Marcelo Grijalva

One pot phytosynthesis of gold nanoparticles using Genipa americana fruit extract and its biological applications.

In this article, rapid one pot synthesis of gold nanoparticles (GNPs) using an eco-friendly extract of Genipa americana L. fruit is described. Electrospray ionization mass spectrometry (ESI-MS) and Fourier transform infrared (FTIR) spectroscopic studies demonstrated that small molecules such as genipin, genipaol, geniposide and ranolazine can act as reducer as well as stabilizers. The monodispersed, spherical GNPs were further characterized by UV-vis spectroscopy at λmax=535 nm, transmission electron microscopy (TEM), dynamic light scattering (DLS) and X-ray diffraction (XRD) analysis. This synthetic approach offers a greener and alternate route to the preparation of GNPs free from toxic chemical components and stable for 6-7 months under room temperature. The green synthesized GNPs showed weak antioxidant efficacy against 1,1-diphenyl-2-picrylhydrazyl and no cytotoxicity against A-549 and HeLa human cancer cell lines, from lung and cervix. This study opens a new industrial scope of G. americana fruit in nanoscience and as surface modified GNPs can be developed into a successful drug carrier for future pharmaceutical products.

In vitro evaluation of silver nanoparticles cytotoxicity on Hepatic cancer (Hep-G2) cell line and their antioxidant activity: Green approach for fabrication and application.

In this article, biosynthesis of silver nanoparticles (AgNPs) using Andean Mora (Rubus glaucus Benth.) leaf has been reported. Different analytical techniques including UV-vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used for the characterization of AgNPs. The initial appearance of color change with the intense surface plasmon resonance (SPR) bands around 440-455 in UV-visible spectra revealing the formation of AgNPs. The TEM image showed the AgNPs to be anisotropic, quasi-spherical in shape with sizes in the range of 12-50nm. On the other hand, XRD studies revealed the formation of face-centered cubic structure for AgNPs. The surface modified AgNPs showed no cytotoxicity at the concentration ranging from 0.01μM to 1.0μM on the Hepatic cancer (Hep-G2) cell line and observed antioxidant efficacy >70% at the concentration 0.05mM/0.20mL against 1, 1-diphenyl-2-picrylhydrazyl. From the results obtained it is suggested that AgNPs could be used effectively in future drug delivery systems and other biomedical concerns.

Molecular and Cellular Effects of Hydrogen Peroxide on Human Lung Cancer Cells: Potential Therapeutic Implications

Lung cancer has a very high mortality-to-incidence ratio, representing one of the main causes of cancer mortality worldwide. Therefore, new treatment strategies are urgently needed. Several diseases including lung cancer have been associated with the action of reactive oxygen species (ROS) from which hydrogen peroxide (H2O2) is one of the most studied. Despite the fact that H2O2 may have opposite effects on cell proliferation depending on the concentration and cell type, it triggers several antiproliferative responses. H2O2 produces both nuclear and mitochondrial DNA lesions, increases the expression of cell adhesion molecules, and increases p53 activity and other transcription factors orchestrating cancer cell death. In addition, H2O2 facilitates the endocytosis of oligonucleotides, affects membrane proteins, induces calcium release, and decreases cancer cell migration and invasion. Furthermore, the MAPK pathway and the expression of genes related to inflammation including interleukins, TNF-α, and NF-κB are also affected by H2O2. Herein, we will summarize the main effects of hydrogen peroxide on human lung cancer leading to suggesting it as a potential therapeutic tool to fight this disease. Because of the multimechanistic nature of this molecule, novel therapeutic approaches for lung cancer based on the use of H2O2 may help to decrease the mortality from this malignancy.

