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1.
Front Cell Infect Microbiol ; 10: 573097, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33330123

RESUMO

Histoplasma capsulatum is a thermodimorphic fungus that causes histoplasmosis, a mycosis of global incidence. The disease is prevalent in temperate and tropical regions such as North America, South America, Europe, and Asia. It is known that during infection macrophages restrict Zn availability to H. capsulatum as a microbicidal mechanism. In this way the present work aimed to study the response of H. capsulatum to zinc deprivation. In silico analyses showed that H. capsulatum has eight genes related to zinc homeostasis ranging from transcription factors to CDF and ZIP family transporters. The transcriptional levels of ZAP1, ZRT1, and ZRT2 were induced under zinc-limiting conditions. The decrease in Zn availability increases fungicidal macrophage activity. Proteomics analysis during zinc deprivation at 24 and 48 h showed 265 proteins differentially expressed at 24 h and 68 at 48 h. Proteins related to energy production pathways, oxidative stress, and cell wall remodeling were regulated. The data also suggested that low metal availability increases the chitin and glycan content in fungal cell wall that results in smoother cell surface. Metal restriction also induces oxidative stress triggered, at least in part, by reduction in pyridoxin synthesis.

2.
Anim Biotechnol ; : 1-11, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-33380273

RESUMO

Cells from different origins behave differently regarding the incorporation of exogenous DNA and formation of transgenic cells. Milk production of recombinant antibody may benefit from efficient transfection protocols to produce transgenic animals. In this context, the objective of this study was to verify the transfection potential of bovine mesenchymal stem cells from Wharton's jelly (MSC-WJ) and adipose tissue (MSC-AT), comparing co-transfection protocols with vectors pBC1-anti-CD3 and pEF-NEO-GFP, using transfection reagents Lipofectamine LTX with Plus Reagent or Xfect. Skin fibroblasts (FIB) were used as the control group. Forty-eight hours after transfection, neomycin was added and cells cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than fibroblasts, for both the Xfect (20.057 ± 1.620,7 and 10.601 ± 702,86, respectively, p < 0.05) and LTX (19.590 ± 113,84 and 10.518 ± 442,65, respectively, p < 0.05). These results, associated with evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than other cells, independent of the kit used. Performing PCR on co-transfected cells demonstrated the presence of anti-CD3, making this approach feasible for future experiments.

3.
Cell Tissue Bank ; 2020 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-33165826

RESUMO

To evaluate factors affecting corneal endothelial cell density (ECD) under enucleation and preservation time studies at Eye Bank of the Federal District of Brazil. We conducted a case-control study collecting data from 1128 corneas where death-to-enucleation time and enucleation-to-preservation time were within 24 h. Low cell count were those corneas with an ECD less than 2000 cells/mm2 and high cell count was defined as those with ECD greater than 2000 cells/mm2. We calculated the independent risk factors related to: cause of death, donor age, death-to-enucleation time, enucleation-to-preservation time and primary graft failure. Correlation analysis was used to assess which parameters influence ECD: death-to-enucleation time, enucleation-to-preservation time, average cell area (AVE), coefficient of variation and percentage of hexagonal cells. Of the total number of corneas, 1004 had ECD data and were selected for the study. 87.4% (n = 877) had high cell counts with 2699 ± 412 cells/mm2. The mean donor age was 38.8 ± 16 years. The most common causes of death were external causes (48.6%, n = 488). Longer times from death-to-enucleation, up to 24 h were not associated with a decrease in ECD (OR 0.58; P = 0.44) or risk of graft survival (P = 0.74). Enucleation-to-preservation intervals greater than 12 h showed increased risk of graft survival (P = 0.04). AVE was the main parameter for ECD (R2 = 0.96, P < 0.001). The overall graft survival rate was 98.2% (n = 761). Donors with 40 years of age and above did not present a higher risk of graft survival (P = 0.09). We suggest that the maximum time from death-to-enucleation should be 24 h, assuming the body has been refrigerated after 6 h; and from enucleation-to-preservation time of 12 h, followed by proper processing and cornea morphology examination.

