Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.507
Filtrar
1.
PLoS Genet ; 16(4): e1008555, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32271760

RESUMO

Loss of the XPF-ERCC1 endonuclease causes a dramatic phenotype that results in progeroid features associated with liver, kidney and bone marrow dysfunction. As this nuclease is involved in multiple DNA repair transactions, it is plausible that this severe phenotype results from the simultaneous inactivation of both branches of nucleotide excision repair (GG- and TC-NER) and Fanconi anaemia (FA) inter-strand crosslink (ICL) repair. Here we use genetics in human cells and mice to investigate the interaction between the canonical NER and ICL repair pathways and, subsequently, how their joint inactivation phenotypically overlaps with XPF-ERCC1 deficiency. We find that cells lacking TC-NER are sensitive to crosslinking agents and that there is a genetic interaction between NER and FA in the repair of certain endogenous crosslinking agents. However, joint inactivation of GG-NER, TC-NER and FA crosslink repair cannot account for the hypersensitivity of XPF-deficient cells to classical crosslinking agents nor is it sufficient to explain the extreme phenotype of Ercc1-/- mice. These analyses indicate that XPF-ERCC1 has important functions outside of its central role in NER and FA crosslink repair which are required to prevent endogenous DNA damage. Failure to resolve such damage leads to loss of tissue homeostasis in mice and humans.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Homeostase , Animais , Sangue , Reagentes para Ligações Cruzadas/farmacologia , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endonucleases/deficiência , Endonucleases/genética , Feminino , Formaldeído/farmacologia , Humanos , Rim/enzimologia , Fígado/enzimologia , Masculino , Camundongos
2.
J Med Chem ; 63(7): 3756-3762, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32109059

RESUMO

Deubiquitinating enzymes (DUBs) are a growing target class across multiple disease states, with several inhibitors now reported. b-AP15 and VLX1570 are two structurally related USP14/UCH-37 inhibitors. Through a proteomic approach, we demonstrate that these compounds target a diverse range of proteins, resulting in the formation of higher molecular weight (MW) complexes. Activity-based proteome profiling identified CIAPIN1 as a submicromolar covalent target of VLX1570, and further analysis demonstrated that high MW complex formation leads to aggregation of CIAPIN1 in intact cells. Our results suggest that in addition to DUB inhibition, these compounds induce nonspecific protein aggregation, providing molecular explanation for general cellular toxicity.


Assuntos
Azepinas/farmacologia , Compostos de Benzilideno/farmacologia , Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Piperidonas/farmacologia , Multimerização Proteica/efeitos dos fármacos , Azepinas/química , Compostos de Benzilideno/química , Linhagem Celular Tumoral , Reagentes para Ligações Cruzadas/química , Reagentes para Ligações Cruzadas/farmacologia , Enzimas Desubiquitinantes/química , Inibidores Enzimáticos/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Piperidonas/química , Proteoma/química , Proteoma/metabolismo , Proteômica
3.
Cell Rep ; 30(4): 1235-1245.e4, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995761

RESUMO

DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Camptotecina/farmacologia , Reparo do DNA/genética , Etoposídeo/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Técnicas Genéticas , Células HeLa , Humanos , Inibidores da Topoisomerase I/farmacologia
4.
Biochemistry ; 59(7): 892-900, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-31977191

RESUMO

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , DNA/metabolismo , Peptídeos/farmacologia , Policetídeos/farmacologia , Reagentes para Ligações Cruzadas/química , DNA/química , DNA/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Reparo do DNA , Desoxirribonuclease IV (Fago T4-Induzido)/química , Escherichia coli/química , Peptídeos/química , Plasmídeos/química , Policetídeos/química
5.
Nucleic Acids Res ; 48(4): 1969-1984, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31863585

RESUMO

One of the key roles of the 12-subunit eukaryotic translation initiation factor 3 (eIF3) is to promote the formation of the 43S and 48S pre-initiation complexes (PICs). However, particular contributions of its individual subunits to these two critical initiation reactions remained obscure. Here, we adapted formaldehyde gradient cross-linking protocol to translation studies and investigated the efficiency of the 43S and 48S PIC assembly in knockdowns of individual subunits of human eIF3 known to produce various partial subcomplexes. We revealed that eIF3d constitutes an important intermolecular bridge between eIF3 and the 40S subunit as its elimination from the eIF3 holocomplex severely compromised the 43S PIC assembly. Similarly, subunits eIF3a, c and e were found to represent an important binding force driving eIF3 binding to the 40S subunit. In addition, we demonstrated that eIF3c, and eIF3k and l subunits alter the efficiency of mRNA recruitment to 43S PICs in an opposite manner. Whereas the eIF3c knockdown reduces it, downregulation of eIF3k or eIF3l increases mRNA recruitment, suggesting that the latter subunits possess a regulatory potential. Altogether this study provides new insights into the role of human eIF3 in the initial assembly steps of the translational machinery.


