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1.
3 Biotech ; 11(2): 49, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33457173

RESUMO

This study presents nanoparticle-based vaccine development for Hepatitis E virus (HEV). Gold nanoparticles (GNP) of average size 12 nm were synthesized by citrate reduction method followed by functionalization with cysteamine hydrochloride for nano-conjugation. Immune response of nano-conjugates of GNP with 26 kDa protein (368-606 amino acids) and 54 kDa protein (112-606 amino acids) were evaluated. In vitro release kinetics of GNP-conjugated 54 kDa (GNP54) and 26 kDa (GNP26) proteins showed slower rate of release of 54 kDa protein as compared to 26 kDa protein. Humoral immune response of mice immunized intramuscularly with GNP54, GNP26 and GNP alone, exhibited HEV-specific IgG titer of 7.9 ± 2.9, 5.686 ± 4.098 and 0.698 ± 0.089, respectively, after 14 days of booster immunization. In addition to this, HEV-specific cell-mediated immune response was demonstrated by splenocyte proliferation assay. Analysis of results using one-way ANOVA, showed statistically significant (p value < 0.05) increase in splenocyte proliferation for GNP54- and GNP26-immunized mice in comparison to GNP alone immunized mice. Stimulation index of HEV ORF2 proteins in GNP54/GNP26-immunized mice were comparable to Concanavalin A-treated positive control. These results indicate GNP-based vaccine as a promising candidate for efficiently mediating both humoral and cell-mediated immune response against HEV.

2.
J Nanosci Nanotechnol ; 20(10): 6057-6062, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32384952

RESUMO

We report a nanoparticles based electrochemical immunosensor to detect and quantify triiodothyronine (T3) hormone. Immunosensor developed using gold nanoparticles and anti-T3 antibody, was employed for quantification of T3 antigen using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) technique. The electrochemical response of the developed immunosensor correlates well with the amount of antigen present in the sample. With increase in antigen concentration the immunocomplex formation on electrode surface increases and hence redox current decreases. The immunosensor shows a lower limit of detection of 1 pg/mL and dynamic range from 1 to 500 pg/mL. Sensitivity of the immunosensor was found to be 29.81 µA/pg/mL/cm².


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Técnicas Eletroquímicas , Eletrodos , Ouro , Hormônios , Imunoensaio , Limite de Detecção
3.
Int J Biol Macromol ; 151: 1240-1249, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31751684

RESUMO

The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity.


Assuntos
Antibacterianos/química , Klebsiella pneumoniae/enzimologia , Serina O-Acetiltransferase/química , Regulação Alostérica/efeitos dos fármacos , Antibacterianos/farmacologia , Clonagem Molecular , Estabilidade Enzimática , Expressão Gênica , Humanos , Cinética , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/genética , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Desnaturação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Serina O-Acetiltransferase/antagonistas & inibidores , Serina O-Acetiltransferase/genética , Serina O-Acetiltransferase/isolamento & purificação , Relação Estrutura-Atividade
4.
Sci Rep ; 9(1): 7866, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133658

RESUMO

The upsurge of drug resistant tuberculosis is major health threat globally. To counteract, antimicrobial peptides are being explored as possible alternatives. However, certain limitations of peptide-based drugs such as potential toxicity, high cost and relatively low stability need to be addressed to enhance their clinical applicability. Use of computer predicted short active motifs of AMPs along with nanotechnology could not only overcome the limitations of AMPs but also potentiate their antimicrobial activity. Therefore, present study was proposed to in silico identify short antimicrobial motif (Pep-H) of human neutrophil peptide-1 (HNP-1) and explore its antimycobacterial activity in free form and using nanoparticles-based delivery systems. Based on colony forming unit analysis, motif Pep-H led to killing of more than 90% M. tb in vitro at 10 µg/ml, whereas, similar activity against intracellularly growing M. tb was observed at 5 µg/ml only. Thereafter, chitosan (244 nm) and gold nanoparticles (20 nm) were prepared for Pep-H with both the formulations showing minimal effects on the viability of human monocyte derived macrophages (MDMs) and RBC integrity. The antimycobacterial activity of Pep-H against intracellular mycobacteria was enhanced in both the nanoformulations as evident by significant reduction in CFU (>90%) at 5-10 times lower concentrations than that observed for free Pep-H. Thus, Pep-H is an effective antimycobacterial motif of HNP-1 and its activity is further enhanced by chitosan and gold nanoformulations.