4.
Biopreserv Biobank ; 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33035068

RESUMO

Preserving genetic material in cryogenic conditions presents a viable alternative for the protection of species' gene variability. However, there is an enormous need to establish and test cryopreservation protocols that are suitable for each diverse cell type to guarantee technical success in the long run. Considering this, fibroblasts from jaguar (Panthera onca), oncilla (Leopardus tigrinus), and pampas cat (Leopardus colocolo) were subjected to cell characterization and then cryopreservation in different cryoprotectant solutions (2.5%, 10% dimethyl sulfoxide [DMSO] or CryoSOfree™). Further testing was conducted to determine each solution's performance in preserving cell viability. In culture, a growth curve to assess cellular growth potential showed that exponential proliferation lasts for about the first 50 hours of in vitro culturing, declining in pace afterward. L. colocolo and L. tigrinus presented no difference in cell viability while using 2.5% DMSO protocols. P. onca cells did not present difference on viability for both concentrations of DMSO. Protocols using CryoSOfree resulted in a decreased viability of P. onca fibroblasts. Morphological differences between fibroblasts among the species were noted under bright field microscopy and scanning electron microscopy. L. colocolo and P. onca cells are fusiform, and L. tigrinus are spherical. All cells presented cytoplasmic projections. Transmission electron microscopy revealed vacuoles and secretion granules, indicating intense cell activity after thawing. Differences found in the efficiency of cryopreservation protocols according to the type of cryoprotectant indicate that species react differently to freezing and thawing processes. This research evaluates key aspects of in vitro protocols for cryopreservation of wild animals, which need to be optimized to guarantee successful cell culturing. More suitable protocols lead to increased efficiency in establishing fibroblast cryobanks and also facilitating the use of wild cats' cells in cloning techniques, contributing directly to preserving wild fauna.

5.
Top Companion Anim Med ; 41: 100463, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32823162

RESUMO

Green iguanas are arboreal lizards, common as pet animals and in captivity. Knowledge of neoplasms in iguanas is scarce, and a challenge to their prevention, treatment, and prognosis. A captive green iguana showed a pigmented nasal exophytic neoplasm. Tumor cells were spindle-shaped to epithelioid with a variable amount of dark-brown or black granular melanin within the cytoplasm, and also presented cytoplasmic positivity for Melan-A and S100. Transmission electron microscopy evidenced intracytoplasmic melanosomes and premelanosomes and provided a definitive diagnosis of a nasal melanophoroma. Full characterization of the clinicopathological and ultrastructural features of the melanophoroma may contribute to the limited knowledge concerning cutaneous neoplasms in green iguanas.

6.
J Nanobiotechnology ; 18(1): 43, 2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32164731

RESUMO

BACKGROUND: Metastasis causes the most breast cancer-related deaths in women. Here, we investigated the antitumor effect of solid lipid nanoparticles (SLN-DTX) when used in the treatment of metastatic breast tumors using 4T1-bearing BALB/c mice. RESULTS: Solid lipid nanoparticles (SLNs) were produced using the high-energy method. Compritol 888 ATO was selected as the lipid matrix, and Pluronic F127 and Span 80 as the surfactants to stabilize nanoparticle dispersion. The particles had high stability for at least 120 days. The SLNs' dispersion size was 128 nm, their polydispersity index (PDI) was 0.2, and they showed a negative zeta potential. SLNs had high docetaxel (DTX) entrapment efficiency (86%), 2% of drug loading and showed a controlled drug-release profile. The half-maximal inhibitory concentration (IC50) of SLN-DTX against 4T1 cells was more than 100 times lower than that of free DTX after 24 h treatment. In the cellular uptake test, SLN-DTX was taken into the cells significantly more than free DTX. The accumulation in the G2-M phase was significantly higher in cells treated with SLN-DTX (73.7%) than in cells treated with free DTX (23.0%), which induced subsequent apoptosis. TEM analysis revealed that SLN-DTX internalization is mediated by endocytosis, and fluorescence microscopy showed DTX induced microtubule damage. In vivo studies showed that SLN-DTX compared to free docetaxel exhibited higher antitumor efficacy by reducing tumor volume (p < 0.0001) and also prevented spontaneous lung metastasis in 4T1 tumor-bearing mice. Histological studies of lungs confirmed that treatment with SLN-DTX was able to prevent tumor. IL-6 serum levels, ki-67 and BCL-2 expression were analyzed and showed a remarkably strong reduction when used in a combined treatment. CONCLUSIONS: These results indicate that DTX-loaded SLNs may be a promising carrier to treat breast cancer and in metastasis prevention.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Docetaxel/farmacologia , Lipídeos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/química , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Portadores de Fármacos/farmacologia , Ácidos Graxos/farmacologia , Feminino , Hexoses/farmacologia , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Tamanho da Partícula , Poloxâmero/farmacologia
7.
Carbohydr Polym ; 227: 115351, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31590861

