Quantitative real-time PCR with SYBR Green detection to assess gene duplication in insects: study of gene dosage in Drosophila melanogaster (Diptera) and in Ostrinia nubilalis (Lepidoptera)

<p>Abstract</p> <p>Background</p> <p>The accurate determination of the number of copies of a gene in the genome (gene dosage) is essential for a number of genetic analyses. Quantitative real time PCR (qPCR) with TaqMan detection has shown advantages over traditional Southern-blot and FISH techniques, however the high costs of the required labeled probes is an important limitation of this method. qPCR with SYBR Green I detection is a simple and inexpensive alternative, but it has never been applied to the determination of the copy number of low copy number genes in organisms with high allelic variability (as some insects), where a very small margin of error is essential.</p> <p>Findings</p> <p>We have tested the suitability of the qPCR with SYBR Green I detection methodology for the detection of low copy number genes in two insects: the genetically well characterized <it>Drosophila melanogaster </it>(Diptera) and the poor genetically characterized <it>Ostrinia nubilalis </it>(Lepidoptera). The system was applied to determine the copy number of: (1) the <it>O. nubilalis cadherin </it>gene, involved in the mode of action of <it>Bacillus thuringiensis </it>toxins, which showed indirect evidence of duplication, and (2) the <it>D. melanogaster BarH1 </it>and <it>BarH2 </it>genes, located within the <it>Bar </it>region of the X chromosome, to clearly determine whether they both are covered by the tandem duplication in the classical <it>Bar </it>(<it>B¹</it>) mutant. Our results showed that the <it>O. nubilalis cadherin </it>gene is an autosomal single copy gene and that <it>BarH1</it>, but not <it>BarH2</it>, is duplicated in the Drosophila <it>B¹</it>mutant.</p> <p>Conclusions</p> <p>This work shows that qPCR with SYBR Green I detection can be specific and accurate enough to distinguish between one and two gene copies per haploid genome of genes with high allelic variability. The technique is sensitive enough to give reliable results with a minimum amount of sample (DNA from individual thoraxes) and to detect gene duplications in tandem.</p

Bacillus thuringiensis Toxins: Functional Characterization and Mechanism of Action

Bacillus thuringiensis (Bt)-based products are the most successful microbial insecticides to date. This entomopathogenic bacterium produces different kinds of proteins whose specific toxicity has been shown against a wide range of insect orders, nematodes, mites, protozoa, and human cancer cells. Some of these proteins are accumulated in parasporal crystals during the sporulation phase (Cry and Cyt proteins), whereas other proteins are secreted in the vegetative phase of growth (Vip and Sip toxins). Currently, insecticidal proteins belonging to different groups (Cry and Vip3 proteins) are widely used to control insect pests and vectors both in formulated sprays and in transgenic crops (the so-called Bt crops). Despite the extensive use of these proteins in insect pest control, especially Cry and Vip3, their mode of action is not completely understood

Activation of Bacillus thuringiensis Cry1I to a 50 kDa stable core impairs its full toxicity to Ostrinia nubilalis

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products

Study of the bacillus thuringiensis Cry1Ia protein oligomerization promoted by midgut brush border membrane vesicles of lepidopteran and coleopteran insects, or cultured insect cells

Bacillus thuringiensis (Bt) produces insecticidal proteins that are either secreted during the vegetative growth phase or accumulated in the crystal inclusions (Cry proteins) in the stationary phase. Cry1I proteins share the three domain (3D) structure typical of crystal proteins but are secreted to the media early in the stationary growth phase. In the generally accepted mode of action of 3D Cry proteins (sequential binding model), the formation of an oligomer (tetramer) has been described as a major step, necessary for pore formation and subsequent toxicity. To know if this could be extended to Cry1I proteins, the formation of Cry1Ia oligomers was studied by Western blot, after the incubation of trypsin activated Cry1Ia with insect brush border membrane vesicles (BBMV) or insect cultured cells, using Cry1Ab as control. Our results showed that Cry1Ia oligomers were observed only after incubation with susceptible coleopteran BBMV, but not following incubation with susceptible lepidopteran BBMV or non-susceptible Sf21 insect cells, while Cry1Ab oligomers were persistently detected after incubation with all insect tissues tested, regardless of its host susceptibility. The data suggested oligomerization may not necessarily be a requirement for the toxicity of Cry1I proteins.This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities, the State Research Agency of Spain and the European FEDER founds (Refs. AGL2015-70584-C2 and RTI2018-095204-B-C21), and by the Generalitat Valenciana (GVPROMETEOII-2015-001). M. Domínguez received a predoctoral fellowship from the Universidad Pública de Navarra, Spain

Quantitative genetic analysis of Cry1Ab tolerance in Ostrinia nubilalis Spanish populations 2

Abstract 2 Tolerance to Bacillus thuringiensis Cry1Ab toxin in Spanish Ostrinia nubilalis 3 populations was analyzed by quantitative genetic techniques, using isolines established 4 from field-derived insects. F1 offspring was tested for susceptibility to trypsin activated 5 Cry1Ab using a concentration that caused a mean larval mortality of 87% (± 17% SD). 6 The progeny of the most tolerant isolines (that had shown mortalities lower than 60%) 7 was crossed to obtain the F2 generation that was exposed to the same Cry1Ab 8 concentration. A clear reduction in mortality (62% ± 17% SD) was observed. The upper 9 limit for heritability was estimated to range between 0.82 and 0.90, suggesting that a 1

Los Aljezares archaeological site (Alicante, Spain) and the MIS 6/5 open-air settlement in the Iberian Peninsula

The record of open-air Middle Palaeolithic sites in the Iberian Peninsula—specifically in the Mediterranean basin—is scarce, hampering the interpretation of the landscape use strategies developed by Neanderthals in this area. In this work, we present Los Aljezares, a new Middle Palaeolithic site found in Pleistocene fluvio-lacustrine deposits in the sedimentary basin of the Vinalopó River. A U/Th age (132 ± 10 ka) from associated carbonate deposits allows us to attribute the site to the uppermost part of the Middle Pleistocene to Late Pleistocene (marine isotope stage 6/5). To date, a total of two levels of human occupation have been identified in which the density of lithic remains is low compared with cave and rock shelter sites in the region. The first results of technology and use-wear, raw material procurement and geological data indicate a settlement in Los Aljezares along a territory characterised by ephemeral channels and their associated palustrine and lacustrine zones. This palaeoenvironmental setting provided biotic and abiotic resources in a transit area between inland and coastal locations.The research has been funded through the following research projects: El pasado lejano: aproximación a la conducta y la ocupación del territorio en el paleolítico valenciano (PROMETEO/2017/060), Estudio del registro climático reciente preservado en depósitos cuaternarios: el caso de Los Aljezares (Aspe, Alicante) y su relación con los depósitos del Medio y Bajo Vinalopó (GRE17-02), Caracterización tecnológica y funcional de los elementos líticos apuntados durante el Paleolítico medio en la región central del mediterráneo ibérico (GV/2021/054) and Síntesis del Paleolítico medio y superior en Valencia y Murcia: aspectos cronológicos, paleoambientales, económicos y culturales (HAR2017-85153-P)

Comprehensive establishment and characterization of orthoxenograft mouse models of malignant peripheral nerve sheath tumors for personalized medicine

Malignant peripheral nerve sheath tumors (MPNSTs) are soft-tissue sarcomas that can arise either sporadically or in association with neurofibromatosis type 1 (NF1). These aggressive malignancies confer poor survival, with no effective therapy available. We present the generation and characterization of five distinct MPNST orthoxenograft models for preclinical testing and personalized medicine. Four of the models are patient-derived tumor xenografts (PDTX), two independent MPNSTs from the same NF1 patient and two from different sporadic patients. The fifth model is an orthoxenograft derived from an NF1-related MPNST cell line. All MPNST orthoxenografts were generated by tumor implantation, or cell line injection, next to the sciatic nerve of nude mice, and were perpetuated by 7-10 mouse-to-mouse passages. The models reliably recapitulate the histopathological properties of their parental primary tumors. They also mimic distal dissemination properties in mice. Human stroma was rapidly lost after MPNST engraftment and replaced by murine stroma, which facilitated genomic tumor characterization. Compatible with an origin in a catastrophic event and subsequent genome stabilization, MPNST contained highly altered genomes that remained remarkably stable in orthoxenograft establishment and along passages. Mutational frequency and type of somatic point mutations were highly variable among the different MPNSTs modeled, but very consistent when comparing primary tumors with matched orthoxenografts generated. Unsupervised cluster analysis and principal component analysis (PCA) using an MPNST expression signature of ~1,000 genes grouped together all primary tumor-orthoxenograft pairs. Our work points to differences in the engraftment process of primary tumors compared with the engraftment of established cell lines. Following standardization and extensive characterization and validation, the orthoxenograft models were used for initial preclinical drug testing. Sorafenib (a BRAF inhibitor), in combination with doxorubicin or rapamycin, was found to be the most effective treatment for reducing MPNST growth. The development of genomically well-characterized preclinical models for MPNST allowed the evaluation of novel therapeutic strategies for personalized medicine

Encuentro Invesderm. Estableciendo Redes de Investigación en Deontología

Datos técnicos: 388 minutos, color, español. Ficha técnica: Gabinete de Presidencia CSIC y Departamento de Comunicación. Emitido en directo el 5 junio 2024INVESDERM es la primera acción conjunta entre la Academia Española de Dermatología y Venereología (AEDV) y el Consejo Superior de Investigaciones Científicas (CSIC) para establecer puentes entre investigadores básicos y clínicos en el área de la Dermatología y la Venereología. Esta reunión pretende ser una puesta en común de algunos investigadores del CSIC y de la AEDV, contando lo que hacen, lo que cada uno puede ofrecer, las carencias detectadas y las posibilidades de interacción en un contexto de investigación traslacional. El programa de INVESDERM2024 se estructura en 4 mesas redondas dinámicas, en las que los moderadores y ponentes intentarán conectar con los asistentes y motivar la interacción entre todos en los espacios. El objetivo es establecer lazos y gérmenes de colaboración entre investigadores básicos y clínicos.N

Sepharadim/conversos and premodern Global Hispanism

Sepharadim participated in the Hispanic vernacular culture of the Iberian Peninsula. Even in the time of al-Andalus many spoke Hispano-Romance, and even their Hebrew literature belies a deep familiarity with and love of their native Hispano-Romance languages. However, since the early sixteenth century the vast majority of Sepharadim have never lived in the Hispanic world. Sepharadim lived not in Spanish colonies defined by Spanish conquest, but in a network of Mediterranean Jewish communities defined by diasporic values and institutions. By contrast, the conversos, those Sepharadim who converted to Catholicism, whether in Spain or later in Portugal, Italy, or the New World, lived mostly in Spanish Imperial lands, were officially Catholic, and spoke normative Castilian. Their connections, both real and imagined, with Sephardic cultural practice put them at risk of social marginalization, incarceration, even death. Some were devout Catholics whose heritage and family history doomed them to these outcomes. Not surprisingly, many Spanish and Portugese conversos sought refuge in lands outside of Spanish control where they might live openly as Jews. This exodus (1600s) from the lands formerly known as Sefarad led to a parallel Sephardic community of what conversos who re-embraced Judaism in Amsterdam and Italy by a generation of conversos trained in Spanish universities. The Sephardic/Converso cultural complex exceeds the boundaries of Spanish imperial geography, confuses Spanish, Portuguese, Catholic, and Jewish subjectivities, and defies traditional categories practiced in Hispanic studies, and are a unique example of the Global Hispanophone