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1.
Life Sci ; 239: 117013, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31678287

RESUMO

AIMS: Liver cancer is one of the leading causes of cancer mortality worldwide. Inspired by the biological structure and function of low-density lipoprotein (LDL), in this study, an ApopB-100 based targeted lipid nanoparticles was synthesized to improve the therapeutic efficacy in liver cancer treatment. MAIN METHODS: The biological composition of ApopB is similar to LDL which can effectively increase the targeting efficiency of nanoparticles in LDL receptor (LDLR)-overexpressed liver tumors. KEYFINDINGS: We have demonstrated that the co-administration of sorafenib (SRF) and Dihydroartemisinin (DHA) could exhibit synergistic anticancer effect in HepG2 liver cancer cells. DHA produced excessive cellular reactive oxygen species (ROS) and induced greater apoptosis of cancer cells. LDL-based SRF/DHA-loaded lipid nanoparticles (LD-SDN) showed remarkable decrease in the cell viability compared to that of either of single drug treated cancer cells. Combination of SRF+DHA resulted in predominant SubG1 proportion of cells. LD-SDN exhibited the highest SubG1 (%) of cells compared to that of any of the individual drugs. Most importantly, robust antitumor response and delayed tumor growth was observed for LD-SDN treated xenograft tumor model. Ki67 proliferation index of LD-SDN (22.1 ± 5.6%) is significantly lesser compared to that of either control (86.2 ± 6.9%) or SRF (75.4 ± 4.89%) or DHA (69.4 ± 6.9%). SIGNIFICANCES: These data provide strong evidence that LDL-mimetic lipid nanoformulations could be utilized as a biocompatible and tumor targeted platform for the delivery of multiple anticancer drugs in cancer treatment.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Lipídeos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas , Receptores de LDL/efeitos dos fármacos , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Artemisininas/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Espécies Reativas de Oxigênio/metabolismo , Sorafenibe/administração & dosagem
2.
Nutrients ; 11(7)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31323989

RESUMO

Human breast milk (HBM) may have beneficial effects on Lactobacillus reuteri DSM 17938 (LR 17938) -mediated immunomodulation. We aimed to determine the effects of HBM on proliferation of LR 17938 in vitro and its associated proteins and metabolites in culture, in order to provide mechanistic insights into the health benefits of LR 17938. LR 17938 was cultured anaerobically in MRS bacterial culture media, HBM (from 6 mothers), and 2 types of cow-milk formula. The colony-forming unit (CFU) was calculated to evaluate LR 17938 growth. Sixteen-hour-fermented supernatants were used for metabolomics, and bacterial lysates were used for proteomics analysis. We found that growth of LR 17938 was 10 times better in HBM than in formula. We detected 261/452 metabolites upregulated when LR 17938 cultured in HBM compared to in formula, mainly participating in the glyoxylate cycle (succinate), urea cycle (citrulline), methionine methylation (N-acetylcysteine), and polyamine synthesis (spermidine). The significantly up-regulated enzymes were also involved in the formation of acetyl-CoA in the glyoxylate cycle and the antioxidant N-acetylcysteine. In conclusion, HBM enhances the growth of LR 17938 compared to formula and promotes LR 17938-associated metabolites that relate to energy and antioxidant status, which may be linked to the physiological effects of L. reuteri.


Assuntos
Lactobacillus reuteri/metabolismo , Leite Humano/química , Probióticos , Proliferação de Células , Meios de Cultura , Humanos , Lactente , Fórmulas Infantis , Lactobacillus reuteri/classificação , Lactobacillus reuteri/efeitos dos fármacos , Lipídeos/farmacologia
3.
ACS Appl Mater Interfaces ; 11(28): 24971-24983, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31264399

RESUMO

A novel "symbiotic self-assembly" strategy that integrates the advantages of biocompatible lipids with a structurally robust polymer to efficiently encapsulate and deliver siRNAs is reported. The assembly process is considered to be symbiotic because none of the assembling components are capable of self-assembly but can form well-defined nanostructures in the presence of others. The conditions of the self-assembly process are simple but have been chosen such that it offers the ability to arrive at a system that is noncationic for mitigating carrier-based cytotoxicity, efficiently encapsulate siRNA to minimize cargo loss, be effectively camouflaged to protect the siRNA from nuclease degradation, and efficiently escape the endosome to cause gene knockdown. The lipid-siRNA-polymer (L-siP) nanoassembly formation and its disassembly in the presence of an intracellular trigger have been extensively characterized experimentally and through computational modeling. The complexes have been evaluated for the delivery of four different siRNA molecules in six different cell lines, where an efficient gene knockdown is demonstrated. The reported generalized strategy has the potential to make an impact on the development of a safe and effective delivery agent for RNAi-mediated gene therapy that holds the promise of targeting several hard-to-cure diseases.


