RESUMO
Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.
Assuntos
Biotina , Melanoma , Oxirredução , Proteômica , Proteômica/métodos , Melanoma/metabolismo , Melanoma/patologia , Humanos , Linhagem Celular Tumoral , Biotina/química , Biotina/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
The use of antimicrobial growth promoters (AGPs) is banned because of problems associated with drug residues in animal products and increased bacterial resistance. The immunization of chickens with specific antigens is a promising strategy for generating specific antibodies that can target a wide range of antibiotic-resistant bacteria and can be used as an alternative to antibiotics. Immunoglobulin Y (IgY) antibodies in a polyclonal antibody (pAb) format, when administered orally, modulate the ruminal microbiome and maintain animal health and performance; however, there are concerns pertaining to protein impurities and biotin concentrations in the samples. Signal amplification strategies involving the noncovalent interaction of biotin with streptavidin is extensively used in diagnosis and scientific research, particularly in enzyme-linked immunosorbent assays (ELISAs). However, the high concentrations of biotin in samples, especially in those derived from rich sources such as egg yolk, can pose challenges and potentially harm the accuracy of diagnostic tests and protein concentration measurements. This study aimed to evaluate the influence of biotin on the measurement of IgY in freeze-dried egg yolk samples obtained from immunized laying hens using immunoassays with biotin-avidin/streptavidin. The detection of IgY in yolk samples using ELISA with streptavidin-biotin binding could lead to misdiagnosis due to biotin interference; the level of interference varies with the specific assay conditions and the concentration of biotin in the yolk samples. An ELISA without streptavidin-biotin binding is advisable to avoid interactions between biotin and target proteins, prevent biotin interference with the results, and achieve more reliable and accurate results.
RESUMO
Electrophotonic (EPh) circuits are novel systems where photons and electrons can be controlled simultaneously in the same integrated circuit, attaining the development of innovative sensors for different applications. In this work, we present a complementary metal-oxide-semiconductor (CMOS)-compatible EPh circuit for biotin sensing, in which a silicon-based light source is monolithically integrated. The device is composed of an integrated light source, a waveguide, and a p-n photodiode, which are all fabricated in the same chip. The functionalization of the waveguide's surface was investigated to biotinylate the EPh system for potential biosensing applications. The modified surfaces were characterized by AFM, optical microscopy, and Raman spectroscopy, as well as by photoluminescence measurements. The changes on the waveguide's surface due to functionalization and biotinylation translated into different photocurrent intensities detected in the photodiode, demonstrating the potential uses of the EPh circuit as a biosensor.
Assuntos
Técnicas Biossensoriais , Biotina , Silício/química , Desenho de Equipamento , Técnicas Biossensoriais/métodos , SemicondutoresRESUMO
Biotin, spermine, and folic acid were covalently linked to the F127 copolymer to obtain a new drug delivery system designed for HY-loaded PDT treatment against B16F10 cells. Chemical structures and binders quantification were performed by spectroscopy and spectrophotometric techniques (1NMR, HABA/Avidin reagent, fluorescamine assay). Critical micelle concentration, critical micelle temperature, size, polydispersity, and zeta potential indicate the hydrophobicity of the binders can influence the physicochemical parameters. Spermine-modified micelles showed fewer changes in their physical and chemical parameters than the F127 micelles without modification. Furthermore, zeta potential measurements suggest an increase in the physical stability of these carrier systems. The phototherapeutic potential was demonstrated using hypericin-loaded formulation against B16F10 cells, which shows that the combination of the binders on F127 copolymer micelles enhances the photosensitizer uptake and potentializes the photodynamic activity.
RESUMO
The role of Malassezia yeasts in dandruff and seborrheic dermatitis is unclear; however, antifungal therapy with ketoconazole is commonly used. We propose that ketoconazole shifts skin lipid profile, affects Malassezia lipid metabolism and favours biotin-producing bacteria. Biotin regulates inflammatory response and cell proliferation, contributing to symptom improvement.
Assuntos
Caspa , Dermatite Seborreica , Malassezia , Antifúngicos/farmacologia , Biotina , Biologia Computacional , Caspa/tratamento farmacológico , Caspa/microbiologia , Dermatite Seborreica/tratamento farmacológico , Dermatite Seborreica/microbiologia , Humanos , Cetoconazol/farmacologia , Metabolismo dos LipídeosRESUMO
PURPOSE: The use of adjuvants can significantly strengthen a vaccine's efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. MATERIALS AND METHODS: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. RESULTS: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around - 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. CONCLUSION: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.
