Genome-wide analysis of murine renal distal convoluted tubular cells for the target genes of mineralocorticoid receptor

Background and objective

Mineralocorticoid receptor (MR) is a member of nuclear receptor family proteins and contributes to fluid homeostasis in the kidney. Although aldosterone-MR pathway induces several gene expressions in the kidney, it is often unclear whether the gene expressions are accompanied by direct regulations of MR through its binding to the regulatory region of each gene. The purpose of this study is to identify the direct target genes of MR in a murine distal convoluted tubular epithelial cell-line (mDCT).

Methods

We analyzed the DNA samples of mDCT cells overexpressing 3xFLAG-hMR after treatment with 10⁻⁷ M aldosterone for 1 h by chromatin immunoprecipitation with deep-sequence (ChIP-seq) and mRNA of the cell-line with treatment of 10⁻⁷ M aldosterone for 3 h by microarray.

Results

3xFLAG-hMR overexpressed in mDCT cells accumulated in the nucleus in response to 10⁻⁹ M aldosterone. Twenty-five genes were indicated as the candidate target genes of MR by ChIP-seq and microarray analyses. Five genes, Sgk1, Fkbp5, Rasl12, Tns1 and Tsc22d3 (Gilz), were validated as the direct target genes of MR by quantitative RT-qPCR and ChIP-qPCR. MR binding regions adjacent to Ctgf and Serpine1 were also validated.

Conclusions

We, for the first time, captured the genome-wide distribution of MR in mDCT cells and, furthermore, identified five MR target genes in the cell-line. These results will contribute to further studies on the mechanisms of kidney diseases.     

Ultrasensitive microchip sensor based on boron-containing polyfluorene nanofilms

A fluorene-based π-conjugated copolymer with on-chain dibenzoborole units was used in the development of a nanocoated gold interdigitated microelectrode array device which successfully detects fluoride in a broad range of concentrations (10(-11)-10(-4) M) in aqueous solution, upon impedance spectroscopy measurements. A calibration curve obtained over this range of concentrations and a new analytical method based on impedance spectroscopy measurements in aqueous solution is proposed. The sensor nanofilm was produced by spin-coating and diagnosed via spectroscopic ellipsometry, AFM, and electrically conductivity techniques. Changes in the conductivity due to the boron-fluoride complex formation seem to be the major mechanism behind the dependence of impedimetric results on the fluoride concentration.     

Occurrence of Cephalonomia stephanoderis Betrem (Hymenoptera: Bethylidae) in coffee plantations of the Brazilian Amazonia

Adults of Cephalonomia stephanoderis Betrem were detected (in November 2003) during the sampling of Coffea canephora Pierre ex A. Froehner berries damaged by Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) in Ouro Preto D'Oeste - Rond?nia, Brazil (10 degrees 45'S and 62 degrees 15'W). From January to July 2004, the parasitoid was monthly sampled in 200 berries damaged by H. hampei. Probably, C. stephanoderis can already exert some parasitism pressure at the coffee berry borer population. The occurrence of this parasitoid in natural conditions points out to another alternative for the biological control of the H. hampei in Rond?nia. This is the first record of C. stephanoderis in coffee plantations of the Brazilian Amazonia.     

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli

Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359 bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni²⁺-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.     

Role of the Sln1‐phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis

The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.     

Genomic analysis and clinical management of adolescent cutaneous melanoma

Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising. We describe the clinical course of a 15‐year‐old female diagnosed with AJCC stage IB non‐ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy. We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas. A somatic BRAF^V600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed. A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma‐predisposing genes. Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable.     

生物多样性管理最小有效面积选取模型与GIS的耦合

人口的增长和自然资源的有限性决定了很难把更多的土地用于生物多样性的保护。通过BMAS(生物多样性管理面积选取)模型与GIS(地理信息系统)的结合,可以用尽可能少的土地资源实现一定水平的生物多样性的保护。该方法初步在西双版纳的勐拉县进行了应用。与现实自然保护区面积的对比,用模型选区自然保护区有显著效果。该方法在保护与发展矛盾尖锐的发展中国家非常适用。     

Relationship of genetically modified crops with the environment and health of the Costa Rican human population

Genetic engineering and the food derived from genetically modified crops (GMCs) have been the center of debate worldwide, as has occurred historically with the advent of new technologies. Questions are derived from the potential impact of GMCs to the environment and the safety of the products to the consumers. In relation to the first inquiry, practice has been oriented to a case-by-case-study, according to the own characteristics of the GMC, in order to minimize its impact in the environment. Scientific studies in diverse latitudes of the world have demonstrated that GMCs in the market showed no adverse effects related to this issue. In relation to food derived from the GMCs, rigorous evaluation protocols have been developed and approved by FAO and WHO to guarantee the innocuousness of these products. Up to the moment, no contraindications for human health have been pointed out for the products that are available today in the market. In the particular case of Costa Rica, the country has established since the 90s a regulatory biosafety framework for the management of the GMCs, safeguarding the biodiversity of the country and the health of consumers. At the same time the country has made significant public and private investments in the field that allowed the country to obtain a leading position in biosafety in the region and genetic engineering research at national research centers. Any attempt to restrict or prohibit these activities in the country, will put in risk the previously described investment, will affect the generation of new knowledge for decision making and the leadership in the field, preventing the benefits derived from this promising technology.