Assuntos
Portadores de Fármacos , Inativação Gênica , Terapia Genética , Lipídeos , Nanopartículas , Polímeros , RNA Interferente Pequeno , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/farmacologia , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Lipídeos/química , Lipídeos/farmacocinética , Lipídeos/farmacologia , Nanopartículas/química , Nanopartículas/uso terapêutico , Polímeros/química , Polímeros/farmacocinética , Polímeros/farmacologia , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , RNA Interferente Pequeno/farmacologia
4.
Colloids Surf B Biointerfaces ; 181: 623-631, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31202972

RESUMO

There is a growing clinical demand for topical itraconazole (ITC) delivery systems because of the expanding potential of the drug for topical fungal and non-fungal applications. Lipid-based nanocarriers offer great promise in this respect. In the present study, a new topical ITC gel based on lipid nanocapsules (LNC) was developed. ITC-LNC were compared to ITC-loaded nanostructured lipid carriers (ITC-NLC) with more established benefits as topical vectors. Both nanocarriers showed high entrapment efficiency (EE > 98%). Compared to ITC-NLC, the ITC-LNC showed a significantly smaller particle size (∼50 vs 155 nm), narrower size distribution (0.09 vs 0.38), faster initial release rate under sink conditions and greater in vitro antifungal activity against Candida albicans (C. albicans) (inhibition zone 29.4 vs 26.4 mm). ITC-LNC and ITC-NLC-based gels significantly enhanced the dermal retention of ITC in excised human skin relative to a conventional ITC gel. Histopathological assessment of a 14-day treatment of induced cutaneous candidiasis in a rat model indicated efficacy of the gel preparations. Fungal elements developed in the superficial epidermal skin layer were cleared by the end of treatment. Equally important, no histopathological changes in the epidermal and dermal layers of rat skin were observed. Findings of this study verified efficacy of topical ITC in the treatment of superficial fungal infections as well as effectiveness of LNC as biomimetic nanocarrier for dermal drug delivery. Combining ITC and LNC would present a bioactive nanocarrier system with good potentials for fungal infections and other skin applications.


Assuntos
Antifúngicos/farmacologia , Candidíase/tratamento farmacológico , Itraconazol/farmacologia , Lipídeos/farmacologia , Pele/efeitos dos fármacos , Administração Cutânea , Adulto , Animais , Antifúngicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Géis/administração & dosagem , Géis/farmacologia , Humanos , Itraconazol/administração & dosagem , Lipídeos/administração & dosagem , Masculino , Pessoa de Meia-Idade , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Tamanho da Partícula , Ratos , Ratos Wistar , Pele/metabolismo , Absorção Cutânea/efeitos dos fármacos , Propriedades de Superfície
5.
Phys Chem Chem Phys ; 21(23): 12530-12539, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31147666

RESUMO

The emergence of antibiotic-resistance is a major concern to global human health and identification of novel antibiotics is critical to mitigate the threat. Mutacin 1140 (MU1140) is a promising antimicrobial lanthipeptide and is effective against Gram-positive bacteria. Like nisin, MU1140 targets and sequesters lipid II and interferes with its function, which results in the inhibition of bacterial cell wall synthesis, and leads to bacteria cell lysis. MU1140 contains a structurally similar thioether cage for binding the lipid II pyrophosphate as for nisin. In addition to lipid II binding, nisin is known to form membrane pores. Membrane pore formation and membrane disruption is a common mode of action for many antimicrobial peptides, including gallidermin, a lantibiotic peptide with similar structural features as MU1140. However, whether and how MU1140 and its variants can form permeable membrane pores remains to be demonstrated. In this work, we explored the potential mechanisms of membrane pore formation by performing molecular simulations of the MU1140-lipid II complex in the bacterial membrane. Our results suggest that MU1140-lipid II complexes are able to form water permeating membrane pores. We find that a single chain of MU1140 complexed with lipid II in the transmembrane region can permeate water molecules across the membrane via a single-file water transport mechanism. The ordering of the water molecules in the single-file chain region as well as the diffusion behavior is similar to those observed in other biological water channels. Multiple complexes of MU1140-lipid II in the membrane showed enhanced permeability for the water molecules, as well as a noticeable membrane distortion and lipid relocation, suggesting that a higher concentration of MU1140 assembly in the membrane can cause significant disruption of the bacterial membrane. These investigations provide an atomistic level insight into a novel mode of action for MU1140 that can be exploited to develop optimized peptide variants with improved antimicrobial properties.