RESUMO

Surfactants have been used as a tool to improve the properties of polymeric nanoparticles (NPs) and to increase the rate of hydrophobic drug release by means of these nanoparticles. In this context, this study evaluated the effect of lecithin on the characteristics of chitosan (CHI) and chondroitin sulfate (CS) nanoparticles, when applied in curcumin (Curc) release. CHI/CS NPs and CHI/CS/Lecithin NPs were prepared by the ionic gelation method, both as standards and containing curcumin. Simultaneous conductimetric and potentiometric titrations were employed to optimize the interaction between the polymers. NPs with hydrodynamic diameter of ∼130 nm and zeta potential of +60 mV were obtained and characterized by HRTEM; their pore size and surface area were also analyzed by BET method, DLS, FTIR, XPS, and fluorescence spectroscopy techniques to assess morphological and surface properties, stability and interaction between polymers and to quantify the loading of drugs. The final characteristics of NPs were directly influenced by lecithin addition, exhibiting enhanced encapsulation efficiency of curcumin (131.8 µg curcumin per mg CHI/CS/Lecithin/Curc NPs). The release of curcumin occurred gradually through a two-stage process: diffusion-controlled dissolution and release of curcumin controlled by dissolution of the polymer. However, the release of curcumin in buffer solution at pH 7.4 was achieved faster in CHI/CS/Lecithin/Curc NPs than in CHI/CS/Curc NPs. in vitro cytotoxic activity evaluation of the curcumin was determined by the MTT assay, observing that free curcumin and curcumin nanoencapsulated in CHI/CS/Curc and CHI/CS/Lecithin/Curc NPs reduced the viability of MCF-7 cells in the 72 h period (by 28.4, 36.0 and 30.7%, P < 0.0001, respectively). These results indicate that CHI/CS/Lecithin NPs have more appropriate characteristics for encapsulation of curcumin.


Assuntos
Quitosana/química , Sulfatos de Condroitina/química , Curcumina/química , Lecitinas/química , Nanopartículas/química , Sobrevivência Celular/efeitos dos fármacos , Quitosana/administração & dosagem , Sulfatos de Condroitina/administração & dosagem , Curcumina/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Liberação Controlada de Fármacos , Humanos , Lecitinas/administração & dosagem , Células MCF-7 , Nanopartículas/administração & dosagem
8.
PLoS One ; 14(12): e0226558, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31835274

RESUMO

Diphylla ecaudata is a hematophagous bat endemic of South America, with food preference for bird blood. Given the lack of information about the reproductive activity of this species, this study aimed to describe the testicular morphology and histomorphometry of D. ecaudata in order to understand its reproductive biology, specially spermatogenesis. The animals were collected in Lajes city, Rio Grande do Norte, Brazil. Following euthanasia, the testes were histologically processed for morphological, morphometric, ultrastructural and immunohistochemical analyses. Their average body weight was 24.64g, with a gonadosomatic index of 0.49%, tubulesomatic index of 0.47%, and a total of 32.20m of seminiferous tubules per gram of testis. The pre-meiotic, meiotic, and post-meiotic phases accounted for 56.20%, 9.30%, and 34.50% of the seminiferous epithelium cycle, respectively. The ultrastructure of spermiogenesis was similar to that described in other mammals and the perforatorium was not observed in the sperm. Androgen receptors were detected in Sertoli cell nuclei and Leydig cell cytoplasm, while aromatase enzyme was detected only in Sertoli cell nuclei. FGF2 and BCL-2 activities were detected in the cytoplasm of zygotene and pachytene primary spermatocytes, as well as round and elongated spermatids. D. ecaudata showed testicular pattern similar to other mammals and characteristics common to other bat species. This species stood out for its high efficiency of Sertoli cells, which presented high capacity to support germ cells, besides the highest sperm production rates among those already recorded. This study is the first step towards the knowledge of D. ecaudata reproduction and the first description of its spermatogenesis.


Assuntos
Biomarcadores/metabolismo , Quirópteros/fisiologia , Espermatogênese , Testículo/fisiologia , Animais , Núcleo Celular/metabolismo , Células Intersticiais do Testículo/citologia , Células Intersticiais do Testículo/metabolismo , Masculino , Túbulos Seminíferos/citologia , Túbulos Seminíferos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Testículo/anatomia & histologia
9.
Rev. bras. oftalmol ; 78(4): 227-232, July-Aug. 2019. tab, graf
Artigo em Inglês | LILACS-Express | LILACS | ID: biblio-1013679

