Insecticidal activity of a protein extracted from bulbs of Phycella australis Ravenna against the aphids Acyrthosiphon pisum Harris and Myzus persicae Sulzer

Aphids cause significant losses in many agricultural crops and in many cases cause repeated insecticide sprays, which increase the risk of resistance. Therefore, other alternatives are needed to control them. The toxic, antireproductive, and feeding deterrent effects of a mannosebinding lectin isolated from bulbs of Phycella australis Ravenna (Amaryllidaceae), named Phycella australis agglutinin (PAA) was assayed on nymphs of the aphids Acyrthosiphon pisum Harris and Myzus persicae Sulzer fed with an artificial diet. After 72 h of PAA exposure, lethal concentration (LC50) values were 109 and 313 μg mL-1 for A. pisum and M. persicae, respectively, while LC90 values were 248 and 634 μg mL-1. Sub-lethal concentrations of PAA significantly reduced the aphid fecundity at a concentration of 80 μg mL-1. Only a total of 5.7 descendants per female were recorded for A. pisum (32% control progeny) and 12.4 for M. persicae (39% control progeny). Acyrthosiphon pisum was strongly deterred by PAA under choice conditions, as after 72 h exposed to 80 μg PAA mL-1 of diet, the feeding deterrent index was 0.91 for A. pisum and only 0.38 for M. persicae. In conclusion, the mannosebinding lectin isolated from bulbs of P. australis showed acute and chronical insecticidal activity against the pea and green peach aphids.

Studying the role of the strawberry Fra protein family in the flavonoid metabolism during fruit ripening

Strawberry fruits are highly appreciated worldwide due to their pleasant flavor and aroma and to the health benefits associated to their consumption. An important part of these properties is due to their content in secondary metabolites, especially phenolic compounds, of which flavonoids are the most abundant in the strawberry fruit. Although the flavonoid biosynthesis pathway is uncovered, little is known about its regulation. The strawberry Fra a (Fra) genes constitute a large family of homologs of the major birch pollen allergen Bet v 1 and for which no equivalents exist in Arabidopsis. Our group has shown that Fra proteins are involved in the formation of colored compounds in strawberries (Muñoz et al., 2010), which mainly depends on the production of certain flavonoids; that they are structurally homologs to the PYR/PYL/RCAR Arabidopsis ABA receptor, and that they are able to bind flavonoids (Casañal et al., 2013). With these previous results, our working hypothesis is that the Fra proteins are involved in the regulation of the flavonoids pathway. They would mechanistically act as the ABA receptor, binding a protein interactor and a ligand to regulate a signaling cascade and/or act as molecular carriers. The main objective of this research is to characterize the Fra family in strawberry and gain insight into their role in the flavonoid metabolism. By RNAseq expression analysis in ripening fruits we have identified transcripts for 10 members of the Fra family. Although expressed in all tissues analyzed, each family member presents a unique pattern of expression, which suggests functional specialization for each Fra protein. Then, our next approach was to identify the proteins that interact with Fras and their ligands to gain knowledge on the role that these proteins play in the flavonoids pathway. To identify the interacting partners of Fras we have performed a yeast two hybrid (Y2H) screening against cDNA libraries of strawberry fruits at the green and red stages. A protein that shares a 95% homology to the Heat stress transcription factor A-4-C like of Fragaria vesca (HSA4C) interacts specifically with Fra1 and not with other family members, which suggests functional diversification of Fra proteins in specific signaling pathways. The Y2H screening is not yet saturated, so characterization of other interacting proteins with other members of the Fra family will shed light on the functional diversity within this gene family. This research will contribute to gain knowledge on how the flavonoid pathway, and hence, the fruit ripening, is regulated in strawberry; an economically important crop but for which basic research is still very limited. References: Muñoz, C, et al. (2010). The Strawberry Fruit Fra a Allergen Functions in Flavonoid Biosynthesis. Molecular Plant, 3(1): 113–124. Casañal, A, et al (2013). The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding Metabolic Intermediates. Journal of Biological Chemistry, 288(49): 35322–35332.

Study of new physiological characteristics as protein receptors (RP55) of light, color, heat, motion, smell, electric, vibration and position located in head, body and abdominal, found in mosquito larvae.

The main objective of the research is to demonstrate new physiological characteristics receptors in the presence of mosquito larvae. 100 larvae of different species were collected and studied for a week in periods of 8-24 hrs. Larvae stages I, II, III and IV have photo-thermo receptors of light and heat housed in the body is divided into head, thorax and abdomen, perceive hot or cold environments, and have fibers in chest or hairs lining your body or abdomen, and a pair of antennae on the head. Stages II and III are more developed than the initial stages. They are attracted by the dark green at the bottom, a pair of eyes that perceive light and color. Have receptors proteins (RP55) that capture motion at a speed the slightest movement of waves in the water. Its nose is not well developed but have chemoreceptors. They adapt to changes in pH in alkaline media, are sensitive to chemical, thermal and mechanical changes nociceptors have electroreceptors or galvanoreceptores sensitive to electrical stimuli, have mechanoreceptors that are sensitive to touch, pain, pressure, gravity, sound. They have a GPS position that seems the guides. It is precisely in the fibers, mushrooms or bristles are recipients along with the micro villi in head, thorax and abdomen.
RESUMEN El objetivo principal de la investigación es demostrar nuevas características fisiológicas como la presencia de receptores en las larvas de mosquitos. Se recolectaron 100 larvas de diferentes especies y se estudiaron por una semana en periodos de 8 a 24 hrs. Las larvas de los estadios I,II,III y IV tienen foto-termo receptores de luz y calor alojados en el cuerpo que se divide en cabeza, tórax y abdomen, perciben ambientes fríos o calientes, así como tienen fibras en tórax o pelos que recubren su cuerpo, y un par de antenas en la cabeza. Los estadios II y III son más desarrollados que las etapas iniciales. Tienen receptores proteicos RP55. Les atrae el color verde oscuro en el fondo, un par de ojos que perciben la luz y color con fotoreceptores. Tienen receptores RP55 de movimiento que captan a una velocidad el más mínimo movimiento de ondas en el agua. Su olfato no está muy desarrollado pero tienen quimioreceptores. Se adaptan a cambios de pH en medios alcalinos, tienen nociceptores sensibles a cambios químicos, térmicos y mecánicos, tienen galvanoreceptores o electroreceptores sensibles a estímulos eléctricos, tienen mecanoreceptores que son sensibles al tacto, dolor, presión gravedad, sonido. Tienen un GPS de posición que pareciera las orienta.

DESIGN, SYNTHESIS AND APPLICATIONS OF FLUORESCENT AND ELECTROCHEMICAL PROBES

“Seeing is believing” the proverb well suits for fluorescent imaging probes. Since we can selectively and sensitively visualize small biomolecules, organelles such as lysosomes, neutral molecules, metal ions, anions through cellular imaging, fluorescent probes can help shed light on the physiological and pathophysiological path ways. Since these biomolecules are produced in low concentrations in the biochemical pathways, general analytical techniques either fail to detect or are not sensitive enough to differentiate the relative concentrations. During my Ph.D. study, I exploited synthetic organic techniques to design and synthesize fluorescent probes with desirable properties such as high water solubility, high sensitivity and with varying fluorescent quantum yields. I synthesized a highly water soluble BOIDPY-based turn-on fluorescent probe for endogenous nitric oxide. I also synthesized a series of cell membrane permeable near infrared (NIR) pH activatable fluorescent probes for lysosomal pH sensing. Fluorescent dyes are molecular tools for designing fluorescent bio imaging probes. This prompted me to design and synthesize a hybrid fluorescent dye with a functionalizable chlorine atom and tested the chlorine re-activity for fluorescent probe design. Carbohydrate and protein interactions are key for many biological processes, such as viral and bacterial infections, cell recognition and adhesion, and immune response. Among several analytical techniques aimed to study these interactions, electrochemical bio sensing is more efficient due to its low cost, ease of operation, and possibility for miniaturization. During my Ph.D., I synthesized mannose bearing aniline molecule which is successfully tested as electrochemical bio sensor. A Ferrocene-mannose conjugate with an anchoring group is synthesized, which can be used as a potential electrochemical biosensor.

Pressure Induced Structural Changes and Gas Diffusion Pathways in Monomeric Fluorescent Proteins

Fluorescent proteins (FPs) are extremely valuable biochemical markers which have found a wide range of applications in cellular and molecular biology research. The monomeric variants of red fluorescent proteins (RFPs), known as mFruits, have been especially valuable for in vivo applications in mammalian cell imaging. Fluorescent proteins consist of a chromophore caged in the beta-barrel protein scaffold. The photophysical properties of an FP is determined by its chromophore structure and its interactions with the protein barrel. Application of hydrostatic pressure on FPs results in the modification of the chromophore environment which allows a systematic study of the role of the protein-chromophore interactions on photophysical properties of FPs. Using Molecular Dynamics (MD) computer simulations, I investigated the pressure induced structural changes in the monomeric variants mCherry, mStrawberry, and Citrine. The results explain the molecular basis for experimentally observed pressure responses among FP variants. It is found that the barrel flexibility, hydrogen bonding interactions and chromophore planarity of the FPs can be correlated to their contrasting photophysical properties at vaious pressures. I also investigated the oxygen diffusion pathways in mOrange and mOrange2 which exhibit marked differences in oxygen sensitivities as well as photostability. Such computational identifications of structural changes and oxygen diffusion pathways are important in guiding mutagenesis efforts to design fluorescent proteins with improved photophysical properties.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.