Assuntos
Fator de Iniciação 3 em Eucariotos/genética , Proteínas Associadas aos Microtúbulos/genética , Ribossomos/genética , Reagentes para Ligações Cruzadas/farmacologia , Formaldeído/farmacologia , Humanos , Ligação Proteica , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Subunidades Ribossômicas Menores de Eucariotos/genética
6.
EBioMedicine ; 50: 81-92, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31735550

RESUMO

BACKGROUND: Pediatric high-grade gliomas (pHGG) are the leading cause of cancer-related death during childhood. Due to their diffuse growth characteristics, chemoresistance and location behind the blood-brain barrier (BBB), the prognosis of pHGG has barely improved in the past decades. As such, there is a dire need for new therapies that circumvent those difficulties. Since aberrant expression of DNA damage-response associated Fanconi anemia proteins play a central role in the onset and therapy resistance of many cancers, we here investigated if FANCD2 depletion could sensitize pHGG to additional DNA damage. METHODS: We determined the capacity of celastrol, a BBB-penetrable compound that degrades FANCD2, to sensitize glioma cells to the archetypical DNA-crosslinking agent carboplatin in vitro in seven patient-derived pHGG models. In addition, we tested this drug combination in vivo in a patient-derived orthotopic pHGG xenograft model. Underlying mechanisms to drug response were investigated using mRNA expression profiling, western blotting, immunofluorescence, FANCD2 knockdown and DNA fiber assays. FINDINGS: FANCD2 is overexpressed in HGGs and depletion of FANCD2 by celastrol synergises with carboplatin to induce cytotoxicity. Combination therapy prolongs survival of pHGG-bearing mice over monotherapy and control groups in vivo (P<0.05). In addition, our results suggest that celastrol treatment stalls ongoing replication forks, causing sensitivity to DNA-crosslinking in FANCD2-dependent glioma cells. INTERPRETATION: Our results show that depletion of FANCD2 acts as a chemo-sensitizing strategy in pHGG. Combination therapy using celastrol and carboplatin might serve as a clinically relevant strategy for the treatment of pHGG. FUNDING: This study was funded by a grant from the Children Cancer-Free Foundation (KIKA, project 210). The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Assuntos
Carboplatina/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Glioma/genética , Glioma/metabolismo , Triterpenos/farmacologia , Adolescente , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Dano ao DNA , Modelos Animais de Doenças , Feminino , Glioma/patologia , Humanos , Masculino , Camundongos , Gradação de Tumores , Proteólise/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Refract Surg ; 35(11): 721-728, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710374

RESUMO

PURPOSE: To determine the relative impact of contact lens- assisted corneal cross-linking (CACXL) and standard protocol CXL (CXL) on regional corneal stiffness using Brillouin microscopy. METHODS: CXL and CACXL were performed on 30 intact fresh porcine eyes (15 per group). Depth profile of stiffness variation and averaged elastic modulus of anterior, middle, and posterior stroma were determined by Brillouin maps. Corneas were cut into strips to conduct mechanical stress-strain tests after Brillouin microscopy to evaluate stiffness difference between CXL and CACXL. Each eye served as its own control. RESULTS: CXL had a greater impact on corneal stiffness, with a maximum increase of 5.74% compared to 3.99% for CACXL (P < .001). CXL increased longitudinal modulus by 7.8% in the anterior, 1.7% in the middle, and -0.7% in the posterior regions compared to CACXL, which increased longitudinal modulus by 5.5% in the anterior (P < .001), 1.2% in the middle (P = .15), and -0.4% in the posterior regions (P = .60). Mechanical stress-strain tests showed that at 10% strain averaged Young's modulus was 5 MPa for CXL and 2.97 MPa for CACXL (P < .001). CONCLUSIONS: Both CACXL and standard protocol CXL induced significant corneal stiffening primarily concentrated in the anterior cornea. CACXL leads to less stiffening compared with CXL. An attenuated but continuous stiffening effect can be observed through the whole cornea for both CACXL and CXL, although CACXL has a smaller stiffness gradient. [J Refract Surg. 2019;35(11):721-728.].