RESUMO

Abstract Objective: The aim of this study is to identify the causes for discarding corneas at the Eye Bank of the Federal District in Brasilia, Brazil, and describe the social and demographic variables and Causa Mortis of cornea donors from 2014 to 2017. Methods: We conducted an exploratory and social-epidemiologic descriptive study regarding cornea donation. The data base information was obtained from the corneal donor's medical records analysis. All of the potential donors' records (cause of death, cause of cornea discard, month of donation, age, gender, and time of death, corneal enucleation and preservation), from 2014 to 2017 were included in the study. Results: We looked at 1,574 corneal donor notifications. Demographic characteristics displayed significant differences in gender distribution (male, 74.8% and female, 25.2%), and the average donor age was 40 ± 15.9 years. 25% of the causes of death were from cardiovascular disease followed by 19.6% from sharp or blunt instrument injury, 14.2% resulted from multiple traumas. We described 3,074 donated corneas from the DF Eye Bank, where 2.6% has not been uptaken. Of those 3,074 corneal tissues, nearly 60% (n=1,836) have been transplanted and 40% (n=1,238) were discarded. Regarding the causes of discard, 68% (n=841) were due to positive or indeterminate serological blood tests and 39% (n=486) because of matureness (expired medium guaranteed period of corneal preservation). Conclusions: Specific issues such as violent causes of death, gender disproportion and total time of corneal processing can be better managed to reduce procurement times, and availability, of corneal tissue for transplantation.


Resumo Objetivo: Identificar as causas do descarte de córneas no Banco de Olhos do Distrito Federal, em Brasília, Brasil, descrever as variáveis sociodemográficas e causa de morte dos doadores de córnea de 2014 a 2017. Métodos: Foi realizado um estudo descritivo exploratório e socioepidemiológico sobre as doações de córnea. As informações da base de dados foram obtidas a partir da análise dos prontuários dos doadores. Todos os registros dos potenciais doadores (causa da morte, causa do descarte, mês de doação, idade, sexo e tempo de morte, enucleação e preservação da córnea), de 2014 a 2017, foram incluídos no estudo. Resultados: Analisamos 1.574 notificações de doadores. Características demográficas apresentaram diferenças significativas na distribuição por sexo (masculino, 74,8% e feminino, 25,2%). A idade média dos doadores foi de 40 ± 15,9 anos. 25% das causas de morte foram de doenças cardiovasculares, seguidas de 19,6% de perfurações por arma de fogo e 14,2% de múltiplos traumas. Descrevemos as 3.074 córneas doadas ao Banco de Olhos do DF e onde apenas 2,6% não foram captadas. Dos 3.074 tecidos da córnea, quase 60% (n = 1.836) foram transplantados e 40% (n = 1.238) foram descartados. Quanto às causas de descarte, 68% (n = 841) foram devidas a exames sorológicos positivos ou indeterminados e 39% (n = 486) por tempo de vencimento (período máximo de preservação da córnea). Conclusões: Questões específicas como causas violentas de morte, desproporção de gênero e tempo total de processamento da córnea podem ser melhor gerenciadas para reduzir o tempo de captação e a disponibilidade de tecido para transplante.

10.
Reprod Domest Anim ; 54(2): 289-299, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30317681

RESUMO

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC-AF), adipose tissue (MSC-AT) and fibroblasts (FIB-control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT-PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC-AF were less hydroxymethylated for both animals. MSC-AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC-AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC-AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.


Assuntos
Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Células-Tronco Mesenquimais/citologia , Acetilação , Animais , Bovinos , Clonagem de Organismos/métodos , Clonagem de Organismos/veterinária , Metilação de DNA , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Histona Acetiltransferases/genética , Histona Desacetilases/genética , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Técnicas de Transferência Nuclear/veterinária
11.
Mater Sci Eng C Mater Biol Appl ; 92: 184-195, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30184741

RESUMO

Remotely assisted drug delivery by means of magnetic biopolymeric nanoplatforms has been utilized as an important tool to improve the delivery/release of hydrophobic drugs and to address their low cargo capacity. In this work, MnFe2O4 magnetic nanoparticles (MNPs) were synthesized by thermal decomposition, coated with citrate and then functionalized with the layer-by-layer (LbL) assembly of polyelectrolyte multilayers, with chitosan as polycation and sodium alginate as polyanion. Simultaneous conductimetric and potentiometric titrations were employed to optimize the LbL deposition and to enhance the loading capacity of nanoplatforms for curcumin, a hydrophobic drug used in cancer treatment. ~200 nm sized biopolymer platforms with ~12 nm homogeneously embedded MNPs were obtained and characterized by means of XRD, HRTEM, DLS, TGA, FTIR, XPS and fluorescence spectroscopy techniques to access structural, morphological and surface properties, to probe biopolymer functionalization and to quantify drug-loading. Charge reversals (±30 mV) after each deposition confirmed polyelectrolyte adsorption and a stable LbL assembly. Magnetic interparticle interaction was reduced in the biopolymeric structure, hinting at an optimized performance in magnetic hyperthermia for magneto-assisted drug release applications. Curcumin was encapsulated, resulting in an enhanced payload (~100 µg/mg). Nanocytotoxicity assays showed that the biopolymer capping enhanced the biocompatibility of nanoplatforms, maintaining entrapped curcumin. Our results indicate the potential of synthesized nanoplatforms as an alternative way of remotely delivering/releasing curcumin for medical purposes, upon application of an alternating magnetic field, demonstrating improved efficiency and reduced toxicity.


