Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

<p>Abstract</p> <p>Background</p> <p>Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean.</p> <p>Results</p> <p>Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes.</p> <p>Conclusions</p> <p>The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean.</p

Understanding the local and remote source contributions to ambient O3 during a pollution episode using a combination of experimental approaches in the Guadalquivir valley, southern Spain

The Guadalquivir Valley is one of three major O3 hotspots in Spain. An airborne and surface measurement campaign was carried out from July 9th to 11th, 2019 to quantify the local/regional O3 contributions using experimental approaches. Air quality and meteorology data from surface measurements, a microlight aircraft, a helium balloon, and remote sensing data (TROPOMI-NO2-ESA) were used to obtain the 3D distribution of O3 and various tracer pollutants. O3 accumulation over 2.5 days started with inputs from oceanic air masses transported inland by sea breezes, which drew O3 and its precursors from a local/regional origin to the northeastern end of the basin. The orographic–meteorological setting of the valley caused vertical recirculation of the air masses inside the valley that caused the accumulation by increasing regional background O3 concentration by 25–30 ppb. Furthermore, possible Mediterranean O3 contributions and additional vertical recirculation through the entrainment zone of the convective boundary layer also contributed. Using particulate matter finer than 2.5 μm (PM2.5), ultrafine particles (UFP), and black carbon (BC) as tracers of local sources, we calculated that local contributions increased regional O3 levels by 20 ppb inside specific pollution plumes transported by the breeze into the valley, and by 10 ppb during midday when flying over an area with abundant agricultural burning during the morning. Air masses that crossed the southern boundaries of the Betic system at mid-altitude (400–1850 m a.s.l.) on July 10th and 11th may have provided additional O3. Meanwhile, a decreasing trend at high altitudes (3000–5000 m a.s.l.) was observed, signifying that the impact of stratospheric O3 intrusion decreased during the campaign

Electrical conductivity measurement: a new technique to detect iatrogenic initial pedicle perforation

Abstract Pedicle screw fixation has achieved significant popularity amongst spinal surgeons for both single and multi-level spinal fusion. Misplacement and pedicle cortical violation occurs in over 20% of screw placement and can result in potential complications such as dysthesia, paraparesis or paraplegia. There have been many advances in techniques available for navigating through the pedicle; however, these techniques are not without drawbacks. A new electrical conductivity-measuring device, previously evaluated on the porcine model to detect the pedicle violation, was evaluated amongst nine European Hospitals to be used in conjunction with the methods currently used in that centre. This new device is based on two original principles; the device is integrated in the drilling or screwing tool. The technology allows real-time detection of perforation through two independent parameters, impedance variation and evoked muscle contractions. Data was collected twofold. Initially, the surgeon was given the device and a comparison was made between the devices ability to detect a breech and the surgeon&apos;s ability to detect one using his traditional methods of pedicle preparation. In the second module of the study, the surgeon was limited to using the electrical conductivity detection device as their sole guide to detect pedicle breaches. A comparison was made between the detection ability of the device and the other detection possibilities. Post-operative fine cut CT scanning was used to detect the pedicle breaches. Overall, the 11 trial surgeons performed a total of 521 pedicle drillings on 97 patients. Initially there were 147 drillings with 23 breaches detected. The detection rate of these breaches were 22/23 for the device compared to 10/23 by the surgeon. Over both parts of the study 64 breaches (12.3%) were confirmed on post-operative CT imaging. The electrical conductivity detection device detected 63 of the 64 breaches (98.4%). There was one false negative and four false positives. This gives the device an overall sensitivity of 98% and specificity of 99% for detecting a pedicle breach. The negative predictive value was 99.8%, with a positive predictive value of 94%. No adverse event was noted with the use of the electrical conductivity device. Electrical conductivity monitoring may provide

Molecular analysis of ex-vivo CD133+ GBM cells revealed a common invasive and angiogenic profile but different proliferative signatures among high grade gliomas