Assuntos
Colágeno/farmacologia , Lentes de Contato , Córnea/fisiopatologia , Doenças da Córnea/tratamento farmacológico , Reagentes para Ligações Cruzadas/farmacologia , Fotoquimioterapia/métodos , Riboflavina/farmacologia , Animais , Córnea/diagnóstico por imagem , Doenças da Córnea/patologia , Doenças da Córnea/fisiopatologia , Modelos Animais de Doenças , Elasticidade , Microscopia/métodos , Fármacos Fotossensibilizantes/farmacologia , Suínos
8.
Mol Vis ; 25: 574-582, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31673223

RESUMO

Purpose: Aim to assess the short-term effect of genipin collagen crosslinking (G-CXL) on corneal structure and biomechanical properties compared with ultraviolet A/riboflavin collagen crosslinking (UVA-CXL) in rabbit corneas. Methods: Right eyes of 40 healthy rabbits were divided into the 0.20% G-CXL group, 0.25% G-CXL group, UVA-CXL group, and control group. Anterior segment optical coherence tomography (ASOCT) and in vivo confocal microscopy (IVCM) were performed before, 7 days after, and 14 days after the CXL treatment. Corneal strips were harvested for tensile strain measurements 7 and 14 days after the CXL treatment. Results: ASOCT showed the demarcation line (DL) in the UVA-CXL group was deeper than in the 0.20% G-CXL group and the 0.25% G-CXL group on day 7 (p=0.014) and day 14 (p=0.012). Nerve and keratocyte density in all CXL groups decreased, but was more obvious in the UVA-CXL group (p<0.001). Endothelial cell loss in the 0.20% G-CXL group, 0.25% G-CXL group, UVA-CXL group, and control group was 11.7%, 6.8%, 32.8%, and 2.0% 14 days after CXL, respectively. Young's modulus and stress in the 0.25% G-CXL group and the UVA-CXL group were statistically significantly higher than in the control group (p<0.05) 7 and 14 days after CXL. No statistically significant differences were observed between the 0.25% G-CXL group and the UVA-CXL group (p>0.05). The DL depth was positively correlated with Young's modulus (r=0.426, p=0.042) and stress (r=0.469, p=0.024). Conclusions: The administration of 0.25% genipin enhances corneal biomechanical properties as long as 14 days after the CXL treatment with low toxicity. The DL exists in CXL-treated corneas, and the depth is related to the biomechanical properties.


Assuntos
Colágeno/farmacologia , Córnea/fisiologia , Reagentes para Ligações Cruzadas/farmacologia , Iridoides/farmacologia , Animais , Fenômenos Biomecânicos/efeitos dos fármacos , Contagem de Células , Córnea/efeitos dos fármacos , Módulo de Elasticidade , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Processamento de Imagem Assistida por Computador , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Coelhos , Tomografia de Coerência Óptica
9.
Nat Commun ; 10(1): 4866, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31653830

RESUMO

Despite the success of current therapies for acute myocardial infarction (MI), many patients still develop adverse cardiac remodeling and heart failure. With the growing prevalence of heart failure, a new therapy is needed that can prevent remodeling and support tissue repair. Herein, we report on injectable recombinant human collagen type I (rHCI) and type III (rHCIII) matrices for treating MI. Injecting rHCI or rHCIII matrices in mice during the late proliferative phase post-MI restores the myocardium's mechanical properties and reduces scar size, but only the rHCI matrix maintains remote wall thickness and prevents heart enlargement. rHCI treatment increases cardiomyocyte and capillary numbers in the border zone and the presence of pro-wound healing macrophages in the ischemic area, while reducing the overall recruitment of bone marrow monocytes. Our findings show functional recovery post-MI using rHCI by promoting a healing environment, cardiomyocyte survival, and less pathological remodeling of the myocardium.


Assuntos
Colágeno Tipo III/farmacologia , Colágeno Tipo I/farmacologia , Coração/efeitos dos fármacos , Infarto do Miocárdio/patologia , Proteínas Recombinantes/farmacologia , Função Ventricular/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Capilares/efeitos dos fármacos , Carbodi-Imidas/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cicatriz/patologia , Vasos Coronários/efeitos dos fármacos , Reagentes para Ligações Cruzadas/farmacologia , Dimetilaminas/farmacologia , Humanos , Macrófagos/efeitos dos fármacos , Camundongos , Monócitos/efeitos dos fármacos , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Succinimidas/farmacologia
10.
Colloids Surf B Biointerfaces ; 184: 110526, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31590049

RESUMO

Nanogels have been applied in protein delivery due to the nanoscale sizes and the crosslinked structures. However, the release of protein molecules from the nanogels without damages to the structures and functionalities is quite a challenging research subject. In this research, responsive self-immolative linker dithioethyl carbamate bond is introduced to connect protein and polymer in the nanogel so that traceless release of protein occurs upon addition of glutathione (GSH) or dithiothreitol (DTT). Thermoresponsive polymer poly(di(ethylene glycol) methyl ether methacrylate-co-2-(2-(2-hydroxyethyl) disulfanyl) ethyl methacrylate) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and was modified with 4-nitrophenyl chloroformate yielding polymer chains with pendant dithioethyl carbonate groups. The dithioethyl carbonate groups were reacted with amine groups of lipases resulting in the formation of dithioethyl carbamate bonds. Meanwhile, biohybrid nanogels were prepared by crosslinking the polymer chains with lipases. The immobilized lipase in the nanogels exhibited enhanced heat and acid resistance. Once the nanogels were treated with GSH or DTT, lipase could be released with no residual groups and most of its bioactivity was recovered.