Assuntos
Alginatos/química , Quitosana/química , Curcumina/química , Compostos Férricos/química , Nanopartículas de Magnetita/química , Compostos de Manganês/química , Materiais Biocompatíveis/química , Sobrevivência Celular/efeitos dos fármacos , Curcumina/metabolismo , Curcumina/farmacologia , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Células MCF-7 , Tamanho da Partícula
12.
J Nanosci Nanotechnol ; 18(1): 522-528, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29768877

RESUMO

Due to the low therapeutic index of different chemotherapeutic drugs used for cancer treatment, the development of new anticancer drugs remains an intense field of research. A recently developed mixture of selenitetriacylglycerides, selol, was shown to be active against different cancer cells in vitro. As this compound is highly hydrophobic, it was encapsulated, in a previous study, into poly(methyl vinyl ether-co-maleic anhydride)-shelled nanocapsules in order to improve its dispersibility in aqueous media. Following this line of research, the present report aimed at enhancing the In Vitro activity of the selol nanocapsules against cancerous cells by decorating their surface with folic acid. It is known that several cancer cells overexpress folate receptors. Stable folic acid-decorated selol nanocapsules (SNP-FA) were obtained, which showed to be spherical, with a hydro-dynamic diameter of 364 nm, and zeta potential of -24 mV. In comparison to non-decorated selol nanocapsules, SNP-FA presented higher activity against 4T1, MCF-7 and HeLa cells. Moreover, the decoration of the nanocapsules did not alter their toxicity towards fibroblasts, NIH-3T3 cells. These results show that the decoration with folic acid increased the toxicity of selol nanocapsules to cancer cells. These nanocapsules, besides enabling to disperse selol in an aqueous medium, increased the toxicity of this drug In Vitro, and may be useful to treat cancer in vivo, potentially increasing the specificity of selol towards cancer cells.

13.
J Nanobiotechnology ; 16(1): 9, 2018 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-29382332

RESUMO

BACKGROUND: In the photodynamic therapy (PDT), the photosensitizer absorbs light and transfers the energy of the excited state to the oxygen in the cell environment producing reactive oxygen species (ROS), that in its turn, may cause cell damage. In the photothermal therapy (PTT), light also is responsible for activating the photothermal agent, which converts the absorbed energy in heat. Graphene oxide is a carbon-based material that presents photothermal activity. Its physical properties allow the association with the photosensitizer methylene blue and consequently the production of ROS when submitted to light irradiation. Therefore, the association between nanographene oxide and methylene blue could represent a strategy to enhance therapeutic actions. In this work, we report the nanographene oxide-methylene blue platform (NanoGO-MB) used to promote tumor ablation in combination with photodynamic and photothermal therapies against a syngeneic orthotopic murine breast cancer model. RESULTS: In vitro, NanoGO-MB presented 50% of the reactive oxygen species production compared to the free MB after LED light irradiation, and a temperature increase of ~ 40 °C followed by laser irradiation. On cells, the ROS production by the nanoplatform displayed higher values in tumor than normal cells. In vivo assays demonstrated a synergistic effect obtained by the combined PDT/PTT therapies using NanoGO-MB, which promoted complete tumor ablation in 5/5 animals. Up to 30 days after the last treatment, there was no tumor regrowth compared with only PDT or PTT groups, which displayed tumoral bioluminescence 63-fold higher than the combined treatment group. Histological studies confirmed that the combined therapies were able to prevent tumor regrowth and liver, lung and spleen metastasis. In addition, low systemic toxicity was observed in pathologic examinations of liver, spleen, lungs, and kidneys. CONCLUSIONS: The treatment with combined PDT/PTT therapies using NanoGO-MB induced more toxicity on breast carcinoma cells than on normal cells. In vivo, the combined therapies promoted complete tumor ablation and metastasis prevention while only PDT or PTT were unable to stop tumor development. The results show the potential of NanoGO-MB in combination with the phototherapies in the treatment of the breast cancer and metastasis prevention.