Assuntos
Escherichia coli , Schistosoma mansoni , Animais , Membrana Externa Bacteriana , Imunidade , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.
Assuntos
Biotina/toxicidade , Proliferação de Células/efeitos dos fármacos , Suplementos Nutricionais/toxicidade , Hormônio Foliculoestimulante/sangue , Espermatogônias/efeitos dos fármacos , Fator de Células-Tronco/metabolismo , Testículo/efeitos dos fármacos , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/genética , Fator de Transcrição Sp3/metabolismo , Espermatogônias/metabolismo , Espermatogônias/patologia , Testículo/metabolismo , Testículo/patologiaRESUMO
During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell-cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell-cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.
Assuntos
Biotina/metabolismo , Infecções por HIV/transmissão , HIV-1/metabolismo , Biotina/análogos & derivados , Linfócitos T CD4-Positivos/virologia , Membrana Celular , Infecções por HIV/virologia , Humanos , Vírion/metabolismo , Montagem de Vírus , Internalização do Vírus , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin-rhizavidin affinity platforms.
Assuntos
Antígenos de Helmintos/biossíntese , Proteínas de Bactérias/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/imunologia , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Cromatografia de Afinidade/métodos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Schistosoma mansoni/química , Schistosoma mansoni/imunologia , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/metabolismo , Esquistossomose mansoni/parasitologiaRESUMO
Purpose: The use of adjuvants can significantly strengthen a vaccine’s efficacy. We sought to explore the immunization efficacy of bacterial outer membrane vesicles (OMVs) displaying the Schistosoma mansoni antigen, SmTSP-2, through a biotin-rhizavidin coupling approach. The rationale is to exploit the nanoparticulate structure and the adjuvant properties of OMVs to induce a robust antigen-specific immune response, in light of developing new vaccines against S. mansoni. Materials and Methods: OMVs were obtained from Neisseria lactamica and conjugated with biotin. The recombinant SmTSP-2 in fusion with the biotin-binding protein rhizavidin (rRzvSmTSP-2) was produced in E. coli and coupled to biotinylated OMVs to generate an OMV complex displaying SmTSP-2 on the membrane surface (OMV:rSmTSP-2). Transmission electron microscopy (TEM) and dynamic light scattering analysis were used to determine particle charge and size. The immunogenicity of the vaccine complex was evaluated in C57BL/6 mice. Results: The rRzvSmTSP-2 protein was successfully coupled to biotinylated OMVs and purified by size-exclusion chromatography. The OMV:rSmTSP-2 nanoparticles showed an average size of 200 nm, with zeta potential around – 28 mV. Mouse Bone Marrow Dendritic Cells were activated by the nanoparticles as determined by increased expression of the co-stimulatory molecules CD40 and CD86, and the proinflammatory cytokines (TNF-α, IL-6 and IL-12) or IL-10. Splenocytes of mice immunized with OMV:rSmTSP-2 nanoparticles reacted to an in vitro challenge with SmTSP-2 with an increased production of IL-6, IL-10 and IL-17 and displayed a higher number of CD4+ and CD8+ T lymphocytes expressing IFN-γ, IL-4 and IL-2, compared to mice immunized with the antigen alone. Immunization of mice with OMV:rSmTSP-2 induced a 100-fold increase in specific anti-SmTSP-2 IgG antibody titers, as compared to the group receiving the recombinant rSmTSP-2 protein alone or even co-administered with unconjugated OMV. Conclusion: Our results demonstrate that the SmTSP-2 antigen coupled with OMVs is highly immunogenic in mice, supporting the potential effectiveness of this platform for improved antigen delivery in novel vaccine strategies.
RESUMO
Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin–rhizavidin affinity platforms.
RESUMO
OBJECTIVE: To detect and to compare the apoptotic effects of intraoperatively topically applied diltiazem, papaverine, and nitroprusside. METHODS: Internal thoracic artery segments of ten patients were obtained during coronary bypass grafting surgery. Each internal thoracic artery segment was divided into four pieces and immersed into four different solutions containing separately saline (Group S), diltiazem (Group D), papaverine (Group P), and nitroprusside (Group N). Each segment was examined with both hematoxylin-eosin and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method in order to determine and quantify apoptosis. RESULTS: Apoptotic cells were counted in 50 microscopic areas of each segment. No significant difference was observed among the four groups according to hematoxylin-eosin staining. However, the TUNEL method revealed a significant increase in mean apoptotic cells in the diltiazem group when compared with the other three groups (Group S=4.25±1.4; Group D=13.31±2.8; Group N=9.48±2.09; Group P=10.75±2.37). The differences between groups were significant (P=0.0001). No difference was observed between the samples of the diabetic and non-diabetic patients in any of the study groups. CONCLUSION: The benefit of topically applied vasodilator drugs must outweigh the potential adverse effects. In terms of apoptosis, diltiazem was found to have the most deleterious effects on internal thoracic artery graft segments. Of the analyzed medical agents, nitroprusside was found to have the least apoptotic activity, followed by papaverine. Diabetes did not have significant effect on the occurrence of apoptosis in left internal thoracic artery grafts.