Assuntos
Antituberculosos/farmacologia , Quitosana/química , Portadores de Fármacos/química , Ouro/química , Mycobacterium tuberculosis/efeitos dos fármacos , alfa-Defensinas/farmacologia , Antituberculosos/química , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Tuberculose/tratamento farmacológico , alfa-Defensinas/química
5.
3 Biotech ; 8(10): 414, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30237961

RESUMO

Hepatitis E virus infection is responsible for acute viral hepatitis and associated with high mortality and still birth in pregnant women in developing countries. We report expression of truncated forms of HEV ORF2 as potential vaccine candidates for nanoparticle-based delivery. These two truncated ORF2 proteins (54 kDa and 26 kDa) have been reported to be highly immunogenic and can be used as nanoparticle-based vaccine candidate. The bacterial expressed protein was purified by affinity chromatography and further confirmed by western blot using anti-HEV antibody. The chitosan nanoemulsion was synthesized using ultrasonic waves. The nanoparticle size was found to be 120-160 nm and the entrapment efficiency of purified truncated ORF2 proteins within these nanoparticles was 70% (26 kDa) and 59% (54 kDa). In cell cytotoxicity analysis, 100 µg/mL nanoemulsion was found suitable for cell viability in both HeLa and THP1 cell lines. Release kinetics of encapsulated proteins at physiological pH 7.4 showed 26-59% and 9.7-40% release of 26 kDa and 54 kDa protein within 1 h that gradually increased with time (48 h). Encapsulated proteins were found to be unstable at pH 1.2.

6.
Biosens Bioelectron ; 106: 78-85, 2018 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-29414093

RESUMO

Assesment of human epidermal growth factor receptor 2 status is a key factor prompting definitive treatment decisions that help in reducing mortality rates associated with breast cancer. In this article, highly sensitive and low-cost impedimetric immunosensor using single-chain fragment variable antibody fragments was developed for quantitative detection of human epidermal growth factor receptor 2 from serum employing gold nanoparticle-modified disposable screen-printed carbon electrodes. The gold nanoparticles facilitate fast electron transfer and offer a biocompatible surface for immobilization of small antibody fragments in an oriented manner, resulting in improved antigen binding efficiency. The single-chain fragment variable antibody fragment-modified screen printed immunosensor exhibits wide dynamic range of 0.01-100 ng mL-1 and detection limit of 0.01 ng mL-1. The advantages offered by this platform in terms of high sensitivity, broad dynamic range and low-cost demonstrates great potential for improved monitoring of human epidermal growth factor receptor 2 levels for the management of breast and other cancers.


Assuntos
Técnicas Biossensoriais , Neoplasias da Mama/sangue , Imunoensaio , Receptor ErbB-2/isolamento & purificação , Anticorpos Imobilizados/imunologia , Carbono/química , Feminino , Ouro , Humanos , Nanopartículas Metálicas/química , Receptor ErbB-2/sangue
7.
Mol Biosyst ; 12(11): 3407-3416, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27714060

RESUMO

Polycystic ovary syndrome (PCOS) is one of the most commonly occurring metabolic and endocrinological disorders affecting women of reproductive age. Metabolomics is an emerging field that holds promise in understanding disease pathophysiology. Recently, a few metabolomics based studies have been attempted in PCOS patients; however, none of them have included patients from the Indian population. The main objective of this study was to investigate the serum metabolomic profile of Indian women with PCOS and compare them with controls. Proton nuclear magnetic resonance (1H NMR) was used to first identify the differentially expressed metabolites among women with PCOS from the Eastern region of India during the discovery phase and further validated in a separate cohort of PCOS and control subjects. Multivariate analysis of the binned spectra indicated 16 dysregulated bins in the sera of these women with PCOS. Out of these 16 bins, 13 identified bins corresponded to 12 metabolites including 8 amino acids and 4 energy metabolites. Amongst the amino acids, alanine, valine, leucine and threonine and amongst the energy metabolites, lactate and acetate were observed to be significantly up-regulated in women with PCOS when compared with controls. The remaining 4 amino acids, l-glutamine, proline, glutamate and histidine were down-regulated along with 2 energy metabolites: glucose and 3-hydroxybutyric acid. Our findings showed dysregulations in the expression of different metabolites in the serum of women with PCOS suggesting the involvement of multiple pathways including amino acid metabolism, carbohydrate/lipid metabolism, purine and pyrimidine metabolism and protein synthesis.