<p>Abstract</p> <p>Background</p> <p>Gliomas are the most common type of primary brain tumours, and in this group glioblastomas (GBMs) are the higher-grade gliomas with fast progression and unfortunate prognosis. Two major aspects of glioma biology that contributes to its awful prognosis are the formation of new blood vessels through the process of angiogenesis and the invasion of glioma cells. Despite of advances, two-year survival for GBM patients with optimal therapy is less than 30%. Even in those patients with low-grade gliomas, that imply a moderately good prognosis, treatment is almost never curative. Recent studies have demonstrated the existence of a small fraction of glioma cells with characteristics of neural stem cells which are able to grow <it>in vitro </it>forming neurospheres and that can be isolated <it>in vivo </it>using surface markers such as CD133. The aim of this study was to define the molecular signature of GBM cells expressing CD133 in comparison with non expressing CD133 cells. This molecular classification could lead to the finding of new potential therapeutic targets for the rationale treatment of high grade GBM.</p> <p>Methods</p> <p>Eight fresh, primary and non cultured GBMs were used in order to study the gene expression signatures from its CD133 positive and negative populations isolated by FACS-sorting. Dataset was generated with Affymetrix U133 Plus 2 arrays and analysed using the software of the Affymetrix Expression Console. In addition, genomic analysis of these tumours was carried out by CGH arrays, FISH studies and MLPA;</p> <p>Results</p> <p>Gene expression analysis of CD133+ vs. CD133- cell population from each tumour showed that CD133+ cells presented common characteristics in all glioblastoma samples (up-regulation of genes involved in angiogenesis, permeability and down-regulation of genes implicated in cell assembly, neural cell organization and neurological disorders). Furthermore, unsupervised clustering of gene expression led us to distinguish between two groups of samples: those discriminated by tumour location and, the most importantly, the group discriminated by their proliferative potential;</p> <p>Conclusions</p> <p>Primary glioblastomas could be sub-classified according to the properties of their CD133+ cells. The molecular characterization of these potential stem cell populations could be critical to find new therapeutic targets and to develop an effective therapy for these tumours with very dismal prognosis.</p

Unveiling the environment and faint features of the isolated galaxy CIG 96 with deep optical and HI observations

Asymmetries in HI in galaxies are often caused by the interaction with close companions, making isolated galaxies an ideal framework to study secular evolution. The AMIGA project has demonstrated that isolated galaxies show the lowest level of asymmetry in their HI integrated profiles, yet some present significant asymmetries. CIG 96 (NGC 864) is a representative case reaching a 16% level. Our aim is to investigate the HI asymmetries of this spiral galaxy and what processes have triggered the star-forming regions observed in the XUV pseudoring. We performed deep optical observations at CAHA 1.23m, 2.2m and VST telescopes. We reach surface brightness (SB) limits of mu_2.2m = 27.5 mag arcsec-2 (Cous R) and mu_VST = 28.7mag arcsec-2 (r) that show the XUV pseudoring of the galaxy in detail. Additionally, a wavelet filtering of the HI data cube from our deep observations with E/VLA telescope allowed us to reach a column density of N_HI = 8.9x10^18 cm -2 (5sigma) (28"x28" beam), lower than in any isolated galaxy. We confirm that the HI extends farther than 4xr_25 in all directions. Furthermore, we detect for the first time two gaseous structures (10^6 Msol) in the outskirts. The g-r colour index image from 1.23m shows extremely blue colours in certain regions of the pseudoring where N_HI>8.5x10^20 cm-2 , whereas the rest show red colours. Galactic cirrus contaminate the field, setting an unavoidable detection limit at 28.5mag arcsec-2 (r). We detect no stellar link within 1degx1deg or gaseous link within 40'x40' between CIG 96 and any companion. The isolation criteria rule out interactions with other similar-sized galaxies for at least 2.7Gyr. Using existing stellar evolution models, the age of the pseudoring is estimated at 1Gyr or older. Undetected previously accreted companions and cold gas accretion remain as the main hypothesis to explain the optical pseudoring and HI features of CIG 96.Comment: 23 pages, 18 figures, 4 table

Determination of bisphenol-type endocrine disrupting compounds in food-contact recycled-paper materials by focused ultrasonic solid-liquid extraction and ultra performance liquid chromatography-high resolution mass spectrometry

Focused ultrasonic solid-liquid extraction (FUSLE) and reverse-phase ultra performance liquid chromatography (UPLC) coupled to a quadrupole-time of flight mass spectrometer (Q-TOF-MS) was applied to the determination of bisphenol-type endocrine disrupting compounds (EDCs) in food-contact recycled-paper materials. Recycled paper is a potential source of EDCs. Bisphenol A (BPA), bisphenol F (BPF) and their derivatives bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) are used for the production of epoxy resins employed in the formulation of printing inks. The FUSLE of bisphenol-type EDCs from packaging is reported for the first time. First, different extraction solvents were studied and methanol was selected. Then, the main FUSLE factors affecting the extraction efficiency (solvent volume, extraction time and ultrasonic irradiation power) were studied by means of a central composite design. The FUSLE conditions selected for further experiments were 20 ml of methanol at ultrasonic amplitude of 100% for 5 s. Finally, the number of extraction cycles necessary for complete extraction was established in two. The analysis of the FUSLE extracts was carried out by UPLC-Q-TOF-MS with electrospray ionization and the determination of the four analytes took place in only 4 min. The FUSLE and UPLC-ESI-QTOF-MS method was validated and applied to the analysis of different food-contact recycled-paper-based materials and packaging. The proposed method provided recoveries from 72% to 97%, repeatability and intermediate precision under 9% and 14%, respectively, and detection limits of 0.33, 0.16, 0.65 and 0.40 g/g for BPA, BPF, BADGE and BFDGE, respectively. The analysis of paper and cardboard samples confirmed the presence of EDCs in these packaging. © 2012 Elsevier B.V