Assuntos
Bacteriocinas/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Simulação de Dinâmica Molecular , Peptídeos/farmacologia , Bacteriocinas/química , Membrana Celular/efeitos dos fármacos , Bactérias Gram-Positivas/citologia , Lipídeos/química , Lipídeos/farmacologia , Testes de Sensibilidade Microbiana , Peptídeos/química , Água/química
6.
Nanoscale ; 11(26): 12517-12529, 2019 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-31188378

RESUMO

Peptide nucleic acids (PNAs) have gained considerable attention due to their remarkable potential in gene editing and targeting-based strategies. However, cellular delivery of PNAs remains a challenge in developing their broader therapeutic applications. Here, we investigated a novel complex made of lipid bicelles and PNA-based carriers for the efficient delivery of PNAs. For proof of concept, PNAs targeting microRNA (miR) 210 and 155 were tested. Comprehensive evaluation of positive as well as negative charge-containing bicelles with PNA : lipid ratios of 1 : 100, 1 : 1000, and 1 : 2500 was performed. The negatively charged bicelles with a PNA : lipid molar ratio of 1 : 2500 yielded a discoidal shape with a uniform diameter of ∼30 nm and a bilayer thickness of 5 nm, while the positively charged bicellar system contained irregular vesicles after the incorporation of PNA. Small-angle X-ray scattering (SAXS) analysis was performed to provide insight into how the hydrophobic PNAs interact with bicelles. Further, flow cytometry followed by confocal microscopy analyses substantiate the superior transfection efficiency of bicelles containing dye-conjugated antimiR PNAs. Functional analysis also confirmed miR inhibition by PNA oligomers delivered by bicelles. The nanodiscoidal complex opens a new pathway to deliver PNAs, which, on their own, are a great challenge to be endocytosed into cells.


Assuntos
Lipídeos , Ácidos Nucleicos Peptídicos , Transfecção , Células HeLa , Humanos , Lipídeos/química , Lipídeos/farmacologia , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , Espalhamento a Baixo Ângulo , Difração de Raios X
7.
PLoS One ; 14(5): e0215760, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31048878

RESUMO

OBJECTIVES: Both maternal HIV infection and antiretroviral therapy are associated with adverse birth outcomes. The role of antenatal nutrient supplements with regard to adverse birth outcomes in HIV infected women exposed to antiretroviral therapy is not well known. We assessed the association between HIV and birth outcomes and explored whether antenatal lipid-based nutrient supplements (LNS) modulated this association. METHODS: We analysed a nested cohort of pregnant Malawian women who received daily LNS, multiple micronutrients (MMN) or iron and folic acid (IFA). Birth weight, length-for-age z-score (LAZ) and weight-for-age z-score (WAZ) were analysed as continuous outcomes and proportion of stunting and small-for-gestational age (SGA) as dichotomous outcomes. RESULTS: 134 HIV infected (46 LNS, 39 MMN, 49 IFA) and 833 HIV uninfected (271 LNS, 287 MMN, 275 IFA) women were included. Maternal HIV infection was associated with a lower mean birth weight (-129g (-209, -48), P = 0.002); LAZ (-0.34 (-0.54, -0.13), P = 0.002) and WAZ (-0.21 (-0.40, -0.02), P = 0.041) and a higher risk of stunting (RR (95% confidence interval), 1.87 (1.24, 2.83), P = 0.003) and SGA (1.66 (1.21, 2.26), P = 0.001) in the newborn. If the women received LNS, HIV was not associated with LAZ (mean difference (95%); -0.02 (-0.35, 0.31), P = 0.918) or newborn stunting (RR (95% CI), 0.84 (0.34, 2.03), P = 0.691). However HIV tended to be associated with LAZ if the women received MMN (-0.42 (-0.80, -0.03), P = 0.053); and was significantly associated with LAZ if the women received IFA (-0.52 (-0.89, -0.14), P = 0.021) and with newborn stunting if they received MMN (2.40 (1.15, 4.98), P = 0.029) or IFA (2.40 (1.26, 4.59), P = 0.024). CONCLUSIONS: Further research to investigate the impact of LNS on various aspects of foetal growth in HIV infected women is warranted.


Assuntos
Suplementos Nutricionais , Desenvolvimento Fetal/efeitos dos fármacos , Infecções por HIV , Lipídeos/química , Lipídeos/farmacologia , Adulto , Peso Corporal/efeitos dos fármacos , Estudos de Coortes , Feminino , Infecções por HIV/metabolismo , Infecções por HIV/fisiopatologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Gravidez
8.
Int J Food Microbiol ; 301: 9-18, 2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31055161