Antimicrobial, antioxidant and anticancer activity of biogenic silver nanoparticles – an experimental report

In the present study, use of a Helicteres isora stem bark extract for the biosynthesis of AgNPs is described. It was observed that the aqueous silver (Ag+) ions, once associated in the stem bark extract, were reduced in solution, thereby leading to formation of the stable AgNPs. These AgNPs were characterized using several techniques. The nanoparticles show a maximum absorbance at 419–431 nm in the ultraviolet-visible spectra. The presence of the steroid sapogenin was identified using Fourier transform-infrared (FTIR) spectroscopy. The reduction of the Ag+ ions to elemental silver was investigated by using X-ray diffraction (XRD). Transmission electron microscopy (TEM) revealed the formation of monodisperse, with low polydispersity, nanoparticles of 25.55 nm, and the presence of elemental silver was confirmed through energy dispersive spectroscopy (EDX) analysis. The AgNPs showed antioxidant activities such as DPPH, hydrogen peroxide and nitric oxide radical scavenging and a reducing power compared to the standard compounds. The antibacterial effect was determined against test strains, showing significant inhibition. The antiproliferative activity of the AgNPs was demonstrated using oral carcinoma (KB) cells with MTT, and confirmed using AO/EtBr, comet assay, DCFH-DA and Rhodamine 123 staining. In the toxicity study, a significant mortality rate was observed against Artemia with an IC50 concentration of 70 μg mL−1 and 108 h exposure. The NPs showed as cytotoxic against Artemia at 108 h, so they are cytotoxic at high concentrations and after prolonged exposure.

Peptides conjugated to silver nanoparticles in biomedicine – a “value-added” phenomenon

Nanotechnology is gaining impetus in the present century and particularly the use of nanoparticles (NPs), whose properties are significantly different from the larger matter. These have found wider and potential applications in the fields of medicine, energy, cosmetics, environment and biomedicine. Among the NPs, silver nanoparticles (AgNPs) are of particular interest for scientists and technologists due to their unique physico-chemical and biological properties. Besides, AgNPs by themselves also possess broad-spectrum microbial activity, which has further expanded their application in both academia and industries. On the other hand, research and drug discovery in the field of peptides is surging. Chemistry and biology of peptides have seen a renaissance in this century as many of the peptide-based therapeutics have entered the market and many more are in the different phases of clinical trials. To fuel this, peptides have also found numerous applications in nanotechnology. Taking advantage of these two scenarios, namely, AgNPs and peptides, conjugation of these entities have emerged as a powerful technique and have opened the doors for a new revolution. Keeping this motivation in mind, we here present a mini-review on the combined concept of AgNPs and peptides.

Biological Effect of Organically Coated Grias neuberthii and Persea americana Silver Nanoparticles on HeLa and MCF-7 Cancer Cell Lines

The aim of this study was to assess the biological effect of organically coated Grias neuberthii (piton) fruit and Persea americana (avocado) leaves nanoparticles (NPs) on cervical cancer (HeLa) and breast adenocarcinoma (MCF-7) cells with an emphasis on gene expression (p53 transcription factor and glutathione-S-transferase GST) and cell viability. UV-Vis spectroscopy analysis showed that synthesized AgNPs remained partially stable under cell culture conditions. HeLa cells remained viable when exposed to piton and avocado AgNPs. A statistically significant, dose-dependent cytotoxic response to both AgNPs was found on the breast cancer (MCF-7) cell line at concentrations above 50 µM. While expression levels of transcription factor p53 showed downregulation in treated MCF-7 and HeLa cells, GST expression was not affected in both cell lines treated. Cell viability assays along with gene expression levels in treated MCF-7 cells support a cancer cell population undergoing cell cycle arrest. The selective toxicity of biosynthesized piton/avocado AgNPs on MCF-7 cells might be of value for novel therapeutics.