Assuntos
Metaboloma , Metabolômica , Reconhecimento Automatizado de Padrão , Síndrome do Ovário Policístico/sangue , Espectroscopia de Prótons por Ressonância Magnética , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Índia , Redes e Vias Metabólicas , Metabolômica/métodos , Reprodutibilidade dos Testes , Adulto Jovem
8.
Enzyme Microb Technol ; 89: 15-30, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27233124

RESUMO

This review focuses on the technological advancements, challenges and trends in immunoassay technologies for ovarian cancer diagnosis. Emphasis is placed on the principles of the technologies, their merits and limitations and on the evolution from laboratory-based methods to point-of-care devices. While the current market is predominantly associated with clinical immunoassay kits, over the last decade a major thrust in development of immunosensors is evident due to their potential in point-of-care devices. Technological advancements in immunosensors, extending from labeled to label-free detection, with and without mediators, for enhancing proficiencies and reliability have been dealt with in detail. Aspects of the utilisation of nanomaterials and immobilization strategies for enhancing sensitivity and altering the detection range have also been addressed. Finally, we have discussed some distinct characteristics and limitations associated with the recently commericalised technologies used for quantitation of relevant ovarian cancer markers.


Assuntos
Neoplasias Ovarianas/diagnóstico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/imunologia , Técnicas Biossensoriais/métodos , Feminino , Humanos , Imunoensaio/métodos , Nanoestruturas , Nanotecnologia , Neoplasias Ovarianas/imunologia
9.
Hum Reprod ; 31(6): 1265-74, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27060172

RESUMO

STUDY QUESTION: Is there any difference at the serum metabolic level between women with recurrent implantation failure (RIF) and women with recurrent implantation success (RIS) when undergoing in vitro fertilization (IVF)? SUMMARY ANSWER: Eight metabolites, including valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea, were found to be significantly up-regulated in women with RIF when compared with women with RIS. WHAT IS KNOWN ALREADY: Despite transfer of three high-grade embryos per cycle, RIF following three or more consecutive IVF attempts occurs in a group of infertile women. Conversely, there is a group of women who undergo successful implantation each cycle, yet have a poor obstetric history. STUDY DESIGN, SIZE, DURATION: This study was conducted over a period of 10 years (January 2004-October 2014). Groups of 28 women with RIF (age ≤40 years and BMI ≤28) and 24 women with RIS (age and BMI matched) were selected from couples with primary infertility reporting at the Institute of Reproductive Medicine, Kolkata, India. Women recruited in the RIF group had history of implantation failure in at least three consecutive IVF attempts, in which three embryos of high-grade quality were transferred in each cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blood samples were collected from both the groups during the implantation window following overnight fasting for at least 10 h (7-10 days post ovulation). Samples were analyzed using a 700 MHz NMR spectrometer and acquired spectra were subjected to chemometric and statistical analysis. Serum levels of endothelial nitric oxide synthase (eNOS) were measured using an enzyme immunoassay technique. MAIN RESULTS AND THE ROLE OF CHANCE: Valine, adipic acid, l-lysine, creatine, ornithine, glycerol, d-glucose and urea were found to be significantly down-regulated in women with RIS when compared with those with RIF, with fold change values of 0.81, 0.82, 0.79, 0.80, 0.78, 0.68, 0.76 and 0.74, respectively. Further, serum eNOS was found to be significantly lower in women with RIF when compared with RIS (P < 0.05), indicating possible impairment in nitric oxide production. Metabolites, mostly related to energy metabolism, lipid metabolism and the arginine metabolic pathway were found to be considerably altered and are likely to be associated with the RIF phenomenon. However, the interplay between these molecules in RIF is complex and holds merit for further exploration. LIMITATIONS, REASONS FOR CAUTION: In-depth studies of the arginine metabolic pathway in endometrial tissues seem necessary to validate our findings. A limitation of the present study is that the metabolic level changes, eNOS and nitric oxide levels have not been investigated in the endometrial tissues of the two groups of women. It would be interesting to investigate whether there exists a direct link between metabolic dysregulation and genetic factors that affects implantation in RIF women. WIDER IMPLICATIONS OF THE FINDINGS: We speculate that tissue metabolomics can provide an improved understanding of the metabolic dysfunction associated with RIF. The identification of serum metabolic marker(s) in women with RIS may help with strategies of early therapeutic intervention, which may improve the chances of implantation significantly in women otherwise susceptible to IVF failure. STUDY FUNDING/COMPETING INTERESTS: One of the authors, S.R.C. acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India [No: 9/81(1228)/14, EMR-I] for financial support.