Assuntos
Reagentes para Ligações Cruzadas/química , Lipase/química , Nanogéis/química , Polímeros/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/síntese química , Reagentes para Ligações Cruzadas/farmacologia , Lipase/metabolismo , Camundongos , Estrutura Molecular , Células NIH 3T3 , Tamanho da Partícula , Polímeros/síntese química , Polímeros/farmacologia , Propriedades de Superfície
11.
Mater Sci Eng C Mater Biol Appl ; 104: 109902, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31500033

RESUMO

OBJECTIVE: This study sought to promote the adhesion, proliferation and differentiation of rat bone marrow mesenchymal stem cells by constructing a neurotrophin-3 (NT-3) sustained-release system cross-linked with an acellular spinal cord scaffold. METHODS: 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) chemistry combined with chemical extraction was used to construct an acellular spinal cord scaffold. The decellularization completion was validated. An EDC cross-linking method was used to construct the NT-3 cross-linked acellular spinal scaffold. ELISA was used to verify sustained release of NT-3; the dorsal root ganglion method was used to verify the biological activity of the sustained-release NT-3. DAPI staining was used to confirm the adhesion of the cultured rat bone marrow mesenchymal stem cells (P3) to the NT-3 scaffold, and cell counting kit-8 (CCK-8) analysis was used to verify the cellular proliferation after 24 h and 48 h of culture. Immunohistochemistry was used to confirm the differentiation of the bone marrow cells into neuron-like cells. RESULTS: An NT-3 sustained-release system cross-linked to an acellular spinal cord scaffold was successfully constructed. Sustained-release NT-3 could persist for 35 days and had biological activity for at least 21 days. It could promote the adhesion, proliferation and differentiation of rat bone marrow mesenchymal stem cells. CONCLUSION: As a composite scaffold, an NT-3 sustained-release system cross-linked with an acellular spinal cord scaffold has potential applications for tissue engineering.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/farmacologia , Células-Tronco Mesenquimais/citologia , Neurotrofina 3/farmacologia , Medula Espinal/fisiologia , Tecidos Suporte/química , Animais , Adesão Celular/efeitos dos fármacos , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Preparações de Ação Retardada/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Ratos Sprague-Dawley , Medula Espinal/efeitos dos fármacos
12.
Biomater Sci ; 7(11): 4738-4747, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31502601

RESUMO

Current nanomedicine suffers from a big challenge due to the fact that most of the nanocarrier systems lack the desired tumor penetration depth, thereby limiting their clinical translation. Unlike the nanomaterials with a similar size or shape, microgels display excellent softness, fluidity and deformability, as well as stimuli-responsiveness in the tumor microenvironment. Herein, we report the synthesis of temperature-responsive poly(N-vinylcaprolactam)/oligo (ethylene glycol) acrylate/glycidyl methacrylate (PVCL/OEGA/GMA) microgels with different hydrodynamic radii (100-500 nm), crosslinking densities, 2-methoxyethyl acrylate (MEA) contents and OEGA chain lengths using a precipitation polymerization method and the investigation of the microgels in terms of their tumor penetration capability using a multicellular tumor spheroid (MCTS) model. The prepared microgels were well characterized with different techniques. We show that regardless of the size, crosslinking density, MEA content and OEGA chain length, all microgels display the desired cytocompatibility in the given concentration range. In vitro cellular uptake data reveal that similar to 2-dimensional (2-D) adherent cells, microgels with a smaller size display more enhanced cellular uptake than those having a larger size in the 3-D MCTS model. Likewise, 3-D MCTS penetration results indicate that the PVCL/OEGA/GMA microgels with the smallest radius of 100 nm exhibit the deepest penetration length. We then selected the microgels with a radius of 200 nm but with different physicochemical parameters to investigate their cellular uptake and tumor penetration behavior. Our data show that microgels with varying crosslinking densities, MEA contents and OEGA chain lengths do not have any appreciable changes in terms of their cellular uptake and penetration in the 3-D MCTS model. Our study provides new insights for the design of different microgel-based systems for further cancer theranostic applications.