Assuntos
Diltiazem/uso terapêutico , Artéria Torácica Interna , Nitroprussiato/uso terapêutico , Papaverina/uso terapêutico , Vasodilatadores/uso terapêutico , Diltiazem/farmacologia , Humanos , Nitroprussiato/farmacologia , Papaverina/farmacologia , Vasodilatadores/farmacologiaRESUMO
Abstract Objective: To detect and to compare the apoptotic effects of intraoperatively topically applied diltiazem, papaverine, and nitroprusside. Methods: Internal thoracic artery segments of ten patients were obtained during coronary bypass grafting surgery. Each internal thoracic artery segment was divided into four pieces and immersed into four different solutions containing separately saline (Group S), diltiazem (Group D), papaverine (Group P), and nitroprusside (Group N). Each segment was examined with both hematoxylin-eosin and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method in order to determine and quantify apoptosis. Results: Apoptotic cells were counted in 50 microscopic areas of each segment. No significant difference was observed among the four groups according to hematoxylin-eosin staining. However, the TUNEL method revealed a significant increase in mean apoptotic cells in the diltiazem group when compared with the other three groups (Group S=4.25±1.4; Group D=13.31±2.8; Group N=9.48±2.09; Group P=10.75±2.37). The differences between groups were significant (P=0.0001). No difference was observed between the samples of the diabetic and non-diabetic patients in any of the study groups. Conclusion: The benefit of topically applied vasodilator drugs must outweigh the potential adverse effects. In terms of apoptosis, diltiazem was found to have the most deleterious effects on internal thoracic artery graft segments. Of the analyzed medical agents, nitroprusside was found to have the least apoptotic activity, followed by papaverine. Diabetes did not have significant effect on the occurrence of apoptosis in left internal thoracic artery grafts.
Assuntos
Humanos , Papaverina/uso terapêutico , Vasodilatadores/uso terapêutico , Nitroprussiato/uso terapêutico , Diltiazem/uso terapêutico , Artéria Torácica Interna , Papaverina/farmacologia , Vasodilatadores/farmacologia , Nitroprussiato/farmacologia , Diltiazem/farmacologiaRESUMO
Approximately 90% of bladder carcinomas are of the urothelial carcinoma type, which are characterized by high rates of recurrence and predisposition to progress to invasive tumors, representing one of the most costly neoplasms for health systems. Intravesical chemotherapy is a standard for the treatment of non-invasive bladder cancer. However, chemotherapy is usually aggressive and cytotoxic, which increases the death rates caused by cancer. Heterocyclic compounds which exhibit favorable pharmacokinetic and pharmacodynamic properties may enhance drug affinity for a target protein by targeting the treatment. Thus, this work presents the synthesis, characterization, and in vitro biological evaluation of new antioxidant (inhibition of lipid peroxidation, scavenging of free radical DPPH, and thiol peroxidase-like activity) and antiproliferative chalcogenobiotin derivatives and tests them against bladder carcinoma 5637 cells. A prominent response was obtained for the selected compounds, with tellurium biotin derivatives displaying effective antioxidant and antiproliferative activity. The effective compounds also demonstrated no toxicity in in vitro or in vivo studies.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Calcogênios/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Bexiga Urinária/efeitos dos fármacos , Antineoplásicos/síntese química , Antineoplásicos/química , Antioxidantes/síntese química , Antioxidantes/química , Compostos de Bifenilo/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Calcogênios/síntese química , Calcogênios/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Estrutura Molecular , Picratos/antagonistas & inibidores , Relação Estrutura-Atividade , Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/patologiaRESUMO
Antibodies against the HIV-1 2F5 epitope are known as one of the most powerful and broadly protective anti-HIV antibodies. Therefore, vaccine strategies that include the 2F5 epitope in their formulation require a robust method to detect specific anti-2F5 antibody production by B cells. Towards this goal, we have biotinylated a previously reported computer-designed protein carrying the HIV-1 2F5 epitope aiming the further development of a platform to detect human B-cells expressing anti-2F5 antibodies through flow cytometry. Biophysical and immunological properties of our devised protein were characterized by computer simulation and experimental methods. Biotinylation did not affect folding and improved protein stability and solubility. The biotinylated protein exhibited similar binding affinity trends compared to its unbiotinylated counterpart and was recognized by anti-HIV-1 2F5 antibodies expressed on the surface of patient-derived peripheral blood mononuclear cells. Moreover, we present a high affinity marker for the identification of epitope-specific B cells that can be used to measure the efficacy of vaccine strategies based on the HIV-1 envelope protein.