RESUMO

A large amount of GRAS (generally recognized as safe) salts and concentrations were evaluated in in vitro tests (inhibition of mycelial growth on PDA dishes) against Lasiodiplodia theobromae, the causal agent of citrus Diplodia stem-end rot. Ammonium carbonate (AC, 0.2%), potassium sorbate (PS, 2.0%), potassium carbonate (PC, 0.2%), sodium methylparaben (SMP, 0.1%), sodium ethylparaben (SEP, 0.1%), sodium benzoate (SB, 2.0%), and potassium silicate (PSi, 2.0%) were selected as the most effective. Disease control ability of edible composite coatings formulated with hydroxypropyl methylcellulose (HPMC), beeswax (BW), and these selected antifungal GRAS salts was assessed in in vivo experiments with 'Ortanique' mandarins and 'Barnfield' oranges artificially inoculated with L. theobromae. Coatings containing 2% PS, 0.1% SEP, or 2% SB were the most effective reducing disease severity (up to 50% reduction) and were also applied to non-inoculated and cold-stored 'Barnfield' oranges to determine their effect on postharvest fruit quality. After periods of 21 and 42 d at 5 °C followed by 7 d of shelf life at 20 °C, coatings containing SEP and SB significantly reduced weight loss and did not adversely affect the physicochemical quality attributes (firmness, soluble solid content, titratable acidity, and ethanol and acetaldehyde content) and sensory flavor with respect to uncoated control fruit. Although the internal gas concentration (CO2 level) of coated fruit increased, the coatings did not induce off-flavors.


Assuntos
Ascomicetos/efeitos dos fármacos , Citrus/microbiologia , Microbiologia de Alimentos/métodos , Conservação de Alimentos/métodos , Derivados da Hipromelose/farmacologia , Sais/farmacologia , Antifúngicos/farmacologia , Frutas/microbiologia , Lipídeos/química , Lipídeos/farmacologia
9.
Cells ; 8(5)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091801

RESUMO

Lipid emulsion (LE) therapy has been used to reduce overdose of bupivacaine (BPV)-induced cardiotoxicity. The TWIK-related potassium channel-1 (TREK-1) is inhibited by BPV and activated by polyunsaturated fatty acids, which are the main component in LE. These pharmacological properties inspired us to investigate whether the TREK-1 channel is associated with cell viability of H9c2 cardiomyoblasts affected by BPV and LE. Consistent with previous studies, BPV-induced cell death was reduced by LE treatment. The reduction in the TREK-1 expression level by BPV was alleviated by LE. The BPV cytotoxicity highly decreased in TREK-1 overexpressed cells but was the opposite in TREK-1 knocked-down cells. TREK-1 channel activators and inhibitors increased and decreased cell viability, respectively. BPV-induced depolarization of the plasma and mitochondrial membrane potential and increase in intracellular Ca2+ level were blocked by LE treatment. BPV-induced depolarization of membrane potential was reduced in TREK-1 overexpressed cells, indicating that TREK-1 channels mediate setting the resting membrane potentials as a background K+ channel in H9c2 cells. These results show that TREK-1 activity is involved in the BPV cytotoxicity and the antagonistic effect of LE in H9c2 cells and suggest that TREK-1 could be a target for action of BPV and LE.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Lipídeos/farmacologia , Mioblastos Cardíacos/efeitos dos fármacos , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Animais , Bupivacaína/química , Cardiotoxicidade/tratamento farmacológico , Linhagem Celular , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mioblastos Cardíacos/citologia
10.
Methods Mol Biol ; 1974: 151-159, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31099001

RESUMO

Systemic delivery of RNA interference (RNAi) payloads for manipulation of gene expression in lymphocytes holds a great potential as a novel therapeutic modality for hematological malignancies and autoimmune disorders. However, lymphocytes are among the most difficult cells to transfect with RNAi, as they are resistant to conventional transfection reagents and are dispersed throughout the body, making it a challenge to successfully deliver these payloads via systemic administration route. We have developed a strategy to target lymphocytes and deliver RNAi payloads in a cell-specific manner to induce therapeutic gene silencing. This approach utilizes antibodies that decorate lipid nanoparticle surfaces to home into lymphocyte subsets. This approach opens new avenues for discovery of new drug targets and potentially for therapeutics.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Inativação Gênica , Lipídeos/genética , RNA Interferente Pequeno/genética , Humanos , Lipídeos/química , Lipídeos/farmacologia , Linfócitos/efeitos dos fármacos , Nanopartículas/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia
11.
Methods Mol Biol ; 1974: 303-328, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31099012