Assuntos
Técnicas de Ablação , Grafite/química , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/patologia , Azul de Metileno/química , Nanopartículas/química , Fototerapia , Animais , Apoptose , Peso Corporal , Linhagem Celular Tumoral , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Luminescência , Neoplasias Mamárias Animais/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Nanopartículas/ultraestrutura , Metástase Neoplásica , Fotoquimioterapia , Espécies Reativas de Oxigênio , Carga Tumoral
14.
J Nanosci Nanotechnol ; 18(6): 3832-3843, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29442716

RESUMO

In this study, we report the synthesis and characterization of a new rhodium(II) succinate complex (Rh2(suc)4) and its immobilization on lauric acid bilayer-coated maghemite nanoparticles (MGH-2L/Rh2(suc)4) and subsequent adsorption with bovine serum albumin (MGH-2L/Rh2(suc)4/BSA). Rh2(suc)4 has been characterized by elemental analysis, potentiometric titration, TGA, MS, FTIR and UV-Vis analysis. The maghemite phase was confirmed by XRD, and a diameter of 10 nm was obtained by Sherrer equation. The VSM experiment showed superparamagnetic properties. TEM showed nanoparticles with a spherical shape and a mean diameter of 8.5±0.4 and 9.1 ± 0.4 nm for MGH-2L/Rh2(suc)4 and MGH-2L/Rh2(suc)4/BSA, respectively. FTIR and TGA confirmed the immobilization of Rh2(suc)4 and bovine serum albumin adsorption on superparamagnetic iron oxide. Hydrodynamic size (DH) and zeta potential (ζ) measurements were made in aqueous, NaCl and DMEM media. DH for dispersions was lower in aqueous medium, but increased in saline and DMEM media. In aqueous and saline media, ζ was not altered for MGH-2L and MGH-2L/Rh2(suc)4, but was significantly lower for MGH-2L/Rh2(suc)4/BSA. Therefore, MGH-2L/Rh2(suc)4/BSA was the most stable dispersion, meaning that BSA coating prevents aggregation more than lauric acid bilayer coating. MGH-2L/Rh2(suc)4 and MGH-2L/Rh2(suc)4/BSA dispersions induced cytotoxicity in breast carcinoma (MCF-7) and fibroblast cells in culture, and this effect was higher than that exerted by free Rh2(suc)4 and more specific to breast carcinoma cells than to fibroblasts. Therefore, we suggest that these dispersions have an important potential for future clinical applications and, thus, they should be considered a platform to enhance Rh2(suc)4 cytotoxicity, specifically in breast carcinoma.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Ácidos Láuricos , Nanopartículas Metálicas , Ródio , Ácido Succínico , Compostos Férricos , Humanos , Nanopartículas , Soroalbumina Bovina , Succinatos , Células Tumorais Cultivadas
15.
Future Sci OA ; 3(4): FSO232, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29134119

RESUMO

Aim: During infection development in the host, Paracoccidioides spp. faces the deprivation of micronutrients, a mechanism called nutritional immunity. This condition induces the remodeling of proteins present in different metabolic pathways. Therefore, we attempted to identify membrane proteins and their regulation by zinc in Paracoccidioides lutzii. Materials & methods: Membranes enriched fraction of yeast cells of P. lutzii were isolated, purified and identified by 2D LC-MS/MS detection and database search. Results & conclusion: Zinc deprivation suppressed the expression of membrane proteins such as glycoproteins, those involved in cell wall synthesis and those related to oxidative phosphorylation. This is the first study describing membrane proteins and the effect of zinc deficiency in their regulation in one member of the genus Paracoccidioides.

16.
Oncol Lett ; 12(4): 2581-2589, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27698831

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive type of human primary brain tumor. The standard treatment protocol includes radiotherapy in combination with temozolomide (TMZ). Despite advances in GBM treatment, the survival time of patients diagnosed with glioma is 14.5 months. Regarding tumor biology, various types of cancer cell overexpress the ether à go-go 1 (Eag1) potassium channel. Therefore, the present study examined the role of Eag1 in the cell damage caused by TMZ on the U87MG glioblastoma cell line. Eag1 was inhibited using a channel blocker (astemizole) or silenced by a short-hairpin RNA expression vector (pKv10.1-3). pKv10.1-3 (0.2 µg) improved the Eag1 silencing caused by 250 µM TMZ, as determined by reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. Additionally, inhibiting Eag1 with the vector or astemizole (5 µM) reduced glioblastoma cell viability and sensitized cells to TMZ. Cell viability decreased by 63% for pKv10.1-3 + TMZ compared with 34% for TMZ alone, and by 77% for astemizole + TMZ compared with 46% for TMZ alone, as determined by MTT assay. In addition, both the vector and astemizole increased the apoptosis rate of glioblastoma cells triggered by TMZ, as determined by an Annexin V apoptosis assay. Collectively, the current data reveal that Eag1 has a role in the damage caused to glioblastoma by TMZ. Furthermore, suppression of this channel can improve the action of TMZ on U87MG glioblastoma cells. Thus, silencing Eag1 is a promising strategy to improve GBM treatment and merits additional studies in animal models of glioma.