Assuntos
Antineoplásicos/farmacologia , Caprolactama/análogos & derivados , Reagentes para Ligações Cruzadas/farmacologia , Microgéis/química , Polímeros/farmacologia , Esferoides Celulares/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Caprolactama/química , Caprolactama/farmacologia , Carbocianinas/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/síntese química , Reagentes para Ligações Cruzadas/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Microscopia de Fluorescência , Estrutura Molecular , Nanomedicina , Imagem Óptica , Tamanho da Partícula , Polímeros/química , Propriedades de Superfície , Temperatura
13.
Ophthalmic Plast Reconstr Surg ; 35(6): 600-603, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31348113

RESUMO

PURPOSE: A follow-up experimental study on the exposure of animal tarsal plate to ultraviolet-A radiation aimed at establishing an optimum range for safe irradiation conditions. METHODS: Sheep tarsus specimens were excised postmortem and then subjected to irradiation with ultraviolet-A rays (wavelength 365 nm) at higher irradiances than those reported in an initial study, using a laboratory radiation source. The mechanical properties (tensile strength and Young's modulus) of irradiated and nonirradiated samples were evaluated in a mechanical tester. The test and control specimens were examined histologically with an aim to assess the effects of radiation upon the meibomian glands and tarsal collagen networks, and to establish a safe range for the exposure irradiance level. RESULTS: As expected, irradiation induced both stiffening and strengthening of the tarsal plate specimens. At an irradiance of 50 mW/cm for 3-minute exposure, these effects were at their maximum level, after which a decline in mechanical characteristics were observed. No destruction of the tarsal connective tissue or the meibomian glands were noticed up to an irradiance of 125 mW/cm for 3-minute exposure, corresponding to a fluence of 22.5 J/cm. Histology revealed that the collagen network surrounding the glands were packed more compactly following irradiation. At a fluence of 45 J/cm, massive destruction of periglandular collagen-rich network and meibocytes were demonstrated histologically. CONCLUSIONS: The study indicates that irradiation of tarsal collagen leading to tissue stiffening shall be carried out at levels of fluence between 10 and 15 J/cm, a region that is deemed safe. The exposure time can be adjusted according to the surgeon's decision.Safe irradiation conditions are established for the exposure of ex vivo ovine tarsus to ultraviolet-A radiation as a potentially effective treatment for eyelid laxity in human patients.


Assuntos
Colágeno/metabolismo , Reagentes para Ligações Cruzadas/farmacologia , Doenças Palpebrais/tratamento farmacológico , Pálpebras , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Riboflavina/farmacologia , Animais , Pálpebras/efeitos dos fármacos , Pálpebras/fisiologia , Pálpebras/efeitos da radiação , Glândulas Tarsais/efeitos dos fármacos , Glândulas Tarsais/efeitos da radiação , Ovinos , Raios Ultravioleta
14.
Cornea ; 38(11): 1390-1394, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31335524

RESUMO

PURPOSE: To investigate the relationship between corneal collagen cross-linking (CXL) and the number of corneal transplants required for the treatment of keratoconus (KCN) in 2 major Canadian provinces. METHODS: This is a retrospective review of all corneal transplantation performed in Ontario and British Columbia over an 18-year period (1998-2016). Data were collected at the Eye Bank of Canada-Ontario/British Columbia Divisions. The primary outcome was to determine the change in proportion and absolute number of corneal transplants required for treatment of KCN since the introduction of CXL in Canada in 2008. RESULTS: A total of 31,943 grafts were included. Overall, the mean age of participants was 39.3 ± 2.2 years, with our cohort being composed of 28% of women and 72% of men. The results showed a significant decrease in the proportion of total transplants required for KCN between 1998 and 2016 [1998-2008 (pre-CXL), range: 14.77%-12.63%; 2009-2016 (post-CXL), range: 12.98%-5.50%, P < 0.001]. However, there was no change in the absolute number of grafts performed for KCN over this time (pre-CXL: 179 ± 26 grafts; post-CXL: 198 ± 27 grafts; P = 0.5), whereas the total number of grafts (pre-CXL: 1318 ± 183 grafts; post-CXL: 2181 ± 404; P < 0.001) and endothelial keratoplasties (pre-CXL: 59 ± 108; post-CXL: 966 ± 431 grafts; P < 0.001) increased significantly. In addition, there were no changes in penetrating keratoplasty/deep anterior lamellar keratoplasty (DALK) performed for indications other than KCN (pre-CXL: 1080 ± 157; post-CXL: 1017 ± 92; P > 0.5). CONCLUSIONS: Although there has been a significant decrease in the proportion of corneal graft rates for KCN since the introduction of CXL as a factor of all transplants performed for all indications, this result is most likely because of an increase in endothelial keratoplasties rather than decreased transplants performed for definitive treatment.