Assuntos
Implantação do Embrião , Metabolômica , Adulto , Transferência Embrionária , Feminino , Fertilização In Vitro , Humanos , Análise Multivariada , Óxido Nítrico Sintase Tipo III/sangue , Resultado do Tratamento
10.
Biosens Bioelectron ; 78: 396-403, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26655179

RESUMO

This paper addresses the question - Is EDC-NHS activated gold nanoparticles modified electrode surface the best available option for antibody immobilization for immunosensor fabrication? Is there any other alternative covalent immobilization strategy for orthogonal orientation of antibody, ensuring enhanced sensitivity of immunosensors? Does EDC-NHS activation of carboxyl functionalized nanoparticles surface really leads to orthogonal or directed immobilization of antibody? Gold nanoparticles synthesized using L-Asparagine as reducing and stabilization agent were employed for orthogonal immobilization of antibody for immunosensor fabrication. Anti-CA125 antibody was used as a model system for immunosensor fabrication. A comparative evaluation of immunosensors fabricated using L-Asparagine stabilized gold nanoparticles and citrate stabilized gold nanoparticles via different immobilization strategies/chemistries was done. The three strategies involved immobilization of Anti-CA125 antibody - (1) after EDC-NHS activation of citrate stabilized gold nanoparticles, (2) directly onto citrate stabilized gold nanoparticles and (3) directly onto L-Asparagine stabilized gold nanoparticles modified electrode surfaces. Comparative evaluation of Impedimetric response characteristics showed 2.5 times increase in sensitivity (349.36 Ω/(IU/mL)/cm(2)) in case of third strategy as compared to first (147.53 Ω/(IU/mL)/cm(2)) and twice that of second strategy (166.24 Ω/(IU/mL)/cm(2)). Additionally, an extended dynamic range of 0-750 IU/mL was observed while for others it was up to 500 IU/mL. Amino acid coated gold nanoparticles ensured orthogonal immobilization, lesser randomization, with 88% of active antibody available for antigen binding as opposed to other two strategies with less than 30% active antibody.


Assuntos
Anticorpos Imobilizados/química , Asparagina/química , Técnicas Biossensoriais , Antígeno Ca-125/isolamento & purificação , Proteínas de Membrana/isolamento & purificação , Antígeno Ca-125/química , Ácido Cítrico/química , Ouro/química , Proteínas de Membrana/química , Nanopartículas Metálicas/química
11.
J Assist Reprod Genet ; 31(9): 1195-204, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24965760

RESUMO

PURPOSE: The aim of this study was to analyze the seminal plasma of patients with idiopathic/male factor infertility and healthy controls with proven fertility by NMR spectroscopy, with a hope of establishing difference in biomarker profiles, if any, between the groups. METHODS: A total of 103 subjects visiting the infertility clinic of Manipal University with normozoospermic parameters, oligozoospermia, asthenozoospermia, azoospermia and teratozoospermia were included. Semen characteristics were analysed by standard criteria. Seminal plasma was subjected to NMR spectroscopy at a 700 MHz (1)H frequency. The resultant data was analyzed by appropriate software. RESULTS: The analysis revealed significant differences between the fertile control group and other forms of male infertility. Interestingly, seminal plasma profile of the idiopathic infertility group showed distinct segregation from the control population as well as other infertile groups. The difference in biomarker profiles between the idiopathic infertility and the rest of the groups combined could originate from either the up-regulation or down regulation of a several compounds, including lysine, arginine, tyrosine, citrate, proline and fructose. CONCLUSION: Our data suggests the presence of a metabolic reason behind the origin of idiopathic infertility. (1)H NMR based metabonomic profiling based on concentration of biomarker lysine has the potential to aid in the detection and diagnosis of idiopathic infertility in an efficient manner.