17.
Ciênc. rural ; 46(10): 1830-1837, Oct. 2016. tab, graf
Artigo em Inglês | LILACS-Express | LILACS | ID: lil-792538

RESUMO

ABSTRACT: Wharton's jelly is a source of mesenchymal stem cells (MSCs) that had not yet been tested for bovine embryo production by nuclear transfer (NT). Thus, the objective of this study was to isolate, characterize and test MSCs derived from Wharton's jelly for embryo and pregnancy production by NT in cattle. The umbilical cord was collected during calving and cells derived from Wharton's jelly (WJCs) were isolated by explant and cultured in Dulbecco's Modified Eagle Medium. Skin Fibroblasts (FB) were isolated after 6 months of life. Morphological analysis was performed by bright field and scanning electron microscopy (SEM) during cell culture. Phenotypic and genotypic characterization by flow cytometry, immunocytochemistry, RT-PCR and differentiation induction in cell lineages were performed for WJC. In the NT procedure, oocytes at the arrested metaphase II stage were enucleated using micromanipulators, fused with WJCs or FB and later activated artificially. SEM micrographs revealed that WJCs have variable shape under culture. Mesenchymal markers of MSCs (CD29+, CD73+, CD90+ and CD105+) were expressed in bovine-derived WJC cultures, as evidenced by flow cytometry, immunocytochemistry and RT-PCR. When induced, these cells differentiated into osteocytes, chondrocytes and adipocytes. After classification, the WJCs were used in NT. Blastocyst formation rate by NT with WJCs at day 7 was 25.80±0.03%, similar to blatocyst rate with NT using skin fibroblasts (19.00±0.07%). Pregnancies were obtained and showed that WJCs constitute a new cell type for use in animal cloning.


RESUMO: A geleia de Wharton é uma fonte de células tronco mesenquimais (CTMs) que ainda não havia sido testada para a produção de embriões bovinos por transferência nuclear (TN). O objetivo deste estudo foi isolar, caracterizar e testar as CTMs derivadas da geleia de Wharton para produção de embriões e gestações por transferência nuclear em bovinos. O cordão umbilical foi coletado durante o nascimento e as células derivadas da geleia de Wharton (CGWs) foram isoladas por explante e cultivadas em Dulbecco's Modified Eagle Medium. Fibroblastos (FB) da pele foram isolados após 6 meses de vida. As análises morfológicas foram realizadas pelas microscopias de campo claro e eletrônica de varredura durante o cultivo celular. Caracterização fenotípica e genotípica por citometria de fluxo, imunocitoquímica, RT-PCR e indução da diferenciação em linhagens celulares foi realizada com as CGWs. No procedimento de TN, ovócitos no estágio de metáfase II foram enucleados usando micromanipuladores, fusionados com CGWs ou FB e então ativados artificialmente. Micrografias de microscopia de varredura revelaram que CGWs tiveram forma variada sob cultivo. Os marcadores mesenquimais de CTMs (CD29+, CD73+, CD90+ and CD105+) foram expressos em cultura de CGWs bovina, como evidenciado por citometria de fluxo, imunocitoquímica e RT-PCR. Quando induzidas, estas células diferenciaram-se em osteócitos, condrócitos e adipócitos. Após classificação, as CGWs foram utilizadas na TN. A taxa de formação de blastocistos por TN com CGWs no sétimo dia de cultivo foi de 25,80±0,03%, similar a produção de blastócitos por TN com fibroblastos de pele (19,00±0,07). Gestações foram obtidas e mostraram que CGWs constituem um novo tipo celular para ser usado na clonagem animal.

18.
Fungal Biol ; 120(10): 1209-24, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27647238

RESUMO

Paracoccidioidomycosis is an endemic disease in Latin America, caused by thermo dimorphic fungi of the genus Paracoccidioides. Although previous proteome analyses of Paracoccidioides spp. have been carried out, the nuclear subproteome of this pathogen has not been described. In this way, we aimed to characterize the nuclear proteome of Paracoccidioides species, in the yeast form. For that, yeast cells were disrupted and submitted to cell fractionation. The purity of the nuclear fraction was confirmed by fluorescence and electron microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 867 proteins. In order to support our enrichment method for nuclear proteins, bioinformatics analysis were applied that allowed the identification of 281 proteins with nuclear localization. The analysis revealed proteins related to DNA maintenance, gene expression, synthesis and processing of messenger and ribosomal RNAs, likewise proteins of nuclear-cytoplasmic traffic. It was also possible to detect some proteins that are poorly expressed, like transcription factors involved in important roles such as resistance to abiotic stress, sporulation, cellular growth and DNA and chromatin maintenance. This is the first descriptive nuclear proteome of Paracoccidioides spp. that can be useful as an important platform base for fungi-specific nuclear processes.