RESUMO

The objective of this study is to prepare vaginal suppository containing chemotherapeutic agent and genetic material that can be applied locally for cervical cancer. Cervical cancer is one of the most life-threatening types of cancer among women and is generally resistant to chemotherapy. Paclitaxel has been selected as chemotherapeutic agent, and siRNA that inhibits the Bcl-2 oncogene has been selected as the genetic material for simultaneous vaginal delivery. For this purpose, three different solid lipid nanoparticles (SLNs) were prepared that include Bcl-2 siRNA and paclitaxel and paclitaxel/Bcl-2 siRNA combination separately, and these SLN formulations were dispersed in vaginal suppositories prepared with PEG 6000. First, the physicochemical properties of SLNs, their cytotoxicities on HeLa cell lines, and the transfection ability of siRNA-incorporated SLN on the cells have been examined. Afterward, the release of SLNs from the three different vaginal suppositories prepared has been determined via horizontal diffusion chamber system. The loaded amount to the SLNs and release amount from suppositories of paclitaxel have been determined via HPLC, whereas stability, loading, and release amount of siRNA has been determined via gel retardation system and UV spectrophotometer.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , RNA Interferente Pequeno/genética , Neoplasias do Colo do Útero/terapia , Feminino , Células HeLa , Humanos , Lipídeos/química , Lipídeos/farmacologia , Nanopartículas/uso terapêutico , Paclitaxel/química , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , Supositórios/química , Supositórios/uso terapêutico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
12.
Mar Drugs ; 17(6)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31142027

RESUMO

Recent studies have shown that marine algae represent a great source of natural compounds with several properties. The lipidic extract of the seaweed Chaetomorpha linum (Chlorophyta, Cladophorales), one of the dominant species in the Mar Piccolo of Taranto (Mediterranean, Ionian Sea), revealed an antibacterial activity against Vibrio ordalii and Vibrio vulnificus, common pathogens in aquaculture, suggesting its potential employment to control fish and shellfish diseases due to vibriosis and to reduce the public health hazards related to antibiotic use in aquaculture. This extract showed also an antioxidant activity, corresponding to 170.960 ± 16. mmol Trolox equivalent/g (oxygen radical absorbance capacity assay-ORAC) and to 30.554 ± 2.30 mmol Trolox equivalent/g (Trolox equivalent antioxidant capacity assay-TEAC). The chemical characterization of the extract, performed by 1D and 2D NMR spectroscopy, highlighted the presence of free, saturated (SAFAs), unsaturated (UFAs) and polyunsaturated (PUFAs) fatty acids. The high content of ω-6 and ω-3 PUFAs confirmed also by gas chromatography indicates the potentiality of this algal species in the production of fortified food. The antibacterial activity seems related to the presence of linolenic acid present at high density, while the antioxidant activity could be likely ascribable to molecules such as carotenoids and chlorophylls (characterized also by thin-layer chromatography), known for this property. The presence of polyhydroxybutyrate, a biopolymer with potentiality in the field of biodegradable bioplastics was also detected. The exploitation of C. linum for a future biotechnological application is also encouraged by the results from a first attempt of cultivating this species in an integrated multi-trophic aquaculture (IMTA) system.


Assuntos
Clorófitas/química , Lipídeos/química , Lipídeos/farmacologia , Compostos Fitoquímicos/química , Antibacterianos/química , Antibacterianos/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Bactérias/efeitos dos fármacos , Clorófitas/crescimento & desenvolvimento , Cromatografia em Camada Delgada , Ácidos Graxos/análise , Espectroscopia de Ressonância Magnética , Mar Mediterrâneo , Compostos Fitoquímicos/farmacologia , Pigmentos Biológicos/química
13.
BMC Res Notes ; 12(1): 225, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30987672

RESUMO

OBJECTIVE: Delivery of constructs for silencing or over-expressing genes or their modified versions is a crucial step for studying neuronal cell biology. Therefore, efficient transfection is important for the success of these experimental techniques especially in post-mitotic cells like neurons. In this study, we have assessed the transfection rate, using a previously established protocol, in both primary cortical cultures and neuroblastoma cell lines. Transfection efficiencies in these preparations have not been systematically determined before. RESULTS: Transfection efficiencies obtained herein were (10-12%) for neuroblastoma, (5-12%) for primary astrocytes and (1.3-6%) for primary neurons. We also report on cell-type specific transfection efficiency of neurons and astrocytes within primary cortical cultures when applying cell-type selective transfection protocols. Previous estimations described in primary cortical or hippocampal cultures were either based on general observations or on data derived from unspecified number of biological and/or technical replicates. Also to the best of our knowledge, transfection efficiency of pure primary neuronal cultures or astrocytes cultured in the context of pure or mixed (neurons/astrocytes) population cultures have not been previously determined. The transfection strategy used herein represents a convenient, and a straightforward tool for targeted cell transfection that can be utilized in a variety of in vitro applications.


