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Morphology and Solubility Products of Calcium Arsenates Found in Arsenic Contaminated Soils in an Abandoned Smelter
Get PDF"The dissolution of calcium arsenates in residues released on the premises of an inactive smelter has caused high levels of arsenic pollution in the adjacent down-gradient 6 km of a perched aquifer, reaching up to 158 mg/L of dissolved arsenic, and releasing a total of 7.5 tons of arsenic in a year. We characterized the morphology of the calcium arsenates responsible of this pollution and determined their thermodynamic solubility products by (1) following the dissolution of arsenic from soil samples in batch reactors until equilibrium was reached and (2) modeling dissolution data using the geochemical code PHREEQC. Solubility product calculations took into account soil solution chemistry. Our results provide critical thermodynamic data to better understand interactions between water and arsenic containing minerals in the environment.
Expresión y análisis de actividad biológica de un péptido antimicrobiano Protegrina-1 producido en tabaco por dos sistemas de transformación genética
Get PDFTesis (Doctorado en Ciencias en Biología Molecular)"El uso inadecuado de antibióticos para tratar enfermedades infecciosas se está convirtiendo en un problema de salud pública, al favorecer la emergencia de cepas bacterianas con resistencia a los antibióticos de uso común. Por lo tanto, los antibióticos alternativos son esenciales para estos tratamientos. Los péptidos
antimicrobianos, como la protegrina-1 (PG-1), son una interesante alternativa para hacer frente a este problema. La PG-1 es un péptido antimicrobiano de amplio espectro de origen porcino. En este estudio, se utilizó un sistema de expresión transitoria llamado magnICON, así como la transformación de cloroplastos, con el fin de expresar PG-1 en tabaco (Nicotiana tabacum). En los experimentos de magnICON se utilizaron ensayos de reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) para detectar el replicón viral activo, generando un amplicón de 290 pb, en tanto que en el análisis de transferencia de ARN por la técnica de Northern blot se detectó un producto de 6.1 kb. El análisis de proteína soluble total por medio de geles de SDS/PAGE reveló la presencia de una banda correspondiente al peso molecular de la PG-1 (2.1 kDa), que estaba ausente en el extracto silvestre de tabaco. Se realizaron ensayos antimicrobianos usando el extracto de proteínas de tabaco transformadas transitoriamente, los cuales
demostraron que la actividad del péptido (PG-1) en los tejidos de la planta era factible y contribuyó a la inhibición de 53.2% de Klebsiella pneumoniae, 70.2% de Staphylococcus aureus, 56.6 % de Escherichia coli, 72% de Mycobacterium bovis BCG, y 70% de los cultivos de Candida albicans, lo cual no se detectó en
extractos silvestres de tabaco. Además se expresó la protegrina-1 (PG1) por biobalística en cloroplasto de tabaco. Se usó un vector bicistrónico modificado a partir del vector pKCZ, con el promotor Prrn y la región 5'UTR de la proteína 10 del fago T7, así como el gen de PG-1 fusionado traduccionalmente a la ubiquitina
(Ub::PG-1). El gen (aadA) se incluyó como un marcador de selección. Se bombardearon hojas jóvenes de tabaco, y tras dos rondas de selección se obtuvieron probables plantas transplastómicas que fueron evaluadas por RT-PCR para confirmar la transcripción del transgén. La producción del péptido Ub::PG-1
se confirmó por ensayos de Western-blot usando un anticuerpo anti-ubiquitina. El extracto proteico redujo el crecimiento de los cultivos de K. pneumoniae en un 87.5%, de S. aureus en un 78.7%, de E. coli en un 66.8%, de M. bovis en un 88.8% y de Clavibacter michiganensis (Cmm) en un 57.8%. Además, se realizó un
ensayo de infección contra Cmm en plantas de tabaco transplastómicas que expresan la PG-1; los resultados mostraron una clara disminución de los síntomas desarrollados, en comparación con la planta silvestre. Por lo tanto, la PG-1 producida tanto en células de plantas de tabaco infiltradas como en cloroplastos transformados, es funcionalmente activa y capaz de reducir el crecimiento de varios patógenos humanos y de plantas.""Mammalian infectious diseases are widespread, and some are becoming difficult to control due to inappropriate use of antibiotics. This has contributed to the incidence of bacterial strains with resistance to commonly used antibiotics. Thus, effective alternative antibiotics are essential for treatment of infectious diseases. Antimicrobial peptides are viable alternatives to address this problem. Among those, protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide. In this study, a magnICON and chloroplast transformation systems were used to express the PG-1 peptide in Nicotiana tabacum. For the transient expression system mediated by Agrobacterium (magnICON), reverse-transcriptase polymerase chain reaction (RTPCR) and Northern blot analyses of transformed N. tabacum were employed to detect viral replicons, 290 bp and 6.1 kb. SDS/PAGE revealed the presence of a band corresponding to the molecular weight of PG-1 (2.1 kDa), which was absent in wild-type N. tabacum. Antimicrobial/antifungal assays of protein extracts from transiently transformed N. tabacum were performed, and these demonstrated that PG-1 peptide activity in these plant tissues was viable and contributed to inhibition of 53.2 % of Klebsiella pneumoniae, 70.2 % of Staphylococcus aureus, 56.6 % of Escherichia coli, 72 % of Mycobacterium bovis BCG, and 70 % of Candida albicans cultures. No inhibition of any of these fungal and bacterial pathogens was detected when wild-type N. tabacum extracts were used. In addition we expressed the protegrin-1 (PG1) encoded by a synthetic gene optimized in its codon-usage for tobacco chloroplast expression (N. tabacum var. Petit Havana), using a biolistic method. A bicistronic vector modified from pKCZ was obtained with the Prrn promoter and 5'UTR region of the protein 10 from phage T7, as well as the Protegrin-1 (PG-1) gene fused to ubiquitin. The (aadA) gene was included as a selection marker. Particle bombardment was used in tobacco young leaves and after two rounds of selection, transplastomic plants were obtained. Putative transplastomic lines were evaluated by RT-PCR. The presence of ubiquitinprotegrin protein production was confirmed by Western-blot assay. The protein extract reduced the growth cultures of K. pneumoniae 87.5%, S. aureus 78.7%, E. coli 66.8%, M. bovis 88.8% and Clavibacter michiganensis (Cmm) 57.8%.
Identification of individual and few layers of WS2 using Raman Spectroscopy
Get PDF"The Raman scattering of single-and few-layered WS2 is studied as a function of the number of S-W-S layers and the excitation wavelength in the visible range (488, 514 and 647 nm). For the three excitation wavelengths used in this study, the frequency of the A(1g)(C) phonon mode monotonically decreases with the number of layers. For single-layer WS2, the 514.5 nm laser excitation generates a second-order Raman resonance involving the longitudinal acoustic mode (LA(M)). This resonance results from a coupling between the electronic band structure and lattice vibrations. First-principles calculations were used to determine the electronic and phonon band structures of single-layer and bulk WS2. The reduced intensity of the 2LA mode was then computed, as a function of the laser wavelength, from the fourth-order Fermi golden rule. Our observations establish an unambiguous and nondestructive Raman fingerprint for identifying single-and few-layered WS2 films.
Interacción Dehidrina-Dehidrina: el caso de la OpsDHN1 de Opuntia streptacantha.
Get PDFTesis (Maestría en Ciencias en Biología Molecular)"Factores de estrés tales como la sequía, la salinidad y las bajas temperaturas provocan un déficit hídrico en las plantas, limitando la productividad de los cultivos a nivel mundial. Para hacer frente a dichas condiciones las plantas han desarrollado mecanismos fisiológicos, bioquímicos y moleculares que dirigen el ajuste osmótico induciendo sistemas de reparación, chaperonas y proteínas LEA. Las dehidrinas (DHNs) están involucradas en la resistencia al estrés abiótico en plantas, sin embargo, el mecanismo exacto a través del cual ejercen sus funciones es desconocido. Recientemente, nosotros reportamos la caracterización bioinformática, molecular y funcional de una DHN acida del tipo SK3 (OpsDHN1) de nopal (Opuntia streptacantha). Demostramos que el gen OpsDHN1 está involucrado en la respuesta a estrés abiótico en O. Streptacantha y que la sobreexpresión de dicho gen confiere tolerancia a estrés por frio en plantas de Arabidopsis thaliana. Posteriormente, nuestro grupo encontró que la proteína OpsDHN1 puede interactuar consigo misma en el sistema split ubiquitin en levadura. En este trabajo se caracterizó la interacción OpsDHN1-OpsDHN1 utilizando el sistema dos híbridos split ubiquitin en levadura. Para ello construimos diversas versiones que excluyen regiones definidas de la proteína OpsDHN1 y analizamos si son importantes para la dimerización. Nuestros resultados muestran que la región entre los aminoácidos 97-248 de la OpsDHN1, que incluye los tres segmentos K y el sitio de unión a metales, son claves para la interacción OpsDHN1-OpsDHN1. Estos hallazgos contribuyen al entendimiento del mecanismo molecular de estas proteínas, en donde dicha interacción podría jugar un papel importante en su actividad como chaperona.""Stress factors such as drought, salinity, and low temperatures cause water deficit in plants, limiting worldwide crop production. Plants have evolved physiological, biochemical, and molecular mechanisms that induce osmotic adjustment, such as chaperones and LEA proteins. Dehydrins (DHNs) are involved in the resistance to abiotic stress in plants; however, the exact mechanism by which DHNS exerts their functions is unknown. Recently, we reported the bioinformatics, molecular, and functional characterization of an acidic SK3 DHN isolated from Opuntia streptacantha (OpsDHN1). We show that the OpsDHN1 gene is involved in the response to abiotic stress and gene overexpression confers tolerance to cold stress in Arabidopsis thaliana. Subsequently, our research group found that OpsDHN1 protein could interact with itself in the yeast split ubiquitin system. In this study, we characterized the OpsDHN1-OpsDHN1 interaction using the two-hybrid system in yeast split ubiquitin. For this aim we generated several versions that exclude defined regions of the OpsDHN1 protein and analyze if these regions are important for dimerization. Our results show that the region between amino acids 97-248 of OpsDHN1, which includes three K-segments and the metal binding site are key to OpsDHN1-OpsDHN1 interaction. These outcomes contribute to understanding the molecular mechanism of these proteins, where this interaction may play an important role in chaperone activity.
Germination of species of the tamaulipan thornscrub in a gradient of altitude
Get PDF"El cambio climático se considera una amenaza para la biodiversidad, en especial, para aquellas especies que se localizan en ecosistemas frágiles. La presente investigación tuvo como objetivo estudiar el efecto de la altitud en la germinación y el crecimiento de 10 especies de diferentes poblaciones (localidades) que ocurren en el Matorral Espinoso Tamaulipeco. La información generada permitirá pronosticar los efectos potenciales del cambio climático en la regeneración natural, así como sus posibles desplazamientos a mayores altitudes y definir cuáles son adecuadas para futuras plantaciones. Se escarificaron y sembraron las semillas en tres altitudes: 350, 550 y 1 600 m. La germinación y crecimiento de las plántulas se monitorearon por 30 días. Se observó que Caesalpinia mexicana, Ehretia anacua y Parkinsonia aculeata registraron el porcentaje de germinación mayor en las tres condiciones altitudinales. Las semillas de algunas procedencias de Prosopis glandulosa, Caesalpinia mexicana, Ehretia anacua, Acacia berlandieri y Parkinsonia aculeata presentaron mayor germinación. Lepidium virginicum y Acacia berlandieri registraron, en promedio, los valores más altos a 1 600 msnm. Los resultados sugieren que algunos taxa pueden germinar por encima de su intervalo de distribución actual y quizá tengan la capacidad de desplazarse hacia altitudes superiores por efecto del cambio climático.""Climate change has been considered a threat to biodiversity, especially for those species that are located in fragile ecosystems. The aim of this study was to determine the effect of elevation in seed germination and seedling growth of ten species from different locations that occur in the Tamaulipan thornscrub. The information generated here will help predict potential effects of climate change in natural regeneration, as well as potential plant migration to higher elevations and adequate altitudes for future plantations. Seeds of all species and locations were stratified and sowed in three elevations: 350, 550 and 1 600 m asl. Germination and seedling growth were followed for 30 days. Caesalpinia mexicana, Ehretia anacua and Parkinsonia aculeata had the highest germination percentages in the three altitudes. The seeds of several provenances of Prosopis glandulosa, Caesalpinia mexicana, Ehretia anacua, Acacia berlandieri and Parkinsonia aculeata showed higher germination. Lepidium virginicum and Acacia berlandieri had the highest germination values at 1 600 masl. These results suggest that some species can germinate above their current distribution range, and might have the ability to migrate upslope as a consequence of climate change.
Active sinking at the bottom of the Rincón de Parangueo Maar (Guanajuato, México) and its probable relation with subsidence faults at Salamanca and Celaya
Get PDF"Rincon de Parangueo is a Quaternary maar that had a perennial lake until the 1980s. The lake was gradually desiccated as a consequence of drawdown in the Salamanca-Valle de Santiago regional aquifer and now functions as a playa-lake. In contrast with the features observed in other crater-lakes in the region (La Alberca, Cíntora, and San Nicolás), which also dried up at the same time, the bottom of the Rincón crater displays clear evidence of active deformation associated with mass movement of lake sediments towards the depocenter inside the crater. The most conspicuous topographic feature is a 10 – 12 m high scarp parallel to the former lake coast. The scarp is produced by an annular shaped normal fault system, down towards the depocenter. Evidence of active mass movement is observed along the topographic scarp. Rotational slides associated with rollover anticlines and local grabens produced by antithetic faults are common on the eastern and northern parts of the scarp. Planar slides with open folds at their base occur at western part of the lake basin. Evaporites (trona, thermonatrite, eitelite, halite, and silvite) are abundant in the playa-lake sediments. Their presence makes us believe that a mass removal process is acting as a consequence of salt dissolution and infiltration of the brine towards the aquifer. This process, probably in conjunction with lake sediment compaction and/or diatreme subsidence, may explain the significantly higher fault displacement rate observed inside the crater ( ≈ 50 cm/year ) in comparison with active faults elsewhere in the Salamanca-Valle de Santiago aquifer ( ≈ 6 cm/year ).""Rincón de Parangueo es un maar cuaternario que hasta los años ochenta contenía un lago perenne en su interior. El lago fue gradualmente desecado como consecuencia de sobre-explotación del acuífero regional Salamanca-Valle de Santiago y ahora funciona como un lago-playa. A diferencia de lo que se observa en otros lagos-cráter de la región (La Alberca, Cíntora y San Nicolás) que también fueron desecados en la misma época, en el fondo del cráter de Rincón se pueden apreciar evidencias claras de deformación activa relacionadas al movimiento en masa de los sedimentos lacustres hacia el depocentro en el cráter. El rasgo topográfico más conspicuo es un escarpe de 10 – 12 m de alto, cercano a la antigua línea de costa del lago, que refleja a un sistema de fallas normales escalonadas, con forma anular y bloques hundidos hacia el depocentro. En el escarpe se observan procesos de remoción en masa que incluyen principalmente deslizamientos rotacionales en la porción oriental y septentrional del escarpe y deslizamientos planares al occidente. Asociados a los movimientos rotacionales hay pliegues roll-over y fallas antitéticas que producen grábenes pequeños cerca de los planos principales de falla. Pliegues abiertos y domos se presentan al pie de los deslizamientos planares en la porción occidental del lago desecado. La abundancia de evaporitas (trona, termonatrita, eitelita, halita y silvita) en el fondo del lago asociadas al hecho de que ahora funciona como lago-playa, nos hacen suponer que existe un proceso de remoción de masa causado por la disolución de las sales e infiltración de la salmuera hacia el acuífero. Este proceso, posiblemente asociado a compactación de los sedimentos lacustres y/o de la diatrema que posiblemente subyace al maar, puede explicar la diferencia significativa entre la tasa de desplazamiento en las fallas adentro del cráter ( ≈ 50 cm/año ) con aquellas en otros sitios del acuífero Salamanca-Valle de Santiago ( ≈ 6 cm/año ).
Construcción de vectores para la generación de cepas Knockdown y sobreexpresantes con proteinas etiquetadas en el hongo filamentoso Trichoderma atroviride.
No full textTesis (Maestría en Ciencias en Biología Molecular)"El hongo filamentoso Trichoderma atroviride es utilizado como agente de biocontrol debido a que induce la resistencia sistémica a patógenos en plantas, produce antibióticos y enzimas líticas. Adicionalmente, este hongo se utiliza como modelo fotomorfogénico debido a que conidia en respuesta a un pulso de luz azul. Las proteínas BLR-1, BLR-2 y TGF-1 juegan papeles muy importantes en la respuesta a la luz azul. A pesar de la importancia de éste hongo en la ciencia básica y la aplicada, los estudios a nivel molecular han sido limitados debido a la carencia de herramientas moleculares para su estudio. En el presente trabajo generamos vectores para el reemplazo de genes esenciales o que provoquen fenotipos aberrantes en T. atroviride. Los vectores portan el promotor inducible Pqa-2 del hongo Neurospora crassa (que responde a la presencia del ácido quínico en el medio de cultivo), el terminador TtrpC del hongo Aspergillus nidulans, el marcador de selección hph y las etiquetas FLAG o c-Myc para la obtención de proteínas etiquetadas en sus extremos amino o carboxilo. Los genes blr-2 y tgf-1, fueron clonados en algunas de las cuatro variantes de los vectores, las cuales fueron utilizadas para transformar a T. atroviride, obteniendo un set de cepas portadoras del gen blr-2 o cepas co-transformadas con los genes blr-2 y tgf-1. El análisis de las transformantes mostró fenotipos de hiperconidiación. La conidiación se potenció conforme se incrementó la concentración del inductor en el medio de cultivo, lo que sugirió la posible inducción de la actividad del Pqa-2 modulando la expresión del gen blr-2 o de los genes blr-2 y tgf-1. Estos indican fuertemente que los vectores generados son funcionales en T. atroviride y muy probablemente lo sean en otros hongos filamentosos.""The filamentous fungus Trichoderma atroviride is used as a biocontrol agent due that it induces the systemic resistance to pathogens in plants, produces and secrets a number of antibiotics and lysing enzymes. In addition, this fungus is also used as a photomorphogenic model due that it conidiates in response to a blue light pulse. The BLR-1, -2 and TGF-1 proteins play important roles in blue light response. Despite the importance of this fungus in basic and applied science, its molecular studies have been scarce, because of the lacking of molecular tools. In this work we generated molecular vectors for the replacement of essential genes or those that provokes aberrant phenotypes in T. atroviride. These vector harbors the Pqa-2 inducible promoter from fungus Neurospora crassa, that responds to the addition of quinic acid in the culture medium, them also carry the TtrpC terminator from the fungus Aspergillus nidulans, the hygromycin phosphotransferase selection cassette and sequences that encode for FLAG and c-Myc epitopes to obtain tagged proteins in their respective amino or carboxy termini. The blr-2 or tgf-1 genes were cloned in some of the four variants of the vectors, which were used to transform T. atroviride, obtaining a set of strains bearing the blr-2 gen or both the blr-2 and the tgf-1 genes. Analysis of the transformants showed hyperconidiation phenotypes, which was enhanced as the concentration of quinic acid was increased in the culture medium, which suggested the transcriptional modulation of blr-2 or tgf-1 and blr-2 together under the control of Pqa-2. These data hardly suggest that the system based on Pqa-2 and TtrpC is functional in T. atroviride and probably in other filamentous fungi.
SERS properties of different sized and shaped gold nanoparticles biosynthesized under different environmental conditions by Neurospora crassa extract
Get PDF"Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60 degrees C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these 'green' synthesized gold nanoparticles might have potential applicability in the field of biological sensing.
Novel fusion protein derived from vasostatin 30 and vasoinhibin II-14.1 potently inhibits coronary endothelial cell proliferation
No full text"Angiogenesis has been considered an important target for cancer therapy. The inhibition of angiogenesis represents a promising strategy for anti-cancer treatment, tumor growth inhibition, and metastasis. Vasostatin 30 (Vs30), and the 14.1 kDa vasoinhibin (Vi-II-14.1) are two peptides with remarkable anti-tumor and anti-angiogenic effect. The aim of this study was to produce a novel fusion protein between Vs30 and Vi-II-14.1, denominated VS_VI, to obtain a new protein with higher biological activity. The protein fusion genes were cloned into a T7 promoter-based vector, expressed in Escherichia coli BL21-SI and purified by affinity column chromatography. In vitro assays showed that the recombinant fusion protein inhibited rat coronary endothelial cell proliferation at 65.5 % at 10 nM, whereas recombinant Vs30 and Vi-II-14.1 inhibited at 33 and 50.5 % respectively, at the same concentration. The results showed that VS_VI is significantly more active than the Vs30 and Vi-II-14.1 separately. In addition, a practical classification of the vasoinhibins based on the peptide origin and theoretical molecular weight is proposed. This is the first study to produce a new fusion protein derived from Vs30 and Vi-II-14.1, both of them proposed as promising therapeutic agents.