Assuntos
Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Paracoccidioides/metabolismo , Proteoma/genética , Núcleo Celular/química , Núcleo Celular/genética , Biologia Computacional , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Paracoccidioides/química , Paracoccidioides/genética , Transporte Proteico , Proteoma/química , Proteoma/metabolismo , Espectrometria de Massas em Tandem
19.
Cell Tissue Bank ; 17(4): 543-553, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27329292

RESUMO

Vital to patient safety is the accurate assessment and minimization of risk for human immunodeficiency virus (HIV), Hepatitis C (HCV), and Hepatitis B (HBV) virus transmission by deceased donor organ and tissue transplantation. The pathogens are tested by serological kits based on enzyme-linked immunosorbent assay (ELISA), chemiluminescence (CLIA) and eletrochemiluminescence (ECLIA) immunoassays. Organ transplantation is a highly successful life-saving treatment in Brazil, but the Brazilian Health Surveillance Agency currently mandates that all deceased organ donors are screened for HIV, HCV and HBV following living donor policies. In this review, six ELISA (Wama®, Bio-Rad®, Biomerieux®, DiaSorin®, Acon Biotech® and Biokit®), three CLIA (Abbott®, Siemens®, Diasorin®) and one ECLIA (Roche®) were utilized for evaluating the effectiveness of those serological tests for deceased donors in Brazil according to manufacturer's guidelines. NAT for HIV, HCV and HBV can assist with detection of pre-seroconversion for those infections, and only Cobas® TaqScreen MPX® test, the Tigris System® Procleix Ultrio Assay® and the Bio-Manguinhos® HIV/HCV/HBV NAT are commercially available. Between all the tests, only the manufacturer Abbott® and Cobas® TaqScreen MPX® test are currently validated for cadaver samples.


Assuntos
Infecções por HIV/diagnóstico , HIV/isolamento & purificação , Hepacivirus/isolamento & purificação , Vírus da Hepatite B/isolamento & purificação , Hepatite B/diagnóstico , Hepatite C/diagnóstico , Autopsia , Brasil , Cadáver , Infecções por HIV/sangue , Hepatite B/sangue , Hepatite C/sangue , Humanos , Testes Sorológicos/métodos , Doadores de Tecidos , Obtenção de Tecidos e Órgãos
20.
Sci Rep ; 6: 24612, 2016 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-27273152

RESUMO

The genome of a novel group II alphabaculovirus, Perigonia lusca single nucleopolyhedrovirus (PeluSNPV), was sequenced and shown to contain 132,831 bp with 145 putative ORFs (open reading frames) of at least 50 amino acids. An interesting feature of this novel genome was the presence of a putative nucleotide metabolism enzyme-encoding gene (pelu112). The pelu112 gene was predicted to encode a fusion of thymidylate kinase (tmk) and dUTP diphosphatase (dut). Phylogenetic analysis indicated that baculoviruses have independently acquired tmk and dut several times during their evolution. Two homologs of the tmk-dut fusion gene were separately introduced into the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, which lacks tmk and dut. The recombinant baculoviruses produced viral DNA, virus progeny, and some viral proteins earlier during in vitro infection and the yields of viral occlusion bodies were increased 2.5-fold when compared to the parental virus. Interestingly, both enzymes appear to retain their active sites, based on separate modeling using previously solved crystal structures. We suggest that the retention of these tmk-dut fusion genes by certain baculoviruses could be related to accelerating virus replication and to protecting the virus genome from deleterious mutation.


Assuntos
Genoma Viral , Nucleopoliedrovírus/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Pirofosfatases/metabolismo , Proteínas Virais/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , DNA Viral/química , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Nucleopoliedrovírus/classificação , Nucleopoliedrovírus/fisiologia , Núcleosídeo-Fosfato Quinase/química , Núcleosídeo-Fosfato Quinase/genética , Nucleotídeos/biossíntese , Nucleotídeos/química , Fases de Leitura Aberta/genética , Filogenia , Estrutura Terciária de Proteína , Pirofosfatases/química , Pirofosfatases/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Células Sf9 , Spodoptera , Sequências de Repetição em Tandem/genética , Proteínas Virais/genética , Replicação Viral
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