Physiol Cell Physiol ; 321(1): C26-C37, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33909501

RESUMO

In vitro models of muscle aging are useful for understanding mechanisms of age-related muscle loss and aiding the development of targeted therapies. To investigate mechanisms of age-related muscle loss in vitro utilizing ex vivo human serum, fasted blood samples were obtained from four old (72 ± 1 yr) and four young (26 ± 3 yr) men. Older individuals had elevated levels of plasma CRP, IL-6, HOMA-IR, and lower concentric peak torque and work-per-repetition compared with young participants (P < 0.05). C2C12 myotubes were serum and amino acid starved for 1 h and conditioned with human serum (10%) for 4 h or 24 h. After 4 h, C2C12 cells were treated with 5 mM leucine for 30 min. Muscle protein synthesis (MPS) was determined through the surface sensing of translation (SUnSET) technique and regulatory signaling pathways were measured via Western blot. Myotube diameter was significantly reduced in myotubes treated with serum from old, in comparison to young donors (84%, P < 0.001). MPS was reduced in myotubes treated with old donor serum, compared with young serum before leucine treatment (32%, P < 0.01). MPS and the phosphorylation of Akt, p70S6K, and eEF2 were increased in myotubes treated with young serum in response to leucine treatment, with a blunted response identified in cells treated with old serum (P < 0.05). Muscle protein breakdown signaling pathways did not differ between groups. In summary, we show that myotubes conditioned with serum from older individuals had decreased myotube diameter and MPS compared with younger individuals, potentially driven by low-grade systemic inflammation.


Assuntos
Envelhecimento/genética , Meios de Cultura/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/genética , Biossíntese de Proteínas/efeitos dos fármacos , Adulto , Idoso , Envelhecimento/metabolismo , Animais , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Linhagem Celular , Meios de Cultura/química , Humanos , Resistência à Insulina , Interleucina-6/sangue , Interleucina-6/genética , Leucina/farmacologia , Masculino , Camundongos , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais
18.
Exp Cell Res ; 403(2): 112599, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33848551

RESUMO

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) create an unlimited cell source for basic and translational research. Depending on the maturity of cardiac cultures and the intended applications, obtaining hiPSC-CMs as a single-cell, monolayer or three-dimensional clusters can be challenging. Here, we defined strategies to replate hiPSC-CMs on early days (D15-30) or later more mature (D60-150) differentiation cultures. After generation of hiPSCs and derivation of cardiomyocytes, four dissociation reagents Collagenase A/B, Collagenase II, TrypLE, EDTA and five different extracellular matrix materials Laminin, iMatrix-511, Fibronectin, Matrigel, and Geltrex were comparatively evaluated by imaging, cell viability, and contraction analysis. For early cardiac differentiation cultures mimicking mostly the embryonic stage, the highest adhesion, cell viability, and beating frequencies were achieved by treatment with the TrypLE enzyme. Video-based contraction analysis demonstrated higher beating rates after replating compared to before treatment. For later differentiation days of more mature cardiac cultures, dissociation with EDTA and replating cells on Geltrex or Laminin-derivatives yielded better recovery. Cardiac clusters at various sizes were detected in several groups treated with collagenases. Collectively, our findings revealed the selection criteria of the dissociation approach and coating matrix for replating iPSC-CMs based on the maturity and the requirements of further downstream applications.


Assuntos
Técnicas de Cultura de Células , Meios de Cultura/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Adulto , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Reprogramação Celular/genética , Colágeno/farmacologia , Colagenases/farmacologia , Meios de Cultura/química , Combinação de Medicamentos , Feminino , Fibronectinas/farmacologia , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Insulina/análogos & derivados , Insulina/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Laminina/farmacologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Cultura Primária de Células , Proteoglicanas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
19.
Int J Mol Sci ; 22(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917790

RESUMO

Breast cancer remains the most common type of cancer, occurring in middle-aged women, and often leads to patients' death. In this work, we applied a cold atmospheric pressure plasma (CAPP)-based reaction-discharge system, one that is unique in its class, for the production of CAPP-activated media (DMEM and Opti-MEM); it is intended for further uses in breast cancer treatment. To reach this aim, different volumes of DMEM or Opti-MEM were treated by CAPP. Prepared media were exposed to the CAPP treatment at seven different time intervals and examined in respect of their impact on cell viability and motility, and the induction of the apoptosis in human non-metastatic (MCF7) and metastatic (MDA-MB-231) breast cancer cell lines. As a control, the influence of CAPP-activated media on the viability and motility, and the type of the cell death of the non-cancerous human normal MCF10A cell line, was estimated. Additionally, qualitative and quantitative analyses of the reactive oxygen and nitrogen species (RONS), generated during the CAPP operation in contact with analyzed media, were performed. Based on the conducted research, it was found that 180 s (media activation time by CAPP) should be considered as the minimal toxic dose, which significantly decreases the cell viability and the migration of MDA-MB-231 cells, and also disturbs life processes of MCF7 cells. Finally, CAPP-activated media led to the apoptosis of analyzed cell lines, especially of the metastatic MDA-MB-231 cell line. Therefore, the application of the CAPP system may be potentially applied as a therapeutic strategy for the management of highly metastatic human breast cancer.


Assuntos
Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Gases em Plasma/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/química , Feminino , Humanos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo
20.
Nat Commun ; 12(1): 2452, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907191

RESUMO

The cell cycle is the process by which eukaryotic cells replicate. Yeast cells cycle asynchronously with each cell in the population budding at a different time. Although there are several experimental approaches to synchronise cells, these usually work only in the short-term. Here, we build a cyber-genetic system to achieve long-term synchronisation of the cell population, by interfacing genetically modified yeast cells with a computer by means of microfluidics to dynamically change medium, and a microscope to estimate cell cycle phases of individual cells. The computer implements a controller algorithm to decide when, and for how long, to change the growth medium to synchronise the cell-cycle across the population. Our work builds upon solid theoretical foundations provided by Control Engineering. In addition to providing an avenue for yeast cell cycle synchronisation, our work shows that control engineering can be used to automatically steer complex biological processes towards desired behaviours similarly to what is currently done with robots and autonomous vehicles.


Assuntos
Ciclo Celular/genética , Ciclinas/genética , Retroalimentação Fisiológica , GTP Fosfo-Hidrolases/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Algoritmos , Automação Laboratorial , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo Celular/efeitos dos fármacos , Meios de Cultura/química , Meios de Cultura/farmacologia , Ciclinas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Genes Reporter , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Organismos Geneticamente Modificados , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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