Genetic diversity of the HLA system in human populations from the Sierra (Andean), Oriente (Amazonian) and Costa (Coastal) regions of Ecuador

We studied HLA class I (HLA-A, -B) and class II (HLA-DRB1, -DQB1) alleles by PCR-SSP based typing in a total of 1101 Ecuadorian individuals from three regions of the country, the Coastal region, the Andean region, and the Amazonian region, to obtain information regarding allelic and haplotypic frequencies and their linkage disequilibrium. We find that the most frequent HLA haplotypes with significant linkage disequilibrium in those populations are HLA-A*24∼B*35∼DRB1*04∼DQB1*03:02, A*02∼B*35∼DRB1*04∼DQB1*03:02, A*24∼B*35∼DRB1*14∼DQB1*03:01, A*02∼B*35∼DRB1*14∼DQB1*03:01 and A*02∼B*40:02∼DRB1*04∼DQB1*03:02. The only non-Native American haplotype with frequency >1% shared by all groups was A*29∼B*44∼DRB1*07∼DQB1*02. Admixture estimates obtained by a maximum likelihood method using HLA-B as genetic estimator revealed that the main genetic components for this sample of mixed-ancestry Ecuadorians are Native American (ranging from 52.86% to 63.83%) and European (from 28.95% to 46.54%), while an African genetic component was only apparent in the Coastal region (18.19%). Our findings provide a starting point for the study of population immunogenetics of Ecuadorian populations.

Analysis of Efflux Pump Genes in β-lactam Resistant Clinical Isolates of Pseudomonas aeruginosa from a Tertiary Level Hospital in Ecuador

espanolPseudomonas aeruginosa es un microorganismo responsable de una amplia variedad de infecciones y es uno de los principales patogenos multiresistentes ante antibioticos β-lactamicos. Uno de los principales mecanismos de resistencia en P. aeruginosa constituyen las bombas de eflujo. El objetivo del presente estudio fue caracterizar el mecanismo de bombas de eflujo mediante el estudio de expresion de 4 genes (mexA, mexX, oprJ y oprM) involucrados en este mecanismo en Pseudomonas aeruginosa. Cuarenta aislados clinicos (20 resistentes y 20 sensibles) fueron recolectados en el Laboratorio de Bacteriologia del Hospital “Carlos Andrade Marin” en Quito-Ecuador. Los niveles de expression de los genes seleccionados fueron evaluados por RT-qPCR usando RpsL como gen constitutivo para el analisis de cuantificacion relativa basado en el metodo ΔΔCt ajustado. La importancia de las bombas de eflujo como mecanismos de resistencia fue confirmada ya que el estudio de expresion de los genes relacionados con bombas de eflujo mostro sobreexpresion de ellos en todos los aislados fenotipicamente resistentes. Se realizo ademas un analisis fenotipo/genotipo comparando los resultados del antibiograma (AST) con los perfiles de expresion. La sobreexpresion de mexA (genotipo) mostro correlacion con el fenotipo de resistencia a TPZ, mientras que el genotipo mexX se correlaciono con los fenotipos de resistencia a IPM, MEM y FEP. En conclusion, los patrones de expression de los genes relacionados con bombas de eflujo sugieren la presencia de mecanismos de resistencia basados en transmision horizontal que posibilitan la diseminacion de patogenos en el ambiente hospitalario. EnglishPseudomonas aeruginosa is a nosocomial microorganism that causes a wide spectrum of infections and is known as one of the primary multi-resistant microorganisms against β-lactam antibiotics. One of the main resistance mechanisms found in P. aeruginosa is the efflux pumps. This study is aimed at characterizing this mechanism by analyzing the expression of four genes (mexA, mexX, oprJ and oprM) involved in antibiotic efflux pumps in Pseudomonas aeruginosa. Forty clinical isolates (20 resistant, 20 susceptible) were collected from the Bacteriology Laboratory at the Carlos Andrade Marin Hospital, in Quito-Ecuador. Expression levels for the selected genes were assessed by RT-qPCR assays using RpsL as a housekeeping gene for ΔΔCt adjusted relative quantitation analysis. The importance of efflux pumps as a resistance mechanism was corroborated through analysis of efflux pumps genes that showed overexpression in all phenotypically resistant isolates. Furthermore, phenotype/genotype analysis was performed comparing Antibiotic Susceptibility Testing (AST) results with expression profiles. Results for the mexA genotype showed correlation with the TPZ resistance phenotype and the mexX genotype with the IPM, MEM and FEP resistance phenotypes. In conclusion, the expression pattern of the efflux pump genes suggests resistance mechanisms that are due to horizontal transmission or pathogens spreading into the hospital environment.

AmpC, oprD Expression Analysis in β-lactam Resistant Pseudomonas aeruginosa Clinical Isolates 1 from a Tertiary Level Hospital in Ecuador

espanolLos mecanismos innatos y adquiridos de resistencia a los antibioticos en Pseudomonas representan un reto para los medicos que buscan una quimioterapia oportuna y eficaz. Esto es par- ticularmente importante en las areas de cuidados intesnsivos de los hospitales. Este estudio esta dirigido a lograr una comprension a nivel molecular de dos de los mas importantes mecanismos de resistencia a los farmacos en Pseudomonas aeruginosa. Cien aislados clinicos de Pseudomonas aeruginosa se obtuvieron de un hospital de tercer nivel en Quito, Ecuador. Se analizo la expresion de ampC y oprD mediante PCR cuantitativa en tiempo real. Se realizo una comparacion entre los perfiles de expresion ampC y oprD y los fenotipos obtenidos en la prueba de susceptibilidad antimicrobiana (AST), con mas del 50% de los aislados con perfiles concordantes para la expresion ampC y oprD. Nuestros resultados sugieren que la expresion ampC y oprD podria proporcionar informacion util sobre mecanismos de resistencia molecular en cepas que estan circulando en Ecuador. Sin embargo, los estudios a mayor escala pueden aclarar los mecanismos de resistencia a los farmacos para establecer el tratamiento adecuado. EnglishInnate and acquired antibiotic resistance mechanisms in Pseudomonas present a challenge for clini- cians looking for timely and effective chemotherapy. This is particularly important in critical care hospital settings. This study is aimed at achieving a deeper understanding of two of the most important drug resistance mechanisms in Pseudomonas aeruginosa at the molecular level. One hundred clinical isolates of Pseudomonas aeruginosa were obtained from a tertiary level hospital in Quito, Ecuador. Expression of ampC and oprD was analysed through quan- titative real-time PCR assays. A comparison between the ampC and oprD expression profiles and the phenotypes in antimicrobial susceptibility testing (AST) was conducted, with more than 50% of the isolates having concordant profiles for both ampC and oprD expression. Our results suggest that ampC and oprD expression might provide useful information about molecular resistance mechanisms in strains which are circulating in Ecuador. However, larger scale studies could clarify drug resistance mechanisms in order to guide targeted treatment.

Identification of the Mycobacterium tuberculosis Beijing lineage in Ecuador

INTRODUCTION Mycobacterium tuberculosis Beijing lineage isolates are considered to be especially virulent, transmissible and prone to acquire resistances. Beijing strains have been reported worldwide, but studies in Latin America are still scarce. The only multinational study performed in the region indicated a heterogeneous distribution for this lineage, which was absent in Chile, Colombia and Ecuador, although further studies found the lineage in Chile and Colombia. OBJECTIVE To search for the presence of the Beijing lineage in Ecuador, the only country in the region where it remains unreported. MATERIALS AND METHODS We obtained a convenience sample (2006-2012) from two hospitals covering different populations. The isolates were genotyped using 24-MIRU-VNTR. Lineages were assigned by comparing their patterns to those in the MIRU-VNTRplus platform. Isolates belonging to the Beijing lineage were confirmed by allele-specific PCR. RESULTS We identified the first Beijing isolate in Ecuador in an unexpected epidemiological scenario: A patient was infected in the Andean region, in a population with low mobility and far from the borders of the neighboring countries where Beijing strains had been previously reported. CONCLUSION This is the first report of the presence of the Beijing lineage in Ecuador in an unusual epidemiological context that deserves special attention.