Assuntos
Astrócitos/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Plasmídeos/metabolismo , Transfecção/métodos , Animais , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Expressão Gênica , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lipídeos/química , Lipídeos/farmacologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Especificidade de Órgãos , Plasmídeos/química , Cultura Primária de Células
14.
Tuberculosis (Edinb) ; 115: 89-95, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30948182

RESUMO

Persisters of Mycobacterium tuberculosis (Mtb) that fail to form colonies on agar media when de-stressed are termed as differentially detectable (DD) persisters. Since in the host, Mtb primarily survives by utilizing lipids, we used a long-term lipid diet model to induce DD persisters of M. tuberculosis. Persisters were induced by replacing the dextrose-containing medium with one containing fatty acids instead of dextrose (FAM). After 2, 4 or 6 weeks, CFU and most probable number assays were performed; the difference between the two gave an estimate of DD persisters. Since rifampicin has been shown to induce formation of DD persisters in vitro, one set of FAM cultures were also given short-term rifampicin stress after 2, 4 or 6 weeks. Fraction of DD persisters increased with time and rifampicin treatment enhanced the effect of fatty acids, at 2 and 4 weeks. At six weeks, even in the absence of rifampicin, ∼95% population were DD persisters. The DD persisters were vulnerable to drugs interfering with bacterial respiration such as thioridazine, bedaquiline and clofazimine. The study indicates potential formation of DD persisters of Mtb in a lipid-rich microenvironment in the host even before antibiotic therapy.


Assuntos
Antituberculosos/farmacologia , Lipídeos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Meios de Cultura , Ácidos Graxos/farmacologia , Testes de Sensibilidade Microbiana , Modelos Biológicos , Fenótipo , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
15.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022840

RESUMO

A library of 197 endophytic fungi and bacteria isolated from the Amazonian palm tree Astrocaryum sciophilum was extracted and screened for antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Four out of five antibacterial ethyl acetate extracts were also cytotoxic for the MRC-5 cells line. Liquid chromatography coupled to tandem mass spectrometry (UPHLC-HRMS/MS) analyses combined with molecular networking data processing were carried out to allow the identification of depsipeptides and cyclopeptides responsible for the cytotoxicity in the dataset. Specific ion clusters from the active Luteibacter sp. extract were also highlighted using an MRSA activity filter. A chemical study of Luteibacter sp. was conducted leading to the structural characterization of eight fatty acid exhibiting antimicrobial activity against MRSA in the tens of µg/mL range.


Assuntos
Antibacterianos/química , Arecaceae/microbiologia , Endófitos/química , Gammaproteobacteria/química , Lipídeos/química , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Humanos , Lipídeos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Infecções Estafilocócicas/tratamento farmacológico , Espectrometria de Massas em Tandem , Árvores/microbiologia
16.
J Sci Food Agric ; 99(11): 4993-4999, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-30977142

RESUMO

BACKGROUND: Furan is a potential carcinogen that can be formed in various heat-treated foods, including milk beverages. Studies on the formation and mitigation of furan in milk beverages are rare. In the present study, the effects of ingredients on furan formation and the reduction of furan by sugar alcohols and antioxidants of bamboo leaves (AOB) were investigated in a milk beverage model system. RESULTS: The results obtained demonstrated that the Maillard reaction is the major pathway for furan formation in a milk beverage model system, and the type of sugar has a great influence on furan formation. High fructose corn syrup (HFCS 55) was more favorable for furan formation than sucrose. Thermal oxidation of ascorbic acid and lipids significantly enhanced furan generation. Xylitol, sorbitol and mannitol inhibited furan formation in model systems by replacing sucrose or HFCS. The maximum inhibition percentage of furan formation was observed when sucrose/HFCS was substituted completely by xylitol and the inhibition rate was 78.28% and 88.64% separately for the sucrose/HFCS-containing system. AOB significantly inhibited furan formation and the inhibition rate reached 32.13% and 28.52% separately for the sucrose/HFCS-containing system. CONCLUSION: The present study demonstrates that the use of sugar alcohols and AOB could be a feasible way of reducing furan formation in thermally processed milk beverages. © 2019 Society of Chemical Industry.


Assuntos
Antioxidantes/farmacologia , Carcinógenos/síntese química , Furanos/síntese química , Folhas de Planta/química , Sasa , Álcoois Açúcares/farmacologia , Animais , Antioxidantes/química , Bebidas/análise , Furanos/antagonistas & inibidores , Temperatura Alta , Humanos , Lipídeos/farmacologia , Reação de Maillard , Leite/química , Álcoois Açúcares/química
17.
Cell Biochem Funct ; 37(3): 161-168, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30907023