Assuntos
Infertilidade Masculina/metabolismo , Adulto , Biomarcadores/metabolismo , Humanos , Masculino , Metabolômica , Análise Multivariada , Ressonância Magnética Nuclear Biomolecular , Análise do Sêmen/métodos
12.
Eur J Med Chem ; 80: 285-94, 2014 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-24793879

RESUMO

The localization and interaction of six naturally occurring flavones (FLV, 5HF, 6HF, 7HF, CHY and BLN) in DPPC bilayers were studied using DSC and multi-nuclear NMR. DSC results indicate that FLV and 6HF interact with alkyl chains. The (1)H NMR shows interaction of flavones with the sn-glycero region. Ring current induced chemical shifts indicate that 6HF and BLN acquire parallel orientation in bilayers. 2D NOESY spectra indicate partitioning of the B-ring into the alkyl chain region. The DSC, NMR and binding studies indicate that 5HF and 7HF are located near head group region, while 6HF, CHY and BLN are located in the vicinity of sn-glycero region, and FLV is inserted deepest in the membrane.


Assuntos
Membrana Celular/metabolismo , Flavonas/metabolismo , Flavonas/farmacologia , Sequestradores de Radicais Livres/metabolismo , Sequestradores de Radicais Livres/farmacologia , Bicamadas Lipídicas/metabolismo , Compostos de Bifenilo/química , Varredura Diferencial de Calorimetria , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Flavonas/química , Sequestradores de Radicais Livres/química , Humanos , Hidróxidos/química , Espectroscopia de Ressonância Magnética , Picratos/química , Relação Estrutura-Atividade
13.
J Proteome Res ; 13(6): 3100-6, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24738780

RESUMO

In an attempt to find out the association of metabolic dysregulation with poor endometrial receptivity and pregnancy loss, serum metabonomic profiling of women with idiopathic recurrent spontaneous miscarriage (IRSM) is carried out and compared with fertile controls. (1)H nuclear magnetic resonance (NMR)-based metabonomics was used to obtain serum metabolic profiles of 36 women with IRSM and 28 proven fertile women during the window of implantation. The acquired data were analyzed using multivariate principal component analysis, partial least-squares-discriminant analysis, and orthogonal projection to latent structure with discriminant analysis. A clear metabolic differentiation was evident between IRSM and control samples. The distinguishing metabolites, l-lysine, l-arginine, l-glutamine, l-histidine, l-threonine, l-phenylalanine, and l-tyrosine are significantly up-regulated in IRSM as compared to controls. These altered metabolites may be involved in the molecular mechanism of exaggerated inflammatory response and vascular dysfunction associated with poor endometrial receptivity in women with IRSM. The present work proposes a vital association of metabolic dysfunction with the disease pathogenesis.


Assuntos
Aborto Habitual/sangue , Metaboloma , Aminoácidos/sangue , Estudos de Casos e Controles , Implantação do Embrião , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Metabolômica , Gravidez , Análise de Componente Principal
14.
Eur J Med Chem ; 79: 102-9, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24727463

RESUMO

Flavonols are an important class of naturally occurring molecules and are known for their pharmacological activity. The activity is associated with the ability of flavonols to influence membrane-dependent processes. We have investigated the in-vitro anti-proliferative and anti-oxidant activity of galangin (GLN), fisetin (FTN) and quercetin (QTN), which possess variable number of phenolic hydroxyl groups. An attempt has been made to correlate the biological activity of these molecules with their interaction and localization in dipalmitoyl phosphatidyl choline (DPPC) bilayers, using differential dcanning calorimetry (DSC) and nuclear magnetic resonance (NMR) methods. Results indicate that GLN interacts to the alkyl chains of the lipid bilayer involving hydrophobic interactions. FTN and QTN interact with head region and sn-1-glycero region involving hydrogen bonding. Ring current induced chemical shifts of lipid protons, due to intermolecular interaction indicate that GLN acquires a parallel orientation with respect to the bilayer normal whereas FTN and QTN resume a mixed orientation. The membrane binding constants of these molecules are in the order GLN > QTN > FTN. It has been shown that the number and position of hydroxyl groups in these molecules play an important role in membrane binding and thereby in biological activity.