Assuntos
Vacinas contra a AIDS/imunologia , Linfócitos B/metabolismo , Biotinilação , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Leucócitos Mononucleares/metabolismo , Simulação de Dinâmica Molecular , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Simulação por Computador , Epitopos/imunologia , HumanosRESUMO
The high incidence of cancer, necessity of treatment, and prognosis times are urgent issues that need to be addressed. In this work, we present DPPC liposomes coated with F127 triblock copolymers as a promising alternative in drug delivery systems for cancer therapy. The proposed mixed liposomes exhibit adequate size, high stability, and passive targeting that result from the EPR effect. An interesting strategy to obtain both passive and active targeting is the vectorization with a covalent bond between F127 and Biotin (a vitamin). Cancer cells can overexpress Biotin receptors, such as Avidin. Here, we evaluate the cytotoxic effects of the erythrosine-decyl ester (ERYDEC). This is a photosensitizer that can be utilized in photodynamic therapy (PDT) and incorporated in DPPC liposomes coated with F127 (F127/DPPC) and the biotinylated-F127 (F127-B/DPPC). The results showed that DPPC liposomes were efficiently mixed with common F127 or F127B, exhibiting adequate physical properties with simple and low-cost preparation. An HABA/Avidin assay showed the amount of Biotin available at the liposome surface. In addition, ERYDEC interaction with lipid vesicles showed high encapsulating efficiency and slow release kinetics. The ERYDEC monomeric species are represented by high light absorption and high singlet oxygen generation (1O2), which confirm the presence of the drug in its monomeric state, as required for PDT. The ERYDEC/liposome system showed high stability and absence of significant cytotoxic effects (absence of light) in fibroblasts of the Mus musculus cell line. In addition, phototoxicity studies showed that ERYDEC/liposomes were able to inhibit cancer cells. However, in the biotinylated system, the effect was much greater than the common F127 coating. This dramatically decreased the inhibitory concentration of CC50 and CC90. In addition, cellular uptake studies based on fluorescence properties of ERYDEC showed that a two-hour incubation period was enough for the uptake by the cell. Therefore, the new vectorized-coated liposome is a potential system for use in cancer treatments, considering that it is a theranostic platform.
Assuntos
Biotina/química , Liberação Controlada de Fármacos , Fármacos Fotossensibilizantes/farmacologia , Animais , Biotinilação , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Eritrosina/farmacologia , Humanos , Hidrodinâmica , Lipossomos , Tamanho da Partícula , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Poloxâmero/química , Espectrofotometria UltravioletaRESUMO
Biotinidase deficiency is an autosomal recessive inherited metabolic disorder caused by mutations in the BTD gene. Clinical manifestations can be treated and effectively prevented with pharmacological doses of biotin. Nine novel mutations in BTD are reported in 14 children diagnosed by the newborn screening program in Minas Gerais, Brazil, from June 2013 to December 2017. Serum BTD enzyme activity was determined for all cases and some parents. Two of the mutations are deletions and seven missense mutations located in the exonic region of the BTD gene, mostly in exon 4. Two newborns were profoundly biotinidase-deficient (one homozygous p.A534V [c.1601C > T] and another, double heterozygous for a novel mutation p.R211S [c.631C > A] co-inherited with an already described mutation p.T532 M [c.1595C > T]). Two mutations were associated with a partial deficiency of biotinidase (p.F361 V [c.1081 T > G] in two homozygous children, and p.S311 T [c.932G > C] in a compound heterozygous child who co-inherited a known severe mutation p.Y438X [c.1314 T > A]). The remaining five mutations were found in compound heterozygous children. Hence, a definitive conclusion about the degree of biotinidase deficiency is not possible yet. These results emphasize the importance of sequencing the BTD gene as an important tool to gain a better understanding of the correlation between biochemical phenotype and genotype.
Assuntos
Alelos , Deficiência de Biotinidase/diagnóstico , Deficiência de Biotinidase/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Deficiência de Biotinidase/epidemiologia , Brasil/epidemiologia , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética/métodos , Humanos , Recém-Nascido , Masculino , Triagem Neonatal , FenótipoRESUMO
Biotin is a water-soluble vitamin that belongs to the vitamin B complex and which is an essential nutrient of all living organisms from bacteria to man. In eukaryotic cells biotin functions as a prosthetic group of enzymes, collectively known as biotin-dependent carboxylases that catalyze key reactions in gluconeogenesis, fatty acid synthesis, and amino acid catabolism. Enzyme-bound biotin acts as a vector to transfer a carboxyl group between donor and acceptor molecules during carboxylation reactions. In recent years, evidence has mounted that biotin also regulates gene expression through a mechanism beyond its role as a prosthetic group of carboxylases. These activities may offer a mechanistic background to a developing literature on the action of biotin in neurological disorders. This review summarizes the role of biotin in activating carboxylases and proposed mechanisms associated with a role in gene expression and in ameliorating neurological disease.
Assuntos
Biotina/metabolismo , Deficiência de Biotinidase/enzimologia , Biotinidase/metabolismo , Carbono-Carbono Ligases/metabolismo , Aminoácidos/metabolismo , Biotina/deficiência , Deficiência de Biotinidase/genética , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Deficiência Múltipla de Carboxilase/genética , Deficiência Múltipla de Carboxilase/metabolismoRESUMO
Several studies have shown that pharmacological concentrations of biotin decrease serum lipid concentrations and the expression of lipogenic genes. Previous studies on epididymal adipose tissue in mice revealed that 8 weeks of dietary biotin supplementation increased the protein abundance of the active form of AMPK and the inactive forms acetyl CoA carboxylase (ACC)-1 and - 2, and decreased serum free fatty acid concentrations but did not affect lipolysis. These data suggest that pharmacological concentrations of the vitamin might affect fatty acid metabolism. In this work, we investigated the effects of pharmacological biotin concentrations on fatty acid synthesis, oxidation, and uptake in 3T3-L1 adipocytes. Similar to observations in mice, biotin-supplemented 3T3-L1 adipose cells increased the protein abundance of active T172 -AMPK and inactive ACC-1 and -2 forms. No changes were observed in the expression of the transcriptional factor PPARα and carnitine-palmitoyltransferase-1 (CPT-1). Radiolabeled assays indicated a decrease in fatty acid synthesis; an increase in fatty acid oxidation and fatty acid incorporation rate into the lipid fraction between control cells and biotin-supplemented cells. The data revealed an increase in the mRNA abundance of the fatty acid transport proteins Fatp1 and Acsl1 but not Cd36 or Fatp4 mRNA. Furthermore, the abundance of glycerol phosphate acyl transferase-3 protein was increased. Triglyceride content was not affected. Lipid droplet numbers showed an increase and their areas were smaller in the biotin-supplemented group. In conclusion, these data indicate that biotin supplementation causes a decrease in fatty acid synthesis and an increase in its oxidation and uptake. © 2018 BioFactors, 45(2):259-270, 2019.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Biotina/farmacologia , Ácidos Graxos/metabolismo , Células 3T3-L1 , Acetil-CoA Carboxilase/metabolismo , Adipogenia/efeitos dos fármacos , Tecido Adiposo/citologia , Animais , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Camundongos , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Group A rotavirus is the most common cause of acute diarrhea in young children worldwide. A simple and rapid enzyme immunoassay (EIA) has been commonly used to detect rotavirus infection and evaluate rotavirus vaccines. Currently licensed commercial EIA kits have low sensitivity. A more sensitive detection of rotavirus can improve rotavirus diagnostics and vaccine efficacy studies. OBJECTIVE: A biotin-avidin based sandwich EIA was developed and compared with commercial EIA kits for improved detection of viral shedding in fecal samples from infants who received human rotavirus vaccine Rotarix in Mexico. STUDY DESIGN: A monoclonal antibody (mAb: 1D4) specific to human rotavirus group antigen VP6 was prepared and used to develop a biotin-avidin based sandwich EIA. This EIA was employed to test 128 fecal samples from vaccinated infants, in comparison with two commercial EIA kits using RT-PCR as a reference. RESULTS: A new biotin-avidin based sandwich EIA showed specific reaction to group A rotaviruses, but not to other enteric viruses. This new EIA had a detection rate of 36.7% for rotavirus antigen shedding in fecal specimens, which was two times higher (16.4%, 18.0%) than those from two commercial EIA kits. CONCLUSION: The new EIA had specificity and higher sensitivity than commercial kits. This new EIA has the potential to detect rotavirus at lower concentration in clinical specimens and thus should be further evaluated as a more sensitive kit for use in diagnostics and vaccine efficacy and effectiveness studies.