RESUMO

Telmisartan, an angiotensin II receptor blocker, has been widely used for hypertension. It has also been reported to improve insulin sensitivity in animal models of obesity and diabetic patients by targeting to the peroxisome proliferator-activated receptor (PPAR)-γ. High glucose/high lipid (HG/HL)-induced apoptosis of pancreatic ß-cells impairs its function of insulin secretion and is generally believed to be the key factor in the development of diabetes. In this study, we investigated whether telmisartan exerted a protective effect against HG/HL-induced apoptosis and insulin secretion in vitro as well as in vivo; 10-µM telmisartan treatment significantly reduced HG (25 mM) or/and HL (0.4 mM palmitic acid) induced-cell apoptosis and greatly improved insulin secretion in INS-1 pancreatic ß-cells, which is consistent in an obesity rat model induced by HG/HL diets. Furthermore, telmisartan treatment markedly reduced the protein level of GRP78, CHOP, and caspase 12, while increasing anti-apoptotic Bcl-2 protein expression. Moreover, telmisartan treatment significantly reduced intracellular ROS levels. Mechanistically, we demonstrated that PPARγ signaling pathway may be involved in the telmisartan protective effects, which were blocked by a PPARγ blocker, GW9662. In conclusion, the protective effect of telmisartan was mediated by an anti-ER stress-induced apoptotic and anti-oxidative pathway. SIGNIFICANCE OF THIS STUDY: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder worldwide pathologically characterized by hyperglycemia and insulin resistance. Long-term high glucose in the blood has been proposed to induce pancreatic ß-cell loss and is generally believed to be the key factor in the development of diabetes. In the present study, we demonstrated that telmisartan, a common drug used for hypertension treatment, has a protective effect against high glucose/high lipid-induced cell apoptosis and greatly improves the insulin secretion function by inhibiting the oxidative stress and ER stress. Furthermore, this protective effect of telmisartan is mediated by the PPAR-γ signal pathway, which may provide a potential strategy against T2DM.


Assuntos
Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Glucose/antagonistas & inibidores , Insulina/metabolismo , Lipídeos/antagonistas & inibidores , Estresse Oxidativo/efeitos dos fármacos , Telmisartan/farmacologia , Anilidas/farmacologia , Anti-Hipertensivos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Glucose/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Lipídeos/farmacologia , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade , Telmisartan/antagonistas & inibidores
18.
J Vis Exp ; (145)2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30907877

RESUMO

Excess dietary salt intake contributes to inflammation and plays a vital role in the development of hypertension. We previously found that antigen-presenting dendritic cells (DCs) can sense elevated extracellular sodium leading to the activation of the NADPH oxidase and formation of isolevuglandin (IsoLG)-protein adducts. These IsoLG-protein adducts react with self-proteins and promote an autoimmune-like state and hypertension. We have developed and optimized state-of-the-art methods to study DC function in hypertension. Here, we provide a detailed protocol for isolation, in vitro treatment with elevated sodium, and adoptive transfer of murine splenic CD11c+ cells into recipient mice to study their role in hypertension.


Assuntos
Transferência Adotiva , Células Apresentadoras de Antígenos/citologia , Células Dendríticas/citologia , Cloreto de Sódio/farmacologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Antígeno CD11c/metabolismo , Células Dendríticas/efeitos dos fármacos , Hipertensão , Lipídeos/farmacologia , Fenômenos Magnéticos , Masculino , Camundongos Endogâmicos C57BL , Baço/citologia , Sístole/efeitos dos fármacos
19.
Mater Sci Eng C Mater Biol Appl ; 99: 762-769, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30889751

RESUMO

SrCO3 is frequently used as Sr2+ source in ceramic cements, but its application as bioactive coating for metallic implants has not been explored yet. Aiming at rapid osteointegration and because of the well-known Sr2+ effects on bone metabolism, researchers have sought to design Sr2+-containing biomaterials. In this context, developing simple techniques to prepare Sr2+-based coatings is a must nowadays. Here, we describe the use of a bioinspired lipid-mediated approach to grow SrCO3 hybrid films on Ti surfaces at room temperature. To obtain these coatings, we applied the Langmuir-Blodgett technique to deposit phospholipid films with high degree of organization on Ti. In this way, we expected that controlled SrCO3 crystal growth could be templated by the array of nucleation points arising from electrostatic interaction between Sr2+ and the phospholipid polar heads. To control surface composition and the amount of Sr2+ released from the coatings, we also promoted CaCO3 co-precipitation in the hybrid films. We characterized the hybrid coatings in terms of morphology, chemical structure, wettability, and ability to release Sr2+ upon immersion in biological medium. In vitro osteoblast culture on mixed SrCO3/CaCO3 films revealed that the osteogenic response depended on surface composition, as indicated by alkaline phosphatase activity overexpression, which is an early indicator of osteoblast differentiation. Results showed that the mixed SrCO3/CaCO3 hybrid film created a synergic environment for osteoblasts, and that proper Sr2+ release associated with a Ca2+-rich environment might have optimized the Sr2+ anabolic effect. In conclusion, we have proposed a bioinspired and versatile technique to grow hybrid films that can control surface composition and Sr2+ release. Our results open an opportunity to explore the use of SrCO3-based coatings for rapid metallic implant osteointegration.