Assuntos
Colágeno/farmacologia , Córnea/patologia , Reagentes para Ligações Cruzadas/farmacologia , Bancos de Olhos/provisão & distribução , Previsões , Ceratocone/tratamento farmacológico , Ceratoplastia Penetrante/estatística & dados numéricos , Adulto , Colúmbia Britânica , Córnea/efeitos dos fármacos , Córnea/cirurgia , Feminino , Seguimentos , Humanos , Ceratocone/diagnóstico , Ceratocone/cirurgia , Masculino , Ontário , Estudos Retrospectivos , Acuidade Visual
15.
Colloids Surf B Biointerfaces ; 181: 705-713, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31228853

RESUMO

Silk fibroin has been utilized extensively for biomedical purposes, especially the drug delivery systems. This study introduced and characterized three novel α-mangostin loaded crosslinked fibroin nanoparticles (FNPs), using EDC or PEI as a crosslinker, for cancer treatment. All three formulas were spherical particles with a mean size of approximately 300 nm. By varying the type and/or amount of the crosslinkers, particle surface charge was controllable from -15 to +30 mV. Crosslinked FNPs exhibited higher drug entrapment efficiency (70%) and drug loading (7%) than non-crosslinked FNP. FT-IR, XRD, and DSC analytical methods confirmed that α-mangostin was entrapped in FNPs in molecular dispersion form. Compared to the free α-mangostin, the crosslinked FNPs increased the drug's solubility up to threefold. They also showed sustained release characteristics of more than 3 days, and reduced free α-mangostin hematotoxicity by 90%. The α-mangostin loaded FNPs were physicochemically stable for up to 24 h when dispersed in intravenous diluent and for at least 6 months when preserved as lyophilized powder at 4 °C. In terms of anticancer efficacy, on both Caco-2 colorectal and MCF-7 breast adenocarcinoma cell lines, all formulas maintain α-mangostin's apoptotic effect while exhibit greater cytotoxicity than the free drug. In conclusion, α-mangostin loaded crosslinked FNPs show high potential for cancer chemotherapy.


Assuntos
Antineoplásicos/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Fibroínas/farmacologia , Nanopartículas/química , Xantonas/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/química , Sistemas de Liberação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Fibroínas/química , Humanos , Células MCF-7 , Tamanho da Partícula , Propriedades de Superfície , Xantonas/química
16.
Biomater Sci ; 7(9): 3640-3651, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31165794

RESUMO

Tissue regeneration requires scaffolds that exhibit mechanical properties similar to the tissues to be replaced while allowing cell infiltration and extracellular matrix production. Ideally, the scaffolds' porous architecture and physico-chemical properties can be precisely defined to address regenerative needs. We thus developed techniques to produce hybrid fibers coaxially structured with a polycaprolactone core and a 4-arm, polyethylene glycol thiol-norbornene sheath. We assessed the respective effects of crosslink density and sheath polymer size on the scaffold architecture, physical and mechanical properties, as well as cell-scaffold interactions in vitro and in vivo. All scaffolds displayed high elasticity, swelling and strength, mimicking soft tissue properties. Importantly, the thiol-ene hydrogel sheath enabled tunable softness and peptide tethering for cellular activities. With increased photopolymerization, stiffening and reduced swelling of scaffolds were found due to intra- and inter-fiber crosslinking. More polymerized scaffolds also enhanced the cell-scaffold interaction in vitro and induced spontaneous, deep cell infiltration to produce collagen and elastin for tissue regeneration in vivo. The molecular weight of sheath polymer provides an additional mechanism to alter the physical properties and biological activities of scaffolds. Overall, these robust scaffolds with tunable elasticity and regenerative cues offered a versatile and effective platform for tissue regeneration.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , Microfibrilas/química , Poliésteres/farmacologia , Polietilenoglicóis/farmacologia , Compostos de Sulfidrila/farmacologia , Animais , Bovinos , Adesão Celular/efeitos dos fármacos , Reagentes para Ligações Cruzadas/síntese química , Reagentes para Ligações Cruzadas/química , Células Endoteliais/efeitos dos fármacos , Peso Molecular , Poliésteres/química , Polietilenoglicóis/química , Artéria Pulmonar/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Compostos de Sulfidrila/química
17.
Nan Fang Yi Ke Da Xue Xue Bao ; 39(5): 508-514, 2019 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-31140412

RESUMO

OBJECTIVE: To investigate the effect of blocking pannexin-1 against acute kidney injury induced by cisplatin. METHODS: Twenty-six male C57BL/6 mice aged 6-8 weeks were randomly divided into control group, cisplatin model (Cis) group and cisplatin + carbenoxolone treatment group (Cis + CBX). In Cis group and Cis + CBX group, the mice were injected intraperitoneally with 20 mg/kg of cisplatin and with CBX (20 mg/kg) at 30 min before and 24 and 48 h after cisplatin inhjection, respectively. All the mice were sacrificed at 72 h after cisplatin injection, and plasma and kidney samples were collected for testing mRNA and protein expression levels of pannexin-1 in the renal tissue using RT-qPCR and Western blotting and for detecting plasma creatinine and BUN levels; the pathological changes in the renal tissues were observed using Periodic Acid-Schiff staining. The expression of kidney injury molecule 1 (KIM-1) was examined using immunohistochemistry and the mRNA expressions of KIM-1 and neutrophil gelatinase- related lipid transport protein (NGAL) were detected by RT-qPCR to evaluate the injuries of the renal tubules. The infiltration of F4/80-positive macrophages and CD4-positive T cells were observed by immunofluorescence. In the in vitro experiment, human proximal tubule epithelial cell line HK-2 was stimulated with 50 µmol/L cisplatin to establish a cell model of acute kidney injury, and the mRNA and protein expressions of pannexin-1 were detected by RT-qPCR and Western blotting at 4, 6, 12, 18 and 24 h after the stimulation. RESULTS: Compared with the control mice, the cisplatin-treated mice showed significantly up-regulated protein levels (P < 0.05) and mRNA levels (P < 0.005) of pannexin-1 in the kidney tissue. Cisplatin stimulation also caused significant increases in the protein levels (P < 0.005) and mRNA levels (P < 0.005) of pannexin-1 in cultured HK-2 cells. Compared with cisplatin-treated mice, the mice treated with both cisplatin and the pannexin-1 inhibitor CBX showed obviously lessened kidney pathologies and milder renal tubular injuries with significantly reduced plasma BUN and Scr levels (P < 0.01), expressions of KIM-1 and NGAL in the kidney (P < 0.05), and infiltration of F4/80-positive macrophages (P < 0.01) and CD4- positive T cells (P < 0.05) in the kidney tissues. CONCLUSIONS: In cisplatin induced acute kidney injury mice model, Pannexin-1 expression is up-regulated in the kidneys tissue, and blocking pannexin-1 alleviates the acute kidney injury via reducing renal inflammatory cell infiltration.


Assuntos
Lesão Renal Aguda , Cisplatino , Conexinas , Reagentes para Ligações Cruzadas , Proteínas do Tecido Nervoso , Lesão Renal Aguda/tratamento farmacológico , Lesão Renal Aguda/metabolismo , Animais , Cisplatino/farmacologia , Conexinas/efeitos dos fármacos , Conexinas/metabolismo , Reagentes para Ligações Cruzadas/farmacologia , Humanos , Rim , Túbulos Renais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/metabolismo , Distribuição Aleatória
18.
Graefes Arch Clin Exp Ophthalmol ; 257(7): 1443-1452, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31041523

RESUMO

PURPOSE: To evaluate the efficacy of corneal cross-linking (CXL) as adjuvant therapy for the treatment of fungal ulcerative keratitis. METHODS: Forty-one patients with fungal ulcerative keratitis were recruited and assigned into two randomized controlled groups. These groups were treated with CXL combined with antifungal medications (CXL-M) or antifungal medications alone (M). The ulcers were assessed by slit-lamp biomicroscopy, slit-lamp images, in vivo confocal microscopy (IVCM), and anterior segment optical coherence tomography (AS-OCT). The patients were followed up before surgery/first visit (FV), 1 day after surgery, 1 and 2 weeks, and 1, 2, 3, 4, 5, and 6 months after surgery/FV. RESULTS: In the cured patients, the area of corneal ulcers, the duration of ulcer healing, the time to non-observed fungal hyphae by IVCM, the number of antifungal medications, the frequency of administered medications, and the maximum ulcer depth decreased significantly after CXL (all P < 0.05) compared with the M group. There were no significant differences in either corneal thickness or epithelial thickness of ulcers after healing between 5 and 6 months after surgery in the CXL-M group, while these were increased significantly at 6 months compared with 5 months after FV in the M group (both P < 0.05). CONCLUSIONS: In our study, CXL accelerated healing of the fungal ulcers, shortened the treatment duration, and minimized the need for medications and surgery. It appears that CXL is an effective procedure and adjuvant therapy for managing fungal keratitis.


Assuntos
Antifúngicos/farmacologia , Córnea/patologia , Úlcera da Córnea/tratamento farmacológico , Reagentes para Ligações Cruzadas/farmacologia , Infecções Oculares Fúngicas/tratamento farmacológico , Fotoquimioterapia/métodos , Riboflavina/farmacologia , Córnea/microbiologia , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/microbiologia , Infecções Oculares Fúngicas/diagnóstico , Infecções Oculares Fúngicas/microbiologia , Feminino , Seguimentos , Fungos/isolamento & purificação , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Fármacos Fotossensibilizantes/farmacologia , Estudos Retrospectivos , Tomografia de Coerência Óptica , Resultado do Tratamento , Raios Ultravioleta
19.
Graefes Arch Clin Exp Ophthalmol ; 257(7): 1435-1442, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31065848

RESUMO

PURPOSE: To investigate the safety of blue light scleral cross-linking (SXL) by evaluating changes in biological parameters in the retina and choroid in the eyes of rhesus macaques (Macaca mulatta). METHODS: Fifteen 3-year-old macaques (30 eyes) were randomly divided into three groups (n = 5). SXL was performed via riboflavin (0.5%) and blue light (460 nm) at the location of the equatorial sclera. Right eyes served as experimental eyes, and left eyes as control eyes. One quadrant of each right eye was irradiated in group A, two quadrants of each right eye and one quadrant of each left eye were irradiated in group B, and two quadrants of each right eye were irradiated in group C. Optical coherence tomography, optical coherence tomography angiography, and flash electroretinography (f-ERG) examinations were performed at baseline and 1 week, 1 month, 3 months, and 6 months after SXL. Additionally, retinal tissue alterations were detected via transmission electron microscopy at 1 week postoperatively. RESULTS: There were no significant differences between experimental eyes and control eyes in retinal thickness, vessel density of retinal superficial capillary plexus, and choroid thickness in any of the groups at any of the time points investigated (p > 0.05). Significant reductions in f-ERG parameters were detected 1 week postoperatively in the experimental eyes of groups A and C (p < 0.05), but they gradually recovered, and there was no significant difference 1 month postoperatively (p > 0.05). Ultrastructural changes were evident in the retinal layers of SXL eyes. In group B, there were no significant differences between the right and left eyes at any of the follow-up time points investigated. CONCLUSIONS: Blue light SXL can cause transient retina damage. The f-ERG parameters reductions and retinal ultrastructural changes were found at early stage, even though there were not significant changes in retinal thickness, vessel density of retinal superficial capillary plexus, and choroid thickness after blue light SXL. The long-term intraocular safety of the blue light SXL technique should be investigated further.


Assuntos
Corioide/patologia , Colágeno/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Miopia/tratamento farmacológico , Fotoquimioterapia/métodos , Retina/patologia , Riboflavina/farmacologia , Animais , Modelos Animais de Doenças , Eletrorretinografia , Angiofluoresceinografia , Seguimentos , Fundo de Olho , Luz , Macaca mulatta , Miopia/diagnóstico , Miopia/fisiopatologia , Fármacos Fotossensibilizantes/farmacologia , Esclera , Tomografia de Coerência Óptica , Resultado do Tratamento
20.
Colloids Surf B Biointerfaces ; 181: 94-101, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31125923

RESUMO

A cross-linkable gemcitabine (GEM)-containing reduction-sensitive polymeric micelles based on the copolymer poly(PEG-co-CMA-co-GEM) was successfully fabricated. The copolymer which synthesized by one-step radical copolymerization of poly(ethylene glycol) methacrylate (PEGMA), 7-(2-Methylacryloylethoxy)-4-methylcoumarin (CMA), and 2-((2-hydroxyethyl)disulfanyl)ethyl acrylate (HSEA-GEM), endowed the micelles with "stealth" surface in circulation, photo cross-linkable property, and reduction-sensitivity. The designed micelles were fabricated by self-assembly of the copolymer in aqueous solution followed by UV-light induced cross-linking of coumarin moieties to enhance stability, which would not disassemble even below critical micelle concentration (CMC) or in non-selective solvent (DMSO/H2O 1:1). In vitro drug release curve demonstrated that the intracellular-mimicking reductive microenvironment could accelerated the GEM release of the prodrug micelles. These micelles could be effectively internalized by BxPC-3 pancreatic cancer cells according to confocal laser scanning microscopy (CLSM) detection and flow cytometry analysis. Meanwhile, methyl thiazolyl tetrazolium (MTT) assay demonstrated that the cross-linked prodrug micelles could efficiently inhibit the proliferation of BxPC-3 cells.


Assuntos
Antineoplásicos/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Desoxicitidina/análogos & derivados , Sistemas de Liberação de Medicamentos , Metacrilatos/farmacologia , Polietilenoglicóis/farmacologia , Pró-Fármacos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Reagentes para Ligações Cruzadas/síntese química , Reagentes para Ligações Cruzadas/química , Desoxicitidina/química , Desoxicitidina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Metacrilatos/química , Micelas , Estrutura Molecular , Oxirredução , Tamanho da Partícula , Polietilenoglicóis/química , Pró-Fármacos/química , Propriedades de Superfície , Células Tumorais Cultivadas
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA