Genes related to mitochondrial functions are differentially expressed in phosphine-resistant and -susceptible Tribolium castaneum

Background: Phosphine is a valuable fumigant to control pest populations in stored grains and grain products. However, recent studies indicate a substantial increase in phosphine resistance in stored product pests worldwide.Results: To understand the molecular bases of phosphine resistance in insects, we used RNA-Seq to compare gene expression in phosphine-resistant and susceptible laboratory populations of the red flour beetle, Tribolium castaneum. Each population was evaluated as either phosphine-exposed or no phosphine (untreated controls) in triplicate biological replicates (12 samples total). Pairwise analysis indicated there were eight genes differentially expressed between susceptible and resistant insects not exposed to phosphine (i.e., basal expression) or those exposed to phopshine (>8-fold expression and 90 % C.I.). However, 214 genes were differentially expressed among all four treatment groups at a statistically significant level (ANOVA, p < 0.05). Increased expression of 44 cytochrome P450 genes was found in resistant vs. susceptible insects, and phosphine exposure resulted in additional increases of 21 of these genes, five of which were significant among all treatment groups (p < 0.05). Expression of two genes encoding anti-diruetic peptide was 2- to 8-fold reduced in phosphine-resistant insects, and when exposed to phosphine, expression was further reduced 36- to 500-fold compared to susceptible. Phosphine-resistant insects also displayed differential expression of cuticle, carbohydrate, protease, transporter, and many mitochondrial genes, among others. Gene ontology terms associated with mitochondrial functions (oxidation biological processes, monooxygenase and catalytic molecular functions, and iron, heme, and tetrapyyrole binding) were enriched in the significantly differentially expressed dataset. Sequence polymorphism was found in transcripts encoding a known phosphine resistance gene, dihydrolipoamide dehydrogenase, in both susceptible and resistant insects. Phosphine-resistant adults also were resistant to knockdown by the pyrethroid deltamethrin, likely due to the increased cytochrome P450 expression.Conclusions: Overall, genes associated with the mitochondria were differentially expressed in resistant insects, and these differences may contribute to a reduction in overall metabolism and energy production and/or compensation in resistant insects. These data provide the first gene expression data on the response of phosphine-resistant and -susceptible insects to phosphine exposure, and demonstrate that RNA-Seq is a valuable tool to examine differences in insects that respond differentially to environmental stimuli.Peer reviewedEntomology and Plant Patholog

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

<div><p>Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET^® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET^® <i>in vitro</i> performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.</p></div

Overview of ISET^® filtration workflows.

<p>(A) ISET^® workflow for isolation and downstream analysis of fixed Circulating Rare Cells (CRC) from 10 mL of whole blood (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a> section 3 for details). The filter can subsequently be sent by post or stored in biobank for years [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref021" target="_blank">21</a>] for further CRC staining, cyto-morphological analysis and counting, immuno-labeling, <i>in situ</i> hybridization and molecular analyses (with or without laser capture microdissection). (B) ISET^® workflow for dual collection of plasma and enrichment of fixed CRC from whole blood (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a>, section 4 for details). (C) ISET^® workflow for enrichment and downstream analysis of live CRC from whole blood (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#sec002" target="_blank">Methods</a>, section 5 for details). Optionally, single-cells can be isolated by micromanipulation for further analyses or CRC can be purified by immune-magnetic depletion of CD45⁺ cells before further molecular or cell growth assays.</p

Assessment of ISET^® intra-assay accuracy and precision.

<p>50 A549 cells were spiked into 5 mL of blood (n = 3 experiments, 43 to 49 cells per 5 mL). The number of tumor cells found on each spot after ISET^® filtration (each corresponding to the filtration of 1 mL of blood) was recorded. Experiments were done on 5 spots but for intra-assay precision and accuracy only the assessment of the number of cells found on single spots or randomly grouped spots (any 1, any 2, any 3, any 4 spots) is relevant. The cell counting on the combination of all the 5 spots was used as reference. Results show that cell counting on four spots exhibited a representative mean tumor cells value per mL of blood. (A) Bar chart with the mean tumor cell number per spot and corresponding standard error of the mean (error bars) depending on the number of spots analyzed. Error bars are calculated using the standard deviation in different combinations of any 4 spots, any 3 spots, any 2 spots or any 1 spot, respectively. If only one spot is considered, standard deviation is higher than when counting 4 spots, showing that counting on four spots gives a reliable mean tumor cells' value per mL of blood. (B) Table indicating the number of tumor cells found on each spot for each of the five experiments, the 95% confidence interval (CI), the precision (%CV) and the accuracy (%error) depending on the number of spots analyzed (1 to 4) as compared to the analysis on five spots.</p

CTC characterization possibilities after CTC isolation or enrichment by ISET^®.

<p>(A) CTC characterization possibilities after fixed CTC isolation by ISET®. (A1)-Enriched cells are stained on the filter and CCC can be identified by cytopathology [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref016" target="_blank">16</a>] and precisely counted. CTC can also be characterized by simple or multiple immuno-fluorescence-labeling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref011" target="_blank">11</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref018" target="_blank">18</a>], simple or multiple immuno-cytochemistry labeling [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref012" target="_blank">12</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref019" target="_blank">19</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref020" target="_blank">20</a>], or FISH [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref021" target="_blank">21</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref023" target="_blank">23</a>]. (A2) CTC can be characterized by molecular analysis (PCR, next generation sequencing …) after laser microdissection of the filter ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref010" target="_blank">10</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref024" target="_blank">24</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref026" target="_blank">26</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref100" target="_blank">100</a>]). (A3) CTC can be characterized by molecular DNA and RNA analyses without microdissection using sensitive methods for detection of mutation such as Competitive Allele-Specific TaqMan® (CAST)-PCR, co-amplification at lower denaturation temperature (COLD)-PCR, Digital PCR, next generation sequencing, or RT-PCR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref027" target="_blank">27</a>]. (B) CTC characterization possibilities after live CTC enrichment by ISET®. (B1) Enriched CTC are collected in suspension and can be optionally immuno-stained or further enriched by CD45 depletion. CTC can be precisely counted after immune-labeling. (B2) Molecular analysis such as PCR and sanger sequencing, next generation sequencing (this study), RNA analysis, DNA methylation analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref101" target="_blank">101</a>] and proteomic [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169427#pone.0169427.ref102" target="_blank">102</a>] can be targeted to CTC after single cell isolation by micromanipulation (manual or by robot such as CellCelector^TM) or dielectrophoresis (DEPArray^TM). Additionally, mutation detection can be performed without single cell isolation on samples in which CTC have been identified using sensitive mutation-detection methods such as CAST-PCR, COLD-PCR, Digital PCR or next generation sequencing. (B3) Samples can be used for short-term culture, <i>in vivo</i> or <i>in vitro</i> expansion and functional assays.</p

<i>In vitro</i> assay of the repeatability and reproducibility of ISET^® sensitivity tests for isolation of fixed cells.

<p><i>In vitro</i> assay of the repeatability and reproducibility of ISET^® sensitivity tests for isolation of fixed cells.</p

Ion Torrent^TM molecular characterization of single HCT116 and leukocytes enriched from blood using ISET^®.

<p>(A) Non-sense variants mutant allele frequency from Catalogue Of Somatic Mutation In Cancer (COSMIC) database in whole genome amplified DNA from single HCT116 tumor cells (H1 to H3), whole genome amplified DNA single leukocytes (L1 to L3), whole genome amplified bulk HCT116 DNA (WH), unamplified bulk HCT116 DNA (BH) and unamplified bulk extracted DNA from the blood donor (BL). (B) Venn diagram showing the concordance of COSMIC non-sense variants determination in whole genome amplified DNA from single HCT116 tumor cells (H1 to H3) as compared to unamplified bulk HCT116 DNA (bulk HCT116) and whole genome amplified bulk HCT116 DNA (WGA). # indicates SMARCB1 R201* and ## indicate CTNNB1 S45P, NOTCH1 L1574P and RB1 E137*. (C) Venn diagram showing the concordance of COSMIC non-sense variants determination in whole genome amplified DNA from single leukocytes (L1 to L3) as compared to unamplified bulk extracted DNA from the blood donor (bulk).</p

<i>In vitro</i> assay of ISET^® sensitivity using various types of cancer cells.

<p><i>In vitro</i> assay of ISET^® sensitivity using various types of cancer cells.</p

<i>In vitro</i> assay of ISET^® sensitivity for enrichment of live tumor cells.

<p><i>In vitro</i> assay of ISET^® sensitivity for enrichment of live tumor cells.</p