Assuntos
Carbonato de Cálcio/química , Carbonatos/química , Materiais Revestidos Biocompatíveis/farmacologia , Lipídeos/farmacologia , Osteogênese/efeitos dos fármacos , Estrôncio/química , Titânio/farmacologia , Animais , Linhagem Celular , Camundongos , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Difração de Raios X
20.
Cir. plást. ibero-latinoam ; 45(1): 5-10, ene.-mar. 2019. tab
Artigo em Espanhol | IBECS | ID: ibc-182672

RESUMO

Introducción y Objetivo: Dentro de las técnicas quirúrgicas utilizadas para el remodelado corporal están la abdominoplastia y la liposucción. Las variaciones bioquímicas del metabolismo lipídico juegan un rol importante al aplicar estas técnicas quirúrgicas. Los lípidos como componentes insolubles en agua, tienen que ser transportados en el organismo unidos a otras moléculas, las lipoproteínas, tomadas en cuenta en nuestro estudio. Nuestro objetivo general es determinar la variación de fracciones de lípidos en pacientes sometidas a abdominoplastia en el Departamento de Cirugía Plástica y Reconstructiva del Hospital Doctor Salvador Bienvenido Gautier de la República Dominicana entre enero y mayo de 2018. Material y Método: Desarrollamos un estudio descriptivo, prospectivo y de corte transversal, en 15 pacientes de sexo femenino con edades entre los 25-44 años e índice de masa corporal por debajo de 34.9 kg/m2, a las que se les practicó abdominoplastia convencional más liposucción de flancos y se les estudió el perfil lipídico prequirúrgico y a los 30 días de postoperatorio. Resultados: Por debajo de los 35 años de edad, los triglicéridos aumentaron en el postoperatorio y por encima de los 35 disminuyeron. El LDL en pacientes menores de 30 años y mayores de 40 aumentó, y entre 30-40 años disminuyó. Las fracciones lipídicas disminuyeron no significativamente en liposucción de 500 a 1499 ml. y aumentaron en las de 2000 a 2500 ml. a los 30 días de postoperatorio. Los triglicéridos y el colesterol total disminuyeron en pacientes con sobrepeso y obesidad y aumentaron en las de peso normal. El colesterol total fue la fracción lipídica con mayor tasa de disminución a los 30 días de postoperatorio. Conclusiones: La abdominoplastia más liposucción produce una movilización de las fracciones lipídicas, por lo que clínicamente debemos ser moderados en la liposucción e individualizar los casos que superen los 2000 ml. para controlar las fracciones de lípidos y tomar las medidas correctivas necesarias en cada paciente


Background and Objective: Abdominoplasty and liposuction are among the surgical techniques used for body modeling. The biochemical variations of lipid metabolism play an important role when applying these surgical techniques. Lipids as insoluble components in water, have to be transported in the body linked to other molecules called lipopoproteins, which are the fractions taken into account in our study. Our general objective is to determine the variation of lipid fractions in patients undergoing abdominoplasty in Dr. Salvador Bienvenido Gautier Hospital (Dominican Republic) between January- May 2018. Methods: A descriptive, prospective and cross-sectional study was conducted in 15 female patients aged between 25-44 years and a with body mass index below 34.9 kg / m2, who underwent conventional abdominoplasty plus flank liposuction, studying pre-surgical lipid profile the previous day and 30 days post-surgical. Results: In relation to age, triglyceride levels below 35 years showed an increase in post-surgery and above 35 years a decrease was observed. Regarding the level of LDL in patients under 30 years and over 40 an increase was observed and between 30-40 decrease was observed. The lipid fractions decreased but not significantly in the cases of liposuction from 500 to 1499 ml. of aspirated fat, and increased in the cases from 2000 to 2500 ml. 30 days after surgery. Triglycerides and total cholesterol decreased in overweight and obese patients and increased in patients with normal body mass index. Total cholesterol was the lipid fraction with the highest rate of reduction at 30 days post-surgery. Conclusions: Abdominoplasty plus liposuction produces a mobilization of lipid fractions, so clinically we should be moderate in liposuction and individualize cases that exceed 2000 ml. to control the fractions of lipids and take the necessary corrective measures in each patient


Assuntos
Humanos , Feminino , Adulto , Abdominoplastia , Lipoproteínas , Lipídeos/farmacologia , Músculos/química , Metabolismo dos Lipídeos , Estudos Transversais , Índice de Massa Corporal
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