Assuntos
Antioxidantes/farmacologia , Flavonoides/farmacologia , Membranas Artificiais , Quercetina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Antioxidantes/síntese química , Antioxidantes/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Flavonoides/síntese química , Flavonoides/química , Células Hep G2 , Humanos , Células K562 , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Quercetina/síntese química , Quercetina/química , Relação Estrutura-Atividade
15.
Bioorg Med Chem ; 21(21): 6753-62, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24012381

RESUMO

The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer's structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time (1)H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.


Assuntos
Antifúngicos/síntese química , Bicamadas Lipídicas/química , Pirimidinas/química , Triazóis/química , 1,2-Dipalmitoilfosfatidilcolina/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Varredura Diferencial de Calorimetria , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Ácidos Carboxílicos/química , Ésteres , Éteres de Hidroxibenzoatos/síntese química , Éteres de Hidroxibenzoatos/química , Éteres de Hidroxibenzoatos/farmacologia , Bicamadas Lipídicas/metabolismo , Espectroscopia de Ressonância Magnética , Metaboloma , Testes de Sensibilidade Microbiana , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Temperatura de Transição , Triazóis/metabolismo , Triazóis/farmacologia , Voriconazol
16.
Biomed Res Int ; 2013: 329058, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23984345

RESUMO

Accumulating evidence indicates the active role of oxidative stress in the development of endometriosis; however, the mechanism of reactive oxygen species generation is poorly understood. Metabonomics/metabolomics is a scientific discipline that can be used to study changes in metabolite ensembles associated with disease pathophysiology. The present study focuses on the use of proton nuclear magnetic resonance spectroscopy based targeted metabolite profiling approach to explore dysregulation in metabolites expression in women with endometriosis. Further, association of oxidative stress with the metabolite ensembles, if any, is investigated. Using multivariate statistics, partial least square discriminant analysis model was generated which could classify endometriosis patients with sensitivity and specificity of 92.83% and 100%, respectively, and with a classification rate of 96.4%. In conjunction with increased glucose metabolism, citrate and succinate were found to be elevated in endometriosis patients. Higher levels of reactive oxygen species, lipid peroxidation, and advanced oxidation protein products and lower levels of total antioxidant capacity, superoxide dismutase, catalase, and glutathione were also observed. Increased glucose metabolism and defects in the mitochondrial respiratory system are suggested to be the possible sources of excessive reactive oxygen species generation in endometriosis.


Assuntos
Endometriose/metabolismo , Endometriose/patologia , Espectroscopia de Ressonância Magnética , Metabolômica , Estresse Oxidativo , Prótons , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Análise de Componente Principal , Adulto Jovem
17.
Biosens Bioelectron ; 50: 174-9, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23850785

RESUMO

Amalgamation of nanotechnology and biology has opened new horizons for controlled synthesis of nanomaterials of nano and micro-lengthscales for diverse sensing, catalytic and electromechanical applications. Inspired from nature and driven by the need to have nanostructures of desired morphology, microbial architecture has been exploited as a template in the present work. Biocompatible 1-D gold microwires, generated by assembly of amino acid functionalized AuNPs over the proliferating fungal hyphae, served as potential microelectrodes for electron transfer between enzyme and electrode surface. Delocalization of electrons over longer length scales, large surface area provided by assembled AuNPs and high biocompatibility yielded excellent analytical performance characteristics with high sensitivity of 43.2 µA/mM/cm(2) with standard deviation of 0.88% and wide linear range from 5 µM to 20 mM of glucose. The gold microwires thus generated demonstrate appreciable repeatability over 20 cycles in a cyclic voltammogram, and reproducibility with root mean square deviation as low as 1.3%. High stability and biocompatibility attribute these microwires with myriad potential biosensing and catalytic applications in varied domains.


Assuntos
Técnicas Biossensoriais/instrumentação , Fungos/fisiologia , Glucose/análise , Ouro/química , Eletrodos , Fungos/ultraestrutura , Limite de Detecção , Microeletrodos
18.
Mol Biosyst ; 8(12): 3281-7, 2012 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-23079773

RESUMO

Our present study focuses on the identification of predictive biomarkers in serum for the early diagnosis of endometriosis in a minimally invasive manner using (1)H-NMR based metabonomics. PLS-DA modeling of bins obtained from CPMG spectra of serum samples discriminated endometriosis patients from controls with sensitivity and specificity levels of about 80% and 90%, respectively. Compared with those from controls, serum samples from endometriosis patients showed increased levels of lactate, 3-hydroxybutyrate, alanine, leucine, valine, threonine, lysine, glycerophosphatidylcholine, succinic acid and 2-hydroxybutyrate as well as decreased levels of lipids, glucose, isoleucine and arginine. Our work offers valuable information for non-invasive diagnosis of endometriosis and may be of potential benefit to understand pathogenesis of the disease.


Assuntos
Aminoácidos/sangue , Proteínas Sanguíneas/análise , Endometriose/sangue , Endometriose/diagnóstico , Lipídeos/sangue , Metabolômica , Biomarcadores/sangue , Diagnóstico Precoce , Endometriose/metabolismo , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Sensibilidade e Especificidade
19.
Biosens Bioelectron ; 37(1): 30-7, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22608576

RESUMO

Present study concerns modulating the electron transfer properties of gold nanoparticles through amino acid induced coupling among them. In addition to conductivity, the amino functionalization of the nanoparticles results in enhanced activity and operational stability of the biosensor fabricated using the same. Nanoparticles synthesized using amino acid as reducing agent (average diameter-20 nm), incorporate the natural coupling property of amino acids and are seen to align in a chain-like arrangement. The coupling of the individual nanoparticles to form chain like structure was confirmed by both absorption spectroscopy as well as transmission electron microscopy. The glucose biosensor developed by adsorption of glucose oxidase (GOx) enzyme onto these coupled gold nanoparticles showed enhanced efficiency as compared to the one with glucose oxidase immobilized onto gold nanoparticles synthesized using the conventional method (trisodium citrate as reducing agent). The fabricated biosensor demonstrated a wide linear concentration range from 1 µM-5mM and a high sensitivity of 47.2 µA mM(-1) cm(-2). Also, an enhanced selectivity to glucose was observed with negligible interference in the physiological range, from easily oxidizable biospecies, e.g. uric acid and ascorbic acid. Furthermore, the electrochemical biosensor has excellent long term stability- retaining greater than 85% of the biosensor activity up to 60 days.


Assuntos
Técnicas Biossensoriais/métodos , Glucose/análise , Ouro/química , Nanopartículas/química , Serina/química , Aspergillus niger/enzimologia , Transporte de Elétrons , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/química , Glucose Oxidase/metabolismo , Nanopartículas/ultraestrutura , Substâncias Redutoras/química , Sensibilidade e Especificidade
20.
J Pharm Pharmacol ; 64(6): 802-10, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22571258

RESUMO

OBJECTIVES: In search of a novel antifungal agent with high susceptibility and increased antifungal potency it is necessary to increase the overall lipophilicity of these agents. In view of that, we have synthesized different carboxylic acid ester analogues of fluconazole, such as fluconazole-benzoate, fluconazole-p-nitrobenzoate, fluconazole-p-methoxybenzoate and fluconazole-toluate, with varying degrees of lipophilicity. In order to probe molecular level interactions of these molecules with biomembrane, lipid bilayers prepared from l-α-dipalmitoyl phosphatidyl choline (DPPC) as the model membrane were used. METHODS: Multinuclear and multidimensional nuclear magnetic resonance, differential scanning calorimetry and transmission electron microscopy was used to investigate the changes in the thermotropic properties, organization of the membrane and intermolecular interactions. KEY FINDINGS: Fluconazole and its analogues show varying degrees of changes in the DPPC bilayer's architecture and physico-chemical characteristics. This might influence important biological features of fungal biomembranes that could be responsible for their respective antifungal effects. CONCLUSIONS: The study indicates that fluconazole-p-methoxybenzoate is the most active among all analogues and therefore could be the most promising antifungal candidate.


Assuntos
Antifúngicos/farmacologia , Fluconazol/farmacologia , Bicamadas Lipídicas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Antifúngicos/química , Varredura Diferencial de Calorimetria/métodos , Ácidos Carboxílicos/farmacologia , Ésteres/farmacologia , Fluconazol/análogos & derivados , Fluconazol/química , Interações Hidrofóbicas e Hidrofílicas , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética
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