RESUMO
White spot disease caused by the white spot syndrome virus (WSSV) incurs a huge loss to the shrimp farming industry. Since no effective therapeutic measures are available, early detection and prevention of the disease are indispensable. Towards this goal, we previously identified a 12-mer phage displayed peptide (designated as pep28) with high affinity for VP28, the structural protein of the white spot syndrome virus (WSSV). The peptide pep28 was successfully used as a biorecognition probe in the lateral flow assay developed for rapid, on-site detection of WSSV. To unravel the structural determinants for the selective binding between VP28 and pep28, we used bioinformatics, structural modeling, protein-protein docking, and binding-free energy studies. We performed atomistic molecular dynamics simulations of pep28-pIII model totaling 300 ns timescale. The most representative pep28-pIII structure from the simulation was used for docking with the crystal structure of VP28. Our results reveal that pep28 binds in a surface groove of the monomeric VP28 ß-barrel and makes several hydrogen bonds and non-polar interactions. Ensemble-based binding-free energy studies reveal that the binding is dominated by non-polar interactions. Our studies provide molecular level insights into the binding mechanism of pep28 with VP28, which explain why the peptide is selective and can assist in modifying pep28 for its practical use, both as a biorecognition probe and a therapeutic.
Assuntos
Técnicas de Visualização da Superfície Celular , Mapeamento de Epitopos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeos/química , Mapeamento de Interação de Proteínas , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Sítios de Ligação , Mapeamento de Epitopos/métodos , Ligação de Hidrogênio , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismoRESUMO
Bacterial cellulose produced by few but specific microbial genera is an extremely pure natural exopolysaccharide. Besides providing adhesive properties and a competitive advantage to the cellulose over-producer, bacterial cellulose confers UV protection, ensures maintenance of an aerobic environment, retains moisture, protects against heavy metal stress, etc. This unique nanostructured matrix is being widely explored for various medical and nonmedical applications. It can be produced in various shapes and forms because of which it finds varied uses in biomedicine. The attributes of bacterial cellulose such as biocompatibility, haemocompatibility, mechanical strength, microporosity and biodegradability with its unique surface chemistry make it ideally suited for a plethora of biomedical applications. This review highlights these qualities of bacterial cellulose in detail with emphasis on reports that prove its utility in biomedicine. It also gives an in-depth account of various biomedical applications ranging from implants and scaffolds for tissue engineering, carriers for drug delivery, wound-dressing materials, etc. that are reported until date. Besides, perspectives on limitations of commercialisation of bacterial cellulose have been presented. This review is also an update on the variety of low-cost substrates used for production of bacterial cellulose and its nonmedical applications and includes patents and commercial products based on bacterial cellulose.
Assuntos
Celulose/química , Gluconacetobacter/química , Materiais Biocompatíveis/química , Biodegradação Ambiental , Sistemas de Liberação de Medicamentos , Fermentação , Nanocompostos/química , Próteses e Implantes , Engenharia Tecidual , Alicerces Teciduais/químicaRESUMO
Dextran stabilized La(0.7)Sr(0.3)MnO(3) (Dex-LSMO) is an alternative cancer hyperthermia agent holding considerable promise. Here, we have carried out a comparative study on radio frequency (~264 kHz) induced Dex-LSMO mediated heating and extraneous heating (mimicking generalized hyperthermia) in terms of changes in the morphology, proliferation pattern and induction of heat shock proteins in a human melanoma cell line (A375). Our results clearly show that the cellular effects seen with extraneous heating (60 min at 43 °C) could be reproduced by just six minutes of radio frequency induced Dex-LSMO mediated heating. More importantly, the observed enhanced levels of HSP 70 and 90 (molecular markers of heat shock that trigger favorable immunological reactions) seen with Dex-LSMO mediated heating were comparable to extraneous heating. These results suggest the possible utility of Dex-LSMO as a cancer hyperthermia agent.
Assuntos
Dextranos/química , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Hipertermia Induzida , Nanopartículas Metálicas/toxicidade , Ondas de Rádio , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lantânio/toxicidade , Compostos de Manganês , Microscopia de Contraste de Fase , Óxidos/toxicidade , Estrôncio/toxicidade , Fatores de TempoRESUMO
Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds. Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 microg/mL and 100 microg/mL SNP, respectively, although with minor decrease in confluence. IC(50) values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 microg/mL and 449 microg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC(50) SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (i.e. 30 microg/mL and 225 microg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (approximately 1.2 fold) and depletion of lipid peroxidation (approximately 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD ( approximately 1.4 fold) and GSH ( approximately 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 microg/mL in primary fibroblasts, 12.5 microg/mL in primary liver cells) than the necrotic concentration (100 microg/mL in primary fibroblasts, 500 microg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC(50) SNP (resulting in apoptosis) and 2 x IC(50)) cells (resulting in necrosis). These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel (approximately 20 microg/g), indicates preliminary safety of the formulation and warrants further study for possible human application.
Assuntos
Fígado/metabolismo , Nanopartículas Metálicas , Prata/metabolismo , Animais , Apoptose , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Glutationa/metabolismo , Peroxidação de Lipídeos , Fígado/citologia , Fígado/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Superóxido Dismutase/metabolismoRESUMO
A systematic study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with HT-1080 and A431 cells was undertaken as a part of an on-going program in our laboratory to develop a topical antimicrobial agent for the treatment of burn wound infections. Upon exposure to SNP (up to 6.25 microg/mL), morphology of both the cell types remained unaltered. However, at higher concentrations (6.25-50 microg/mL) cells became less polyhedral, more fusiform, shrunken and rounded. IC(50) values for HT-1080 and A431 as revealed by XTT assay were 10.6 and 11.6 microg/mL, respectively. When the cells were challenged with approximately 1/2 IC(50) concentration of SNP (6.25 microg/mL), clear signs of oxidative stress, i.e. decreased GSH ( approximately 2.5-folds in HT-1080, approximately 2-folds in A431) and SOD ( approximately 1.6-folds in HT-1080, 3-folds in A431) as well as increased lipid peroxidation ( approximately 2.5-folds in HT-1080, approximately 2-folds in A431) were seen. Changes in the levels of catalase and GPx in A431 cells were statistically insignificant in both cell types. DNA fragmentation in SNP-exposed cells suggested apoptosis. When the apoptotic thresholds of SNP were monitored with caspase-3 assay the concentrations required for the onset of apoptosis were found to be much lower (0.78 microg/mL in HT-1080, 1.56 microg/mL in A431) than the necrotic concentration (12.5 microg/mL in both cell types). These results can be used to define a safe range of SNP for the intended application as a topical antimicrobial agent after appropriate in vivo studies.
Assuntos
Apoptose/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Catalase/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Nanopartículas Metálicas/química , Tamanho da Partícula , Prata/química , Superóxido Dismutase/metabolismoRESUMO
AIM: Optimization of process parameters for mercury removal by an Hg (II)-reducing Pseudomonas aeruginosa strain. METHODS AND RESULTS: A strain of Ps. aeruginosa was found to reduce 10 mg l(-1) Hg (II) to Hg0 with 70% efficiency in 24 h. To optimize process performance, a statistical tool--Taguchi design of experiments (DOE)--was used to carry out 18 well-defined experiments (L18 Orthogonal array) with eight variable parameters (viz. agitation, temperature, pH, carbon source, medium volume: flask volume ratio and concentrations of Hg (II), ammonium sulfate and yeast extract). When data obtained were analyzed using specialized software for Taguchi design, Qualitek-4 (Nutek Inc., MI, USA), Hg (II) reduction efficiency was predicted to be 95% in 24 h under the optimized process parameters (also suggested by the software). In the validation experiment, Hg (II) removal of 99.29% in 24 h was indeed obtained. CONCLUSIONS: Using Taguchi DOE, Hg (II) reduction (and hence its removal) using Ps. aeruginosa could be improved by 29.3%. SIGNIFICANCE AND IMPACT OF THE STUDY: Taguchi approach could be employed as an efficient and time-saving strategy for parameter optimization